ZZZ KLFKURP FR XN VDOHV#KLFKURP FR XNfiles.alfresco.mjh.group/alfresco_images/pharma/2018/12/... · 2019. 10. 8. · November 2016 Volume 19 Number 4 PEER REVIEW Detecting low-abundance

November 2016 Volume 19 Number 4

www.chromatographyonline.com

PEER REVIEW

Detecting low-abundance

proteins in serum

GC CONNECTIONS

Gas cylinder safety

COLUMN WATCH

A review of HPLC 2016

Troubleshooting TipsWhat causes peaks to elute before

the column dead time?

9\GDF��$OOWLPD��$OOWLPD�+3��3UHYDLO�$SROOR��$OOVHS��$SH[�DQG�*HQHVLVDQDO\WLFDO�+3/&�FROXPQ�UDQJHV

DFTXLUHG�IURP�*UDFH�$OOWHFKE\�+LFKURP�/LPLWHG

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Features6 Determination of Very Low-Abundance Diagnostic Proteins in

Serum Using Immuno-Capture LC–MS/MS

Léon Reubsaet and Trine Grønhaug Halvorsen

This article reviews immuno-capture LC–MS/MS for protein

analysis in serum.

Columns

21 GC CONNECTIONS

Gas Cylinder Safety, Part 1: Hazards and Precautions

John V. Hinshaw

In the first of a two-part series, this month’s “GC Connections”

examines the principal hazards and safety issues surrounding the

compressed gas cylinder.

26 COLUMN WATCH

Highlights from HPLC 2016

David S. Bell

A review of the recent 2016 HPLC conference in San Francisco,

USA.

Departments33 Products

COVER STORY16 LC TROUBLESHOOTING

Readers’ Questions: Early

Eluted Peak

John W. Dolan

What could be causing a peak to

be eluted before the column dead

time?

November | 2016

Volume 19 Number 4


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Kevin Altria

GlaxoSmithKline, Harlow, Essex, UK

Daniel W. Armstrong

University of Texas, Arlington, Texas, USA

Günther K. Bonn

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3BEJPDIFNJTUSZ �6OJWFSTJUZ�PG�*OOTCSVDL �

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Deirdre Cabooter

Department of Pharmaceutical and

Pharmacological Sciences, University of

Leuven, Belgium

Peter Carr

Department of Chemistry, University

of Minnesota, Minneapolis, Minnesota, USA

Jean-Pierre Chervet

Antec Leyden, Zoeterwoude, The

Netherlands

Jan H. Christensen

Department of Plant and Environmental

Sciences, University of Copenhagen,

Copenhagen, Denmark

Danilo Corradini

*TUJUVUP�EJ�$SPNBUPHSBGJB�EFM�$/3 �3PNF �

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Hernan J. Cortes

H.J. Cortes Consulting,

Midland, Michigan, USA

Gert Desmet

Transport Modelling and Analytical

Separation Science, Vrije Universiteit,

Brussels, Belgium

John W. Dolan

LC Resources, Walnut Creek, California,

USA

Anthony F. Fell

Pharmaceutical Chemistry,

University of Bradford, Bradford, UK

Attila Felinger

Professor of Chemistry, Department of

Analytical and Environmental Chemistry,

University of Pécs, Pécs, Hungary

Francesco Gasparrini

Dipartimento di Studi di Chimica e

Tecnologia delle Sostanze Biologica-

mente Attive, Università “La Sapienza”,

3PNF �*UBMZ

Joseph L. Glajch

Momenta Pharmaceuticals, Cambridge,

Massachusetts, USA

Jun Haginaka

School of Pharmacy and Pharmaceutical

Sciences, Mukogawa Women’s

University, Nishinomiya, Japan

Javier Hernández-Borges

Department of Analytical Chemistry,

Nutrition and Food Science University of

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John V. Hinshaw

Serveron Corp., Hillsboro, Oregon, USA

Tuulia Hyötyläinen

VVT Technical Research of Finland,

Finland

Hans-Gerd Janssen

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Sciences, Amsterdam, The Netherlands

Kiyokatsu Jinno

School of Materials Sciences, Toyohasi

University of Technology, Japan

Huba Kalász

Semmelweis University of Medicine,

Budapest, Hungary

Hian Kee Lee

National University of Singapore,

Singapore

Wolfgang Lindner

*OTUJUVUF�PG�"OBMZUJDBM�$IFNJTUSZ �

University of Vienna, Austria

Henk Lingeman

Faculteit der Scheikunde, Free University,

Amsterdam, The Netherlands

Tom Lynch

BP Technology Centre, Pangbourne, UK

Ronald E. Majors

Analytical consultant, West Chester,

Pennsylvania, USA

Debby Mangelings

Department of Analytical Chemistry

and Pharmaceutical Technology, Vrije

Universiteit, Brussels, Belgium

Phillip Marriot

Monash University, School of Chemistry,

Victoria, Australia

David McCalley

Department of Applied Sciences,

University of West of England, Bristol, UK

Robert D. McDowall

McDowall Consulting, Bromley, Kent, UK

Mary Ellen McNally

DuPont Crop Protection,Newark,

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Imre Molnár

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Luigi Mondello

Dipartimento Farmaco-chimico, Facoltà

di Farmacia, Università di Messina,

.FTTJOB �*UBMZ

Peter Myers

Department of Chemistry,

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Department of Chemistry, University of

Waterloo, Ontario, Canada

Colin Poole

Wayne State University, Detroit,

Michigan, USA

Fred E. Regnier

Department of Biochemistry, Purdue

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Harald Ritchie

Trajan Scientific and Medical, Milton

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Kortrijk, Belgium

Pat Sandra

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Kortrijk, Belgium

Peter Schoenmakers

Department of Chemical Engineering,

Universiteit van Amsterdam, Amsterdam,

The Netherlands

Robert Shellie

Trajan Scientific and Medical,

Ringwood, Victoria, Australia

Yvan Vander Heyden

Vrije Universiteit Brussel,

Brussels, Belgium

Editorial Advisory Board

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Determination of proteins in biological samples using liquid

chromatography–mass spectrometry (LC–MS) is gaining

much interest. An increasing amount of approved protein

biopharmaceuticals, not to mention the abuse of these

compounds in sports, demands the ability to determine them with

a high degree of certainty in biological samples. In addition to this

there is a growing interest in determination of endogenous proteins

in biological samples using LC–MS for diagnostic purposes:

concentration increase or decrease of such proteins allows us to

monitor the state of a pathological condition, including cancer.

Generally speaking, proteins are very diverse with respect

to their biological activity; however, their physicochemical

properties are rather uniform. Additionally, in biological samples

proteins can occur in a broad concentration range. The

concentration of albumin in serum is in the medium g/L level,

while the concentration of interleukin-10 in serum is in the high

10-12 g/L level. Many proteins with diagnostic value occur in the

low to very low abundance range (<10-6 g/L). The diagnostic

value of proteins is determined by the ability to quantify them

robustly so that changes in concentration can be interpreted with

good precision and accuracy. The huge span in concentrations

makes this challenging because these proteins need to be

measured in the presence of other endogenous proteins where

the concentration in some cases is a billion (1012) times higher.

%FUFSNJOJOH�.BSLFST�6TJOH�*NNVOPMPHJDBM�.FUIPET�In clinical laboratories around the world, diagnostic

proteins are determined using immunological methods

such as enzyme-linked immunosorbent assay (ELISA) and

radioimmunoassay (RIA). These are techniques that are based

on the selective capture of target proteins using antibodies. After

the capture and washing steps, the target proteins are quantified

by measuring absorbance, fluorescence, or radioactivity. These

techniques are rather simple, do not need require expensive

equipment, can handle many samples at the same time, and

reach detection limits that allow determination of very low

abundant proteins. However, a disadvantage of this approach

is that the methods are prone to false results: both false positive

(caused by cross reactivity) and false negative (caused by high

dose hook effect) results can occur. This can lead to erroneous

diagnosis with the obvious negative consequences. In addition

to this, most of the assays are not able to distinguish between

isoforms of the target proteins and do not allow the determination

and differentiation of several target proteins in the same assay

(multiplexing) (1–3).

-$m.4�#BTFE�GPS�5BSHFUFE�1SPUFJO�"OBMZTJTTo cope with the disadvantages mentioned previously, the use

of LC–MS in target protein analysis has gained more interest.

There are two main strategies to determine proteins using

LC–MS: the top-down approach and the bottom-up approach.

In the top-down approach, proteins are analyzed intact.

Although feasible, this approach does not allow determination

of very low concentrations. A bottom-up approach (4) is

chosen for quantification most often (see Figure 1). This

approach is based on the enzymatic digestion (mostly trypsin)

of the target protein into lesser peptides. When trypsin is

used as an enzyme for this purpose, the resulting peptides

will mainly be doubly charged and will give rise to good peak

intensities in the mass spectrometer. The peak intensity of a

-ÊPO�3FVCTBFU�BOE�5SJOF�(S�OIBVH�)BMWPSTFO

School of Pharmacy, Department of Pharmaceutical Chemistry, University of Oslo, Blindern, Oslo, Norway

5IF�VTF�PG�BOUJCPEJFT�JO�iCPUUPN�VQu�-$m.4�XPSLGMPXT�UP�EFUFSNJOF�MPX�BCVOEBOU�CJPMPHJDBM�BDUJWF�QSPUFJOT�JO�DPNQMFY�IVNBO�TBNQMFT�IBT�JODSFBTFE�JO�SFDFOU�ZFBST��JNNVOP�DBQUVSF�BOBMZTJT�DPNCJOFT�UIF�XPSLGMPX�PG�DPOWFOUJPOBM�JNNVOPMPHJDBM�BTTBZT�XJUI�-$m.4�BOBMZTJT��5IJT�QBQFS�EFTDSJCFT�UZQJDBM�DIBMMFOHFT �TVDI�BT�DSPTT�SFBDUJWJUZ�BOE�UIF�NBTT�TQFDUSPNFUFS�T�EZOBNJD�SBOHF �BT�XFMM�BT�UIF�BEWBOUBHFT�PG�JTPGPSN�EJGGFSFOUJBUJPO�BOE�NVMUJQMFYJOH��"EEJUJPOBMMZ �TPNF�FYQFSJNFOUBM�GPSNBUT�PG�JNNVOP�DBQUVSF�CPUUPN�VQ�-$m.4�BOBMZTJT�PG�CJPMPHJDBM�BDUJWF�QSPUFJOT�JO�DPNQMFY�IVNBO�TBNQMFT�XJMM�CF�EJTDVTTFE�

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,&:�10*/54t� Immuno-capture bottom-up LC–MS offers the

possibility of less false results.

t�Multiplexing analysis allows simultaneous

determination of several biomarkers.

t� Isovariant and isoform differentiation might allow more

personalized diagnosis.

Determination of Very Low-Abundance Diagnostic Proteins in 4FSVN�6TJOH�*NNVOP�$BQUVSF�-$m.4�.4

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tryptic peptide is higher than that of the undigested protein,

which makes the bottom-up approach attractive because it

gives good detection limits. As a single protein will give rise to

many tryptic peptides and hence a complex chromatogram,

it is therefore important to choose the right tryptic peptides for

quantification.

The amino acid sequence of the tryptic peptide chosen for

quantification needs to be highly specific; in other words, no

Check candidate signaturepeptide specificity

Confirm peptide identityusing MS/MS

Quantify peptide withLC–MS/MS using SRM

Specific peptidesNon-specific peptides

Time

Time

m/z

BLASTTrypsin

Run LC–MS

'JHVSF�� Bottom-up approach in LC–MS-based analysis. Proteins are digested into peptides. Specificity of these peptides for its parent protein can be evaluated using Basic Local Alignment Search Tool (BLAST: http://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins). LC–MS/MS analysis will allow peptide identification and quantification.

Add serum = intact hCG

Peak in

ten

sity

Add buffer+ trypsin

IncubateWashDry

Immunocapture and wash Protein digestion Reversed phase LC–MS sample analysis conditions

uSPEAnalysis

LC–MS/MS

= signature peptideintact hCG

LC–MS/MS signal of tryptic peptides

– Add 100 μL serum to well– Incubate and shade: time 1h

– Wash with PBS/Tween– Wash with PBS– Wash with Tris-HCL– Remove wash buffer after each wash

– Add 200 μL 50 mM ABC– Add 4 μL 50 mM DTT– Mix and incubate: 15 min @ 95 oC

– Add 6 μL 200 mM IAA– Mix and incubate: 15 min @ 20 oC

– Add 4 μL 50 ug/mL trypsin– Mix and incubate: overnight @ 37 oC

Column: 50 x 1 mm, 5-μm BioBasic C8 column 300 ÅMobile phase: 20 mM formic acid and acetonitrileGradient elutionFlow: 40 μL/min

Interface: ESI positive modeMass spectrometer: single quadrupoleSIM mode (m/z values shown in Figure 3)

'JHVSF�� A 96-well plate format for the immuno extraction of hCG from serum. Anti-hCG antibodies are immobilized on the wall of a microtitre plate. hCG in serum will be captured by these antibodies and remain in the well after extensive washing. Trypsin will digest the captured hCG, yielding a clean sample, after which μSPE can be injected into the LC–MS system. Typical experimental conditions are given in the lower part of the figure [from (14)].

9XXX�DISPNBUPHSBQIZPOMJOF�DPN


other protein in the sample should produce a peptide with

the same amino acid sequence. Such specific peptides are

designated as signature peptide or proteotypic peptide: the

parent protein can be determined by means of this signature

peptide. This peptide needs to be specific and, preferably, gives

a high peak intensity, has good chromatographic properties, is

the result of a full digest (no cleavage sites left in the peptide),

and is reproducible.

The value of the mass spectrometer becomes clear when

the identity of the signature peptide needs to be confirmed and

the concentration needs to be determined. Confirmation of the

identity is perfomed by both mass (m/z value) and fragmentation

pattern. For this purpose most tandem mass spectrometers

are suited (5). Quantification is preferably performed on a triple

quadrupole mass spectrometer in the selected reaction mode

(SRM) mode because this is still the most sensitive way to

determine peptides; the mass spectrometer is highly selective in

both confirmation and quantification of the signature peptide —

false positives as a result of other cross reacting proteins will be

avoided (6).

$IBMMFOHFT�8IFO�"OBMZ[JOH�1SPUFJOT�JO�4FSVNMeasuring a target protein in a buffered solution with

LC–MS after a bottom-up pre-treatment is often a

straightforward procedure. Detection limits down to the

femtogram level can be reached (of course, this depends on

the sensitivity of the instrument used) without doing specific

clean up and/or enrichment. This ideal situation changes

rapidly when the same target protein needs to be determined

in a biological matrix like serum or urine. Without any special

treatment, such as clean-up or enrichment, a serum sample

will generate a huge amount of peptides as well as many

different peptides when tryptic digestion is performed. This

causes a great deal of trouble for the mass spectrometer

because its ability to determine low concentration and high

concentration simultaneously is limited by a limited dynamic

range. In addition, ion suppression occurs in the electrospray

when the signature peptide of the target protein coelutes with

other, high concentration, peptides. To be able to perform

robust quantitative analysis of target proteins in the very low

abundance range using LC–MS sample preparation, as well

as trypsination, is required.

4BNQMF�1SFQBSBUJPO�5FDIOJRVFT�GPS�5BSHFU�1SPUFJO�-$m.4�"OBMZTJTThere are several techniques described to perform sample

cleanup and enrichment. The choice of the sample preparation

strategy depends very much on the concentration of the target

protein. In some cases the concentration of the target protein is

so high that direct injection of a digested serum sample in the

LC–MS could be performed. When the concentration becomes

lower, sample cleanup, such as depletion (7), precipitation and

restricted access material (RAM) (8), affinity cleanup (9–11), and

molecular imprints (12) is used.

hCG varient

hCG varient VALPALPQVVCNYR m/z=765.42+

m/z=914.52+

m/z=955.52+

m/z=964.02+

hCG nicked 44/45 LQGVLPAPQVVCNYR

hCG β-core GVNPVVSYAVALSCQCAL

hCG intact VLQGVLPAPQVVCNYR

Signature peptide Extracted ion chromatogram

'JHVSF�� Four of the isovariants of hCG. All of these have their specific signature peptides with their specific m/z value and retention behaviour. Within a single analysis these four isovariants can be monitored simultaneously (14).

17500

15000

12500

10500

7500

5000

2500

5.0 7.5 10.0 12.5 15.0 17.5

Time (min)

Inte

nsi

ty

15000

12500

10500

7500

5000

2500

Inte

nsi

ty

T5

T5

cfT9

nT5’

(a)

(b)

20.0 22.5

5.0 7.5 10.0 12.5 15.0 17.5

Time (min)20.0 22.5

'JHVSF�� (a) Chromatogram for the analysis of the urine of a pregnant woman in the third month. The peaks correspond to the signature peptides for intact hCG (T5) and core fragment hCG (cfT9). (b) Chromatogram from the analysis of a cancer patient serum sample. The peaks correspond to the signature peptides for intact hCG (T5) and hCG nicked 44/45 (nT5’). Adapted with permission from reference 14.

-$r($�"TJB�1BDJàD November 2016��


Affinity cleanup can be performed by either using antibodies,

which have a high affinity against the parent protein, or by using

antibodies, which have a high affinity against the signature

peptide (SISCAPA) (13).

In this overview the focus will be on the use of antibodies

directed against the whole protein in the sample cleanup and

enrichment. The article is meant to provide an introduction to

the experimental approach of immuno-capture LC–MS, rather

than being a comprehensive review of all work done in the field.

Immuno-capture LC–MS has much less disadvantages compared

to immunological assays This is because mass spectrometric

detection is much more selective than the immunological assays:

a false positive analysis in an immunological assay, caused by

cross reactivity, will not be false positive in an LC–MS analysis: the

signature peptide for the target protein will not be present in the

cross-reacting protein and will not therefore generate a signal.

There are several ways to use antibodies to enrich target

proteins. In general the principle is the same: the antibody

is immobilized on a solid support, which can be the wall of

a 96-well plate (14), on sorbents packed in a small column

(15), or on small magnetic beads (16). The biological sample

is incubated with the antibodies thereby allowing the target

protein and the antibodies to interact. After this incubation,

the excess of biological sample is washed away. After some

repetitive washing steps the target protein is either eluted and

digested, or digested directly into its tryptic peptides before

final analysis.

)PX�%PFT�*NNVOP�$BQUVSF�'VODUJPO�JO��8FMM�'PSNBU Figure 2 shows the workflow as well as the typical

experimental conditions of targeted LC–MS analysis on

human chorionic gonadotropin (hCG) using antibodies

immobilized on the walls of a 96-well plate. hCG is used as

a marker to diagnose ovarian or testicular cancer. It is also

on the World Anti-Doping Agency’s list of prohibited

substances. It is therefore of interest to be able to measure

this protein. In the procedure shown antibodies (anti-hCG)

are immobilized by incubating a solution in the wells. After

blocking and washing the wells, they are ready to use. In this

example serum is applied to the wells and allowed to incubate

for 1 h. After washing and drying, ammonium bicarbonate

buffer is added to the wells and a standard procedure to

reduce, alkylate, and digest is performed. In other words,

the protein is digested in the well without prior elution of

the target protein from the antibody. Since the volume of

the digestion buffer must be the same as the volume of the

applied serum sample, only cleanup is achieved and no

anti-ProGRP

anti-ProGRP

Addition ofmagnetic beads

to serum

Separation anddetection by

LC–MS/MS (SRM)

ProGRP

100

95

90

85

80

75

70

65

60

55

50

45

40

35

30

25

20

15

10

15 16 17 18 19 20 21 22 23 24 25 26 27 28

Time (min)

Rela

tive A

bu

nd

an

ce

29

22.3

26.8

22.3

15.0

30

5

0

Pro

GR

P iso

form

1LS

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� 5

44.4

IS t

ota

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GR

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form

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59.9

magnetic bead

magnetic bead

ddAddiAddiAddititiontion ffof ofmagnetic beads

to serum

Immunocapture and wash Protein digestion Reversed phase LC–MS sample analysis conditions

– Add 20 μL anti ProGRP-magnetic beads and 1 mL serum to vial– Incubate and shake: time 1h @ 20 oC

– Retain beads with magnet and remove serum

– Wash with PBS/Tween– Wash with PBS– Wash with Tris-HCL– Wash with 50 mM ABC– Remove solutions after each wash

– Add 70 μL 50 mM ABC– Add 5 μL 100 mM DTT– Mix and incubate: 15 min @ 60 oC

– Add 5 μL 400 mM IAA– Mix and incubate: 15 min @ 20 oC

– Add 10 μL 50 ug/mL trypsin– Add 10 μL internal standard– Mix and incubate: overnight @ 37 oC

Column: 50 x 1 mm, 3-μm C8 column 300 ÅMobile phase: 20 mM formic acid and acetonitrileGradient elutionFlow: 40 μL/min

Interface: ESI positive modeMass spectrometer: triple quadrupoleMRM mode (m/z transitions shown in chromatogram)

a

mag

Immuno-capture

Matrixremoval

Trypticdigest

Wash

'JHVSF�� Immuno-capture of ProGRP using anti-ProGRP antibodies coupled to magnetic beads. Large sample volumes can be used in the capture of ProGRP. Using a magnet, the beads, with the captured ProGRP, can be retained. After extensive washing trypsin can be used to digest ProGRP. This digested sample can be injected into the LC–MS system. This approach allows determination of the total amount of ProGRP, as well as quantification of ProGRP isoform 1 and ProGRP isoform 3. Internal standard (IS) for total ProGRP is included in this method (23). Typical experimental conditions are given in the lower part of the figure. The chromatogram shown is that of a spiked serum sample.

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enrichment. The latter might be achieved by a simple μSPE

step using reversed-phased material (C8 or C18) to retain

tryptic peptides (14).

A similar procedure is described for biomarkers like ProGRP

(17) (small cell lung cancer), PSA (18) (prostate cancer), and

protein therapeutics (19).

In the example from Figure 2 the amount of hCG is

determined by measuring the amount of its signature peptide.

Although hCG is often mentioned as one protein, it consists

of an α-chain and a β-chain. The α-chain occurs in hCG and

in other proteins. However, the β-chain is specific for hCG,

and thus does not occur in other proteins. Aside from the fact

that hCG is a heterodimeric protein, variations in the β-chain

occur, giving rise to several isovariants. These isovariants

have mainly the same amino acid sequence but there are

also differences. A conventional immunological method would

allow determination of the amount of total hCG, but would not

be able to differentiate between these variants. One of the

biggest advantages of using immuno-capture LC–MS is that

it can differentiate between various isovariants in a single run.

Figure 3 shows a table of the amino acid sequence of four

of these β-chain isovariants of hCG. Each isovariant has its

own signature peptide with MS/MS fragments and a specific

retention time. From the chromatogram in the same figure it

Addition ofmagnetic beads

ProGRP

anti-ProGRP

magnetic bead

NSE

anti-NSE

magnetic bead

ProGRP

anti-ProGRP

magnetic bead

NSNSNSNSNSEEEEE

ananananantititititi-N-N-NNNSESESESESE

mamamamamagngngngngneteteteteticicicicic b bbbbeaeaeaeaeaddddd

Immuno-capture

Matrixremoval

Trypticdigest

Separation anddetection by

LC–MS/MS (SRM)

Wash

1514 16 17 18 19 20 21 22 23Time (min)

24 25 26 27 28 29

100

90

80

70

60

50

40

30

20

10

Rela

tive A

bu

nd

an

ce

0

Pro

GR

P iso

form

1

IS t

ota

l Pro

GR

P

tota

l Pro

GR

P

Pro

GR

P iso

form

3

NSE a

-su

bu

nit

TIA

PALV

SK

450/6

6

84.4

IS N

SE y

-su

bu

nit

NSE y

-su

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nit

ELP

LYR

395.7

548.3

'JHVSF�� Multiplexing: simultaneous immuno-capture of ProGRP (and its isoforms) and NSE (and its various subunits) using a mixture of anti-ProGRP antibodies coupled to magnetic beads and anti-NSE antibodies coupled to magnetic beads. After the beads are added to the sample, the procedure is similar to that shown in Figure 5. LC–MS analysis allows simultaneous determination of total ProGRP, ProGRP isoform 1, ProGRP isoform 3, NSE α-subunit, and NSE γ-subunit. Internal standards (IS) for total ProGRP and NSE γ-subunit are included in this method (28). Typical experimental conditions for such an experiment are given in the lower part of Figure 5. The only difference is that besides 20 μL of anti ProGRP beads, 20 μL of anti NSE beads are also added to the serum sample. The chromatogram shown is that of a spiked serum sample. m/z transitions for the NSE subunits are shown in the chromatogram.

Isoform 1

MRGRELPLVLLALVLCLAPRGRAVPLPAGGGTVLTKMYPRGNHWAVGHLMGKKSTGESSSVS

ERGSLKQQLREYIRWEEAARNLLGLIEAKENRNHQPPQPKALGNQQPSWDSEDSSNFKDVG

SKGKVGRLSAPGSQREGRNPQLNQQ

Isoform 2


ERGSLKQQLREYIRWEEAARNLLGLIEAKENRNHQPPQPKALGNQQPSWDSEDSSNFKDVG

SKGKVGRLSAPGSQREGRNPQLNQQ

Isoform 3


ERGSLKQQLREYIRWEEAARNLLGLIEAKENRNHQPPQPKALGNQQPSWDSEDSSNFKDLV

DSLLQVLNVKEGTPS

'JHVSF�� Amino acid sequence of three ProGP isoforms. Each isoform contains the sequence NLLGLIEAK (in bold), which is used to determine the total amount of ProGRP. Isoform 1 and 3 each have a detectable signature peptide (LSAPGSQR for isoform 1 and DLVDSLLQVLNVK for isoform 3) that allows differentiation.

��XXX�DISPNBUPHSBQIZPOMJOF�DPN


can be seen that each isovariant produces its own peak. When

using the procedure shown in Figure 2 on urine and serum,

both intact hCG and some of the isovariants are detected

(Figure 4).

Although very clean chromatograms are obtained using

antibodies immobilized on the walls of a 96-well plate, there is a

drawback. Since the volume of the well is approximately

200–250 μL, it is not possible to use sample volumes higher

than this. In some cases it is advantageous to use high volumes

because the concentration and therewith the amount of target

protein can be very low in the biological sample. This may be

circumvented by the immobilization of antibodies on the surface

of magnetic beads.

*NNVOP�$BQUVSF�6TJOH�"OUJCPEJFT�*NNPCJMJ[FE�PO�.BHOFUJD�#FBET�Magnetic beads are small spherical beads with a uniform

size distribution (mainly 2.8 μm). The immuno-active magnetic

beads can be pipetted into a serum sample of which the

volume can be up to 1 mL (or even higher). Extraction of the

beads (and therewith the target protein) is performed using a

magnet (16, 20–22). Figure 5 shows the extraction of ProGRP

(23). Experimental conditions, workflow, and a typical result

are included in this figure. ProGRP is a highly selective and

sensitive biomarker for small cell lung cancer. It is not only used

to diagnose the disease, but also to monitor the success of the

treatment.

As can be seen from this figure, the principle of the

extraction is comparable to that of the 96-well plate extraction.

The biggest differences are the volume of the sample, which

can be increased, and also the enrichment of the target

protein. It is possible to start with 1 mL of serum sample from

which the extraction is performed. This is done by gently

shaking for 1 h (at room temperature [RT]). The magnet is

used to retain the beads when the sample is removed. After

washing the beads, the digestion can be performed in a much

smaller volume such as for 80 μL, which would give a potential

enrichment of 12.5. An additional solid-phase extraction (SPE)

step would provide an even higher enrichment. In this example

digestion is performed directly on-beads, meaning that no

elution of the target protein is performed.

In the case of ProGRP the possibility of using larger sample

volumes is of great importance: the concentration of this protein

usually does not exceed 60 pg/mL and increases in the case

of pathology. With 96-well based immuno-capture the amount

of protein that could be captured was simply too low to be

determined. However, using a whole millilitre of serum allows

a much better detection limit, making it possible to determine

even non-pathological levels.

In addition, in the case of ProGRP, isoforms also occur. The

conventional immunological assays will only be able to determine

the amount of total ProGRP; with the immuno-MS approach

some isoforms can be differentiated. Figure 6 shows a table of

the amino acid sequence of the three known isoforms of ProGRP.

With the chosen approach one can determine both ProGRP

isoform 1 and isoform 3 (for isoform 2, no signature peptide was

identified) in addition to the amount of total ProGRP through a

signature peptide, which is common for all three isoforms.

>ƵĚŐĞƌ�Ɛ�ĞǆƉĞƌƟƐĞ�ĂŶĚ�Yď��ĂƉƉƌŽĂĐŚ�ƚŽ�ĚƌƵŐ�ŐůǇĐŽƐǇůĂƟŽŶ�ĂŶĂůǇƐŝƐ�ĐĂŶ�ŚĞůƉ�ǇŽƵ�ĂĐŚŝĞǀĞ�ƐƵĐĐĞƐƐ��

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www.ludger.com

Are you seeking regulatory approval for your glycosylated biopharmaceutical?

ΎĂƐ�ũƵĚŐĞĚ�ďǇ�'ůŽďĂů�ĂƚĂ�ĨƌŽŵ�ϮϬϭϰ�ŐůŽďĂů�ƐĂůĞƐ

Kits & Reagents ZĞůŝĂďůĞ�ƚĞĐŚŶŽůŽŐǇ�ĨŽƌ�ŐůǇĐĂŶ�ƐĂŵƉůĞ�

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Dr. Louise Royle+HDG�RI�*O\FRSURÀ[email protected]

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Besides immobilization of antibodies on magnetic beads,

there are other formats described in the literature: Griffiths et al.

used agarose beads as a solid support for factor VIII inhibitor

analysis with MS (24); Lin et al. used latex beads as solid support

for the immune-precipitation of several biomarker candidates

(25); Sherma et al. used affinity pipette tips to enrich proteins

before analysis (26); and Baily-Chouriberry et al. used anti-EPO

monoliths for enrichment and subsequent MS analysis (27).

4JNVMUBOFPVT�*NNVOP�$BQUVSF�BOE�%FUFSNJOBUJPO�PG�.VMUJQMF�%JBHOPTUJD�.BSLFSTAnother advantage, as well as a better insight into the

distribution of several isoforms, is that immune-extractions

using magnetic beads can be performed without considerable

volume loss. The patient sample can be re-used for other

analyses: After extraction of the target protein, all the other

components are still present in the serum sample. Therefore,

additional analyses on the same sample can be performed.

Using magnetic beads, these additional analyses can be

combined in a single sample cleanup and enrichment and

analysis (28,29).

Figure 7 shows the multiplexed determination of both

ProGRP and neuron-specific enolase (NSE). Experimental

conditions, workflow, and a typical result are included in

the figure caption. This multiplex is based on merging two

existing validated methods for the determination of ProGRP

(23) and NSE (20). By using a mix of two types of modified

magnetic beads (one coated with anti-ProGRP and one

coated with anti-NSE), these proteins can be extracted from a

serum sample simultaneously. One reason to determine both

proteins is that NSE, like ProGRP, is a diagnostic marker for

small cell lung cancer. In other words, by doing this multiplex

determination, a more robust diagnosis can be performed.

As this dual extraction gives exactly the same results as two

separate extractions, the time gain is also considerable. The

method shown here was used to determine both ProGRP,

its isoforms, and the NSE isoforms in samples from cancer

patients. There was good correlation between data obtained

from the conventional immunological method and the multiplex

LC–MS method. In addition, an isoform distribution was

determined (28). The clinical relevance of this remains to be

explored, however, one can imagine that additional diagnostic

proteins can be added to this test panel, potentially giving a

differentiated diagnosis of the patients’ state.

4FRVFOUJBM�*NNVOP�$BQUVSF�GPS�%JGGFSFOUJBUJPO�PG�)FUFSPEJNFSJD�.BSLFSTThe possibility of re-using the patient sample after the initial

immuno-capture step also opens up the potential for sequential

analysis of biomarkers that cannot be separated by LC–MS/

MS. An example of this is differentiation between intact hCG and

its free hCGβ-subunit. This could be of interest both in cancer

diagnosis and doping analysis. As mentioned earlier, hCG is a

dimer consisting of an α- and a β-subunit, where the β-subunit

is specific for hCG and therefore the signature peptide used

for quantification must be derived from the β-subunit. This

makes differentiation and quantification of intact hCG and

the free β-subunit difficult using a single immuno-capture

step. Currently, sequential immuno-capture is described for

differentiation of intact hCG, hCGβ and hCGβ-core in urine

(only), by first depleting urine for the free hCGβ-variants using

an antibody selective to the β-subunit prior to extracting the

remainder variants using an antibody able to capture all hCG

variants (21). The captured intact and free variants are further

processed and analyzed in two separate runs.

$BMJCSBUJPO�4USBUFHJFT�GPS�"DDVSBUF�1SPUFJO�%FUFSNJOBUJPOAlthough the subject of calibration is not within the scope

of this overview, it is important to be aware that

quantification in targeted protein analysis is not as

straightforward as quantification of small molecules. To

be able to perform an accurate quantitative analysis of

the target protein(s), there are several internal standard

calibration strategies that can be applied: AQUA (Absolute

QUAntification), PSAQ (Protein Standard for Absolute

Quantification) and QconCAT (concatamer of standard

Q-peptides). The goal of these strategies is to eliminate

variations in the analytical process, which comprises

capture, cleanup, digestion, SPE, and an LC–MS step.

Table 1 shows which of the analytical variations are

eliminated per standard calibration strategy.

$PODMVTJPOT�Combining immuno-capture with LC–MS has resulted in an

approach with sensitivity in the same range as immunological

assays. However, it can be said that detection limits are still

slightly better for the immunological assays, although, with

the current development in MS, there will soon no longer

be any difference. Disadvantages of the immuno-capture

LC–MS approach compared to the immunological methods

are high operational costs and the need for highly trained

personnel, both as a result of the LC–MS instrument. One

might argue that this approach is only feasible for exploratory

biomarker research and less suited for low-cost routine

determination. On the other hand, immuno-capture LC–MS

allows various isoforms to be differentiated between without

using isoform specific antibodies. This potentially allows for

more differentiated diagnosis. The possibility of determining

many biomarkers at the same time in the same analysis not

only saves time, but also gives a more robust diagnostic profile

of a patient. Although immuno-capture LC–MS also uses

antibodies in the sample cleanup, it is much less, or not at all,

prone to false positive results because the mass spectrometric

detection is very specific. Efforts are being made to perform

LC–MS analysis without the use of the immuno-capture

as clean-up. Although protein determination is possible, it

5BCMF�� Analytical variation correction

7BSJBUJPO*OUFSOBM�TUBOEBSE

PSAQ QconCAT AQUA

Incomplete capture � � �

Protein loss during washing � � �

Incomplete digestion � � �

Variable digestion � � �

Variable SPE recovery � � �

Injection variability � � �

MS variability � � �

Multiplexing �* � �*

*: multiplexing can be carried out using this standard, however, for each protein

to be determined, an internal standard needs to be added to the sample.


(19) W. Yang, R. Kernstock, N. Simmons, and A. Alak, Bioanalysis �,

307–318 (2015).

(20) S.B. Torsetnes, S.G. Lovbak, C. Claus, H. Lund, M.S. Nordlund, E.

Paus, et al., J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. ��,

125–132 (2013).

(21) G.A. Woldemariam and A.W. Butch, Clin. Chem. ��, 1089–1097 (2014).

(22) M. Vogel, M. Blobel, A. Thomas, K. Walpurgis, W. Schänzer, C.

Reichel, et al., Anal. Chem. 86(24), 12014–12021 (2014).

(23) S.B. Torsetnes, M.S. Nordlund, E. Paus, T.G. Halvorsen, and L.

Reubsaet, J. Proteome Res. ��, 412–420 (2013).

(24) A.E. Griffiths, W. Wang, F.K. Hagen, and P.J. Fay, J. Thromb

Haemost. 9, 1534–1540 (2011).

(25) D. Lin, W.E. Alborn, R.J.C. Slebos, and D.C. Liebler, J. Proteome

Res. ��, 5996–6003 (2013).

(26) N. Sherma, C. Borges, O. Trenchevska, J. Jarvis, D. Rehder, and P.

Oran et al., Proteome Sci. ��, 1–12 (2014).

(27) L. Bailly-Chouriberry, F. Cormant, P. Garcia, M. Lonnberg, S.

Szwandt, U. Bondesson, et al., Analyst. ��, 2445–2453 (2012).

(28) S.B. Torsetnes, M.S. Levernæs, M.N. Broughton, E. Paus, T.G.

Halvorsen, and L. Reubsaet, Anal. Chem. 86, 6983–6992 (2014).

(29) R. Villar-Vázquez, G. Padilla, M.J. Fernández-Aceñero, A. Suárez, E.

Fuente, C. Pastor, et al., Proteomics ��(8), 1280–90 (2016).

-ÊPO�3FVCTBFU�is professor in pharmaceutical analysis at the

School of Pharmacy, University of Oslo, Norway. He works with

mass spectrometry as a diagnostic tool for the determination of

protein biomarkers in complex biological samples. Improvement

of sample handling, sample preparation, and bottom-up LC–MS

strategies are his main focus.

5SJOF�(S�OIBVH�)BMWPSTFO is an associate professor in

pharmaceutical analysis at the School of Pharmacy, University

of Oslo, Norway. Her research interests include development

of new strategies for analysis of protein biomarkers in biological

matrices by LC–MS/MS with a main focus on novel sampling

materials and sample preparation of low abundance markers.

does not come close to the detection limit needed for low

abundance biomarker determination.

All in all, immuno-capture LC–MS is potentially a very

powerful tool in the field of diagnostics.

3FGFSFODFT(1) S. Dodig, Biochemia Medica ��, 50–62 (2009).

(2) N. Jassam, C.M. Jones, T. Briscoe, and J.H. Horner, Ann. Clin.

Biochem. ��, 314–317 (2006).

(3) A.N. Hoofnagle and M.H. Wener, J. Immunol. Methods ��, 3–11

(2009).

(4) R. Aebersold and M. Mann, Nature ��, 198–207 (2003).

(5) A. Doerr, Nat. Meth. ��, 23 (2013).

(6) P. Picotti and R. Aebersold, Nature Methods 9, 555–566 (2012).

(7) L. Anderson and C.L Hunter, Mol. Cell. Proteomics �, 573–588

(2006).

(8) B. Winther, P. Moi, E. Paus, and J.L.E. Reubsaet, J. Sep. Sci. ��,

2638–46 (2007).

(9) D. Nedelkov, Expert Rev. Proteomics �, 631–40 (2006).

(10) B.L. Ackermann and M.J. Berna, Expert Rev. Proteomics �, 175–86

(2007).

(11) W.H. Dunham, M. Mullin, and A.C. Gingras, Proteomics��, 1576–90

(2012).

(12) C. Rossetti, A. Abdel Qader, T.G. Halvorsen, B. Sellergren, and L.

Reubsaet, Anal. Chem. 86, 12291–12298 (2014).

(13) N.L. Anderson, A. Jackson, D. Smith, D. Hardie, C. Borchers, and

T.W. Pearson, Mol. Cell. Proteomics 8, 995–1005 (2009).

(14) H. Lund, S.B. Torsetnes, E. Paus, K. Nustad, L. Reubsaet, and T.G.

Halvorsen, J. Proteome Res. 8, 5241–5252 (2009).

(15) T. Kosaka, R. Okuyama, W. Sun, T. Ogata, T. Harada, and K. Araki,

Anal. Chem. ��, 2050–2055 (2005).

(16) H. Lund, K. Løvsletten, E. Paus, T.G ,Halvorsen, and L. Reubsaet,

Anal. Chem. ��, 7926–7932 (2012).

(17) B. Winther, M.S. Nordlund, E. Paus, L. Reubsaet, and T.G.

Halvorsen, J. Sep. Sci. ��, 2937–2943 (2009).

(18) V. Kulasingam, C.R. Smith, I. Batruch, A. Buckler, and D.A. Jeffery,

J. Proteome Res. �, 640–647 (2008).

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-$r($�"TJB�1BDJà�D November 201616

In a recent instalment of “LC

Troubleshooting” (1) we looked at

problems two readers had with

ghost peaks in gradient runs. This

month, we’ll continue looking at

submitted questions and examine

one submitted by another reader of

this column.

1SPCMFN�XJUI�BO�&BSMZ�1FBLA reader submitted a problem he

observed during a reversed-phase

liquid chromatography (LC) analysis

of a pharmaceutical product.

An unknown peak unexpectedly

appeared at a retention time that was

much too short. For the analysis, he

needed to calculate retention factors

for the peaks of interest, so he had

injected uracil and observed a

retention time of 2.3 min. Everything

was satisfactory when reference

standards were injected, with a

normal and acceptable retention

time for the peak of interest.

However, when the sample was

analyzed, in addition to the normal

appearance for the peak of interest,

an unknown peak was consistently

seen at a retention time of 1.4 min.

This was all the information I was

given. I assume that the method

was isocratic, because retention

factors cannot be calculated from

retention and the column dead time

(t0) with gradients. Also, I assume

a C18 column was used and a

mobile phase of buffer–organic or

water–organic. I have not seen a

chromatogram.

The two most likely causes of

this problem are the presence of a

late-eluted peak that belongs to a

prior chromatogram or the exclusion

of a sample component from the

pores of the column packing. Let’s

consider both of these possibilities.

-BUF�&MVUJPONormally we expect that all the

peaks in the sample will be eluted

before we stop collecting data,

but this is not guaranteed. An

example of the problem that may be

observed is shown in the simulated

chromatogram of Figure 1(a).

You can see that the peak with

a retention time (tR) of ~2.2 min

appears to be much broader than

its neighbours. Whether we’re

looking at a gradient or isocratic

chromatogram, all the peaks in a

narrow region of the chromatogram

should be approximately the same

width. With isocratic separations,

when a peak is much wider than

its neighbours, it is likely that it

arises from a previous injection, but

insufficient time was allowed for it

to be eluted before injection of the

next sample. A simple way to check

this is to extend the run time for

the chromatogram until the peak in

question appears in its proper place,

as is the case for Figure 1(b), where

two broad peaks appear. The first,

at tR ≈ 2.2 min, is from the previous

injection and the peak at tR ≈ 7.2 is

in its proper position with the width

appropriate for this retention time.

This step confirms that the broad

peak in Figure 1(a) belongs to a prior

chromatogram.

Late-eluted peaks can appear

at any time in a chromatogram,

and in the reader’s case, it could

have appeared before t0 if it

originated from an earlier injection.

Usually a visual evaluation of the

chromatogram is enough to predict

if late elution is the problem, but

I did not receive a copy of the

chromatogram in question, so I can

only speculate.

Sometimes the data collection

time is sufficiently short and the

retention of the late-eluted peak is

large enough that it doesn’t appear

in the next chromatogram. An

example of this is seen in Figure 2.

Here, visual inspection should

lead us to suspect that the peak

at ~1.5 min is a late-eluted peak

because it is significantly wider

than its neighbours. When the run

is extended, it does not appear in

the next . . . or the next . . . or the

next chromatogram. I like to use

a simple calculation based on the

plate number (N ) to estimate the

true retention time of such peaks

so that I know where it is likely to

elute normally. Recall that the plate

number is calculated as follows:

N = 5.54(tR/W0.5)2 [1]

where W0.5 is the peak width at

half the peak height. This can be

rearranged to

tR = (W0.5 × N0.5)/5.540.5 [2]

3FBEFST��2VFTUJPOT��Early Eluted Peak+PIO�8��%PMBO �LC Troubleshooting Editor

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LC TROUBLESHOOTING


LC�5306#-&4)005*/(

0 2 4 6 8 10 Time (min)

(a)

(b)

0 1 2 3 4

Time (min)

'JHVSF�� Simulated chromatograms showing problem of late-eluted peak. (a) Chromatogram with broad peak (~2.2 min) out of place; (b) extended run showing broad peak at proper retention (~7.2 min).

'JHVSF�� Chromatogram with broad peak (~1.5 min) from previous injection.

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LC�5306#-&4)005*/(

4BNQMF�&YDMVTJPOReversed-phase LC packings

typically comprise porous silica

particles with a retentive stationary

phase (for example, C18) bonded

to the surface. These particles are

packed into a stainless steel tube

and held in place by porous frits and

endfittings at each end of the column.

Although it is sometimes convenient

to think of the particles as silica

tennis balls with C18 fuzz bonded

to the surface, that is a very poor

description of the particles. A better

model is that of a 5-μm-diameter

popcorn ball, where nanoparticles

(with a diameter of 8–10 nm) of solid

silica form the particle with pores

resulting from the spaces between the

nanoparticles. The resulting particle

has an external surface area that is

<<1% of the total surface area of the

particle (2). For sample molecules to

With equation 1, we can calculate

the plate number of a normally eluted

peak, such as one of the later peaks

in Figure 1(a) or the third peak in

Figure 2. The plate number should

be approximately constant for all

peaks in the chromatogram, so

once we know N, we can use

equation 2 to estimate the true

retention time of the broad peak (the

peak at 2.2 min in Figure 1[a] or the

second peak in Figure 2). Using this

technique, I estimated tR ≈ 7 min for

Figure 1 and tR ≈ 26 min for Figure 2.

Because this is an estimate of

retention, I would not be surprised if

these estimates were off by 10–20%,

but the estimates should help to

locate where the late-eluted peaks

belong.

be retained, they must interact with

the bonded phase on the particle

surface. Because nearly all of the

surface is inside the particle, sample

molecules must diffuse into the pores

of the particle before they can be

retained. If they cannot enter the

pores, they are excluded and will not

be retained.

One of the descriptors of a column

is its dead volume (Vm), which is

'JHVSF�� Conceptual diagram of LC column packed with 14 totally porous particles. Particles comprise nanospheres with pores between them; the interstitial space comprises the region between particles.

0 5 10 15 0 5 10 15 20

Time (min)

(a) (b) B1, B2 ,B3

N

B1

B2

B3

A

A

N

Time (min)

'JHVSF�� Example of ion exclusion with ion pair chromatography. (a) Mixture of an acid (A), three bases (B1, B2, and B3) and a neutral analyte with no ion-pair reagent; (b) same conditions as (a) except 14 mM octane sulfate added to mobile phase. C18 column, 20% methanol–buffer (20 mM phosphate, pH 6) mobile phase, and column temperature of 25 °C. Adapted from data of reference 3.

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LC�5306#-&4)005*/(

the total volume inside the column

comprising the volume within the

particles (the pore volume) and the

volume between the particles (the

interstitial volume). These concepts

can be seen in the cartoon of

Figure 3, where 14 particles are

shown packed into a column. The

dead volume of a column packed

with totally porous particles is

typically 60–65% of the volume of

the empty column. The interstitial

volume is approximately 40% (2), so

the pore volume is 20–25% of the

volume of the empty column. Another

way of looking at this dead volume

distribution is that approximately

60% of the dead volume is interstitial

volume and 40% is pore volume.

The column dead volume can

be measured by injecting an

unretained solute, such as thiourea

or uracil, which are unretained on

reversed-phase columns when the

mobile phase contains more than

~50% organic solvent. Alternatively,

the column dead volume can be

estimated if we know the size of the

column and assume a dead volume

of 60–65% of the empty column. One

easy-to-remember estimate for 4.6 mm

i.d. columns is shown in equation 3:

Vm ≈ 0.01L [3]

where L is the length of the column

in millimetres and Vm is in millilitres.

Thus, a 250 mm × 4.6 mm column

will have Vm ≈ 2.5 mL. For columns

of internal diameters other than

4.6 mm, another volume estimate is

as follows:

Vm ≈ 0.5 L dc2/100 [4]

where dc is the column internal

diameter in millimetres. The same

250 mm × 4.6 mm column will have

Vm ≈ 2.6 mL by equation 4. You

calculate that equation 3 uses ~60%

total porosity and equation 4 uses

~64%, so the estimates are probably

good to ~±10%.

How can we use this information

to help determine if the reader’s

problem could be sample exclusion?

First, it would be nice to know what

size column was being used so we

can confirm that the retention for

uracil is reasonable, but since the

column size was not supplied, we can

use equation 3 or 4 to help us guess.

As we saw above, for a 250 mm

× 4.6 mm column these equations

allow us to estimate Vm ≈ 2.5 mL. At

a flow rate of 1 mL/min, the column

dead time (t0) would be ~2.5 min.

This time is close enough to the

observed retention time for uracil

of 2.3 min to safely assume that the

method uses a 250 mm × 4.6 mm

column operated at 1 mL/min.

Next, we can estimate what the

retention time would be if the sample

was excluded from the pores. In the

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LC�5306#-&4)005*/(

components that put sufficient charge

on the column to exclude sample

components of the same charge.

4VNNBSZWe have seen that a sample peak

that is eluted before the column dead

volume is likely the result of either

late elution from a previous injection

or exclusion from the pores of the

column packing. I was not given

sufficient data to make a definitive

determination of the root cause of this

problem. To help make the decision,

I would like to see a chromatogram.

If the problem peak is broader

than the peaks normally eluted

early in the chromatogram, I would

suspect late-elution as the problem.

I would verify this by allowing the

chromatogram to run for two or three

times as long as normal to see if the

peak is eluted in the expected place.

I could use the techniques derived

from plate number measurements

to estimate the approximate true

retention time of such peaks. If the

problem peak had a width similar

to normally retained peaks, sample

exclusion is a more likely cause. This

suspicion would be reinforced if the

method used ion pairing conditions.

3FGFSFODFT(1) J.W. Dolan, LCGC Europe ��(10),

570–575 (2016).

(2) U.D. Neue, HPLC Columns: Theory,

Technology, and Practice (Wiley-VCH,

1997).

(3) J.H. Knox and R.A. Hartwick, J.

Chromatogr. A ��, 3–21 (1988).

“LC Troubleshooting” Editor +PIO�

%PMBO�has been writing “LC

Troubleshooting” for LCGC for more

than 30 years. One of the industry’s

most respected professionals, John

is currently the Vice President of

and a principal instructor for LC

Resources in Lafayette, California,

USA. He is also a member of LCGC

Asia Pacific ’s editorial advisory

board. Direct correspondence

about this column should go to

[email protected]. To

contact the editorial team please

address any correspondence to:

“LC Troubleshooting”, LCGC Asia

Pacific, Hinderton Point, Lloyd Drive,

Ellesmere Port, CH65 9HQ, UK, or

e-mail the editor-in-chief, Alasdair

Matheson, at alasdair.matheson@

ubm.com

aggregates might be sufficiently large

to be excluded.

Another exclusion mechanism can

be observed if the analyte molecule

has the same charge as the surface of

the packing material. This is commonly

seen in ion-exchange chromatography

(IEC). For example, an anion-exchange

column carries a positive charge so

it can separate negatively charged

analytes (anions). If the sample also

contains cations, the positive charge

of the cationic analyte will be repelled

from the positively charged surface,

so it does not enter the pores and is

excluded from the packing. Normally

we don’t observe this problem with

reversed-phase chromatography,

because the buffer salt concentration

in the mobile phase tends to override

minor ion exclusion effects. However,

if ion pairing is used for a method,

the ion pairing reagent will build up

a net positive or negative charge on

the column surface and can create

ion-exclusion conditions. An example

of this effect is shown in Figure 4 for a

sample of acids, bases, and a neutral

compound (3). In Figure 4(a), a pH 6

buffer–methanol mobile phase is used

with a C18 column and no ion-pair

reagent. In this case, the bases are

charged and poorly retained. The acid

peak is also charged, but has enough

reversed-phase character that it is well

retained. The neutral compound has

intermediate retention. In Figure 4(b),

octane sulfonate is added as an

ion-pair reagent and the column takes

on a net negative charge, so the

bases are well-retained by the added

influence of this charge. The pH is

unchanged, so the acidic component

is still charged. This charge causes

it to be repelled by the net negative

charge on the column surface, so

it is now excluded. The change in

conditions has only a minor influence

on the neutral compound. A similar

situation could occur with the reader’s

sample if the method uses ion-pairing

reagents or other mobile-phase

discussion above, we saw that the

interstitial (nonpore) volume was

~60% of the dead volume, so an

excluded peak would be expected

to be eluted at ~60% of the retention

time for uracil (t0). Therefore we

expect tR ≈ 0.6 × 2.3 min = 1.4 min.

This is the same as the observed

retention time for the unknown peak,

lending support to the hypothesis

that the peak represents a sample

component that is excluded from the

pores.

There are two common reasons

why an analyte might be excluded

from the packing pores. One is

related to sample size and the other

to sample charge. In size-exclusion

chromatography (SEC), sample

molecules are separated by their

relative ease of entering the pores of

the column. If the molecule is very

small relative to the pore diameter, it

can freely enter the pore and will be

retained. If the sample is so large that

it cannot enter the pore, it will not be

retained (excluded). In between these

two extremes are intermediate-sized

molecules that are partially retained

based on their relative size and

therefore ease of pore entry. In

SEC, ideally there is no chemical

interaction, so the earliest possible

peak comprises all molecules too big

to get into the column pores and the

last peak in the chromatogram will

comprise all molecules so small that

they can fully access the pores. In

reversed-phase mode, molecules that

are too big to enter the pores (or to a

certain extent those large enough to

have only partial access to the pores)

will be excluded. These molecules

will be eluted between the retention

represented by the interstitial volume

and t0, depending on their size. As

a rule of thumb, a molecule needs

to have a hydrodynamic radius of

less than one-third the pore diameter

to have full access to the pores.

Typical reversed-phase column

packings for small-molecule analysis

have pore diameters of ~10 nm. For

analysis of large molecules, such as

proteins, packings with ≥30-nm pore

diameters are favoured. If the sample

in question contained a polymer

excipient or other large molecule, it

might be excluded and appear prior

to t0. Another possibility is if the

sample molecules aggregated to form

dimers or larger aggregates, these

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21www.chromatographyonline.com

GC CONNECTIONS

Gas cylinders present several obvious and some less-familiar hazards, including sudden decompression; the risk of explosion or reaction; and possible acute toxicity.

The following is a concept script

for a gas safety video. Readers are

encouraged to find as many safety

violations or bad practices as they

can. Monday morning, 10:02 am, in

a small chromatography lab. While

starting up the gas chromatographs

and lighting their flame detectors,

Sam finds that one of the helium

cylinders in the laboratory has

gone empty over the weekend. He

reaches over the other gas cylinders,

applies a large tank wrench, and

accompanied by a loud hissing

sound, detaches the regulator fitting

from the tank. Letting the regulator

hang by its plastic connecting

tubing, he moves the hydrogen and

air cylinders out of the way into

the space between the laboratory

benches, tilts the empty cylinder

on its bottom edge, and rolls it into

position near the door.

Sam leaves the laboratory and

returns in a moment pushing a

furniture dolly. With a grunt, he tilts

the cylinder sideways onto the dolly

and, pushing it along, saunters down

the corridor whistling the “Heigh-ho,

Heigh-ho” theme from Disney’s

1950s Snow White. His coworker

Amanda looks at him aghast as she

heads into the laboratory.

Ten minutes later Sam returns with

a new cylinder on the dolly. He lifts

the tank up to a vertical position

and the dolly rolls off, banging

against the laboratory bench as

Amanda jumps out of the way.

Without bothering to strap any of

the cylinders in place, Sam ducks

down slightly and cracks open the

new cylinder’s stem valve. Amanda

is startled by a 110-decibel roar as

the escaping gas expresses its new

freedom.

Satisfied with the demonstration,

Sam rolls the tank into position and

reattaches the regulator. Then he

starts to secure the other tanks.

Amanda calls his name out loudly,

“Sam, what do you think you’re

doing?” As he spins around to deliver

a clever reply, his belt buckle catches

one of the dangling gas lines. In slow

motion, the hydrogen tank starts to

head for a horizontal position. Its

valve and regulator glance off the

bench top on the way down. The

cylinder heads for the walls, and in a

flash a bright orange-yellow light fills

the laboratory . . .

Certainly, no one would take all

of the wrong actions that this video

dramatizes, but how many of us

have done just one of them? I’ve

witnessed them all, and I’m guilty

of a few myself from time to time,

especially in exceptional situations

such as setting up a demonstration

in a conference room. I sincerely

hope that everyone in the laboratory

treats flammable solvents and

toxic chemicals with well-deserved

respect and understands the short-

and long-term hazards involved with

handling hazardous materials. So,

what leads some of us to fall short

of giving compressed gas cylinders

the respect they deserve? In terms

of stored potential kinetic energy,

they are bombs waiting to explode;

in terms of suffocation potential or

flammability, they can be just as

much a fire hazard and as potentially

toxic as any number of solvents and

solids.

Periodically, “GC Connections”

reviews gas cylinder safety. It’s

been a while since the topic was last

touched (1,2), so let’s take another

look at the hazards gas cylinders

present and some procedures and

practices that can maximize safety

for those who must work with them.

Cylinder HazardsGas cylinders present several

obvious and some less-familiar

hazards, including sudden

decompression that can propel

a cylinder remarkably quickly

across the laboratory and displace

breathing air; the risk of explosion

or reaction; possible acute toxicity;

heavy-object hazards; and personal

injury from high-pressure gas

streams or cryogenic liquids.

For reference, the Occupational

Safety and Health Administration

(OSHA) regulations 29CFR, Parts

1910.101–105 (3) provide specific

guidelines for the use of compressed

gases in the workplace that should

be followed strictly. An excellent

practical gas-safety document

can be found on-line as well (4).

Gas Cylinder Safety, Part 1: Hazards and PrecautionsJohn V. Hinshaw, GC Connections Editor

Many gas chromatographers are not fully aware of safe practices for handling high-pressure gas cylinders. Gas chromatography (GC) operators should be trained to properly transport, install, connect, and maintain their gas supplies, as well as to deal with emergencies. In the first of a two-part series, this month’s “GC Connections” examines the principal hazards and safety issues surrounding the compressed gas cylinder. Next month’s instalment will present safe procedures for routine cylinder use.


GC CONNECTIONS

These procedures and guidelines

are discussed in more detail in the

second part of this two-part series.

Table 1 lists hazard classes for

commonly used gas chromatography

(GC) gases. Gas chromatographers

do not normally use some of the

common hazardous gases in pure

form such as acetylene, oxygen,

nitrous oxide, or propane. These

gases may be present in laboratories

where other instrumentation is used,

such as atomic absorption (AA) or

atomic emission (AE) spectrometers.

Everyone in the laboratory should be

aware of the extra dangers posed by

chemically reactive, fuel, or oxidizer

gases.

DecompressionThe first thought that comes to mind

when discussing gas cylinders is

their rocket potential. A 1-A size

cylinder of helium contains 8.3 m3

(293 ft3) of room-pressure gas that’s

compressed into a space of less

than 0.5 m3 (2.0 ft3) at a nominal

fill pressure of 18.1 MPa (2640 psi).

European “L” size cylinders contain

slightly more compressed gas.

These cylinders weigh approximately

91 kg (200 lb) when empty, and

the weight of helium contained

in a fully pressurized cylinder is

around 1.4 kg (3 lb). When the

gas pressure is released rapidly

through an opening the size of

the valve stem, the cylinder — if it

accelerates in a straight line — can

reach velocities of close to 30 m/s,

108 km/h, or 66 mph. A 91-kg metal

cylinder hurtling at high velocity

can do tremendous damage almost

instantaneously, and there is nothing

that a person can do to stop it once

a decompression incident starts.

See the sidebar “How Fast Will a

Cylinder Go” for the calculations that

produced this velocity figure.

The thought of a heavy cylinder

careening through the laboratory

walls gets the attention of most lab

workers. This type of accident is

easy to avoid by always restraining

cylinders with appropriate chains

or brackets, transporting them in

cylinder carts, and keeping them

capped at all times unless actually

in use with a regulator or manifold

attached. Any cylinder that is found

to be damaged or has a stuck valve

should be returned immediately to

the supplier. If the damage is to the

cylinder body the supplier should be

notified to come and remove it. Never

try to vent a damaged cylinder.

"TQIZYJBUJPOEven though the cylinder is

restrained, another problem can

occur when the contents of any

large gas cylinder — other than an

air cylinder — are vented rapidly.

The sudden release of over 8 m3 of

unbreathable gas in the laboratory

may reduce the level of oxygen in

the air drastically and present a real

suffocation hazard. Liquefied gases

expand by as much as 1000-fold

when vaporized and can present a

much greater hazard. Liquid nitrogen

Dewars contain enough nitrogen

gas to make a room incapable of

sustaining life if the gas is released

rapidly. Carbon dioxide can cause

immediate unconsciousness followed

by death when breathed in any

significant concentration. It is much

denser than air and will settle in low

unventilated areas. Liquid carbon

dioxide tanks, such as used for GC

oven cooling, can release especially

large quantities of gas during a tank

rupture.

If an event such as this happens,

leave the area immediately,

prevent others from entering the

laboratory, and seek the assistance

of personnel trained in the use of a

self-contained breathing apparatus.

Without the proper breathing

equipment, never try to re-enter a

hazardous area to assist someone

else. Some companies have such

equipment on-site, but many rely

upon emergency services to enter

the affected area. Always make sure

the area has been well ventilated

before returning. Many unnecessary

tragedies have occurred due to

misguided rescue attempts.

Explosion and Fire HazardsIf a hydrogen cylinder vents into

the laboratory in an uncontrolled

manner, even if the leak is through

the pressure-release disc on the

cylinder or regulator, leave the area

immediately, close the doors, pull the

fire alarm to evacuate the building,

and call emergency services. Don’t try

to extinguish flame detectors, or shut

down anything else in the laboratory

— just get out quickly. Hydrogen has

a lower explosive limit (LEL) in air of

4%, so a venting cylinder can easily

Table 1: Hazard classes for commonly used GC gases and other gases that may be

found in the laboratory

Decompression Flammability "TQIZYJBUJPO Toxicity Cryo-Hazard

Nitrogen 9 9 9 (liquefied)

Helium 9 9 9 (liquefied)

Argon 9 9 9 (liquefied)

Air 9

Hydrogen 9 9 9 9 (liquefied)

Carbon

dioxide

9 9 9 9 (liquefied)

Acetylene 9 9 9

Propane 9 9 9

Oxygen 9 9 (accelerates

combustion)

9 9 (liquefied)

Chemical

reagents

(reactive

compressed

gases)

9 9 9 9

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Sampling and switching valves


GC CONNECTIONS

accumulate and explode if an ignition

source is present.

Hydrogen particularly presents a

special hazard because it burns in air

with an invisible flame. Never try to

investigate a possible hydrogen fire

by approaching the suspected flame

area: leave it to the professionals.

Although the combustion byproducts

of hydrogen are nontoxic, the fire

may burn other nearby items such

as plastics, which can produce toxic

combustion byproducts.

High-pressure gas cylinders can

rupture explosively when heated in

a fire. All cylinders include a thermal

fuse that is supposed to melt and

release the cylinder contents in a

semicontrolled manner before the

internal pressure exceeds a safe upper

limit. However, if the cylinder has been

mechanically stressed by falling over

or from the impact of another cylinder,

it can burst before the pressure release

valve can act. A chain-reaction effect

sometimes occurs in large fires in areas

where many cylinders are stored.

ToxicityGC gases generally aren’t toxic.

That is, after a victim has been

removed from an accident area and

has received first aid the immediate

effects of inert gas exposure, such

as dizziness and difficulty breathing,

will rapidly diminish. Chemically

active sample or reaction gases, on

the other hand, can present a real

toxic health hazard and a significant

disposal problem. If even a small

leak of a toxic gas such as carbon

monoxide or ammonia is detected,

leave the area and call in trained

personnel to remove the leaking

cylinder to a safe place.

Each type of gas or gas blend

has an associated material safety

data sheet (MSDS) that must be

sent in advance to the purchaser

who must then keep the information

on file for access by any employee

or emergency response personnel.

MSDSs contain extensive information

about the use, storage, and

disposal of chemicals — including

compressed gases — their toxicity,

and any other relevant information.

Refer to the appropriate MSDS when

you have any questions about a

particular material.

Many years ago I saw lecture

bottles of methyl bromide, hydrogen

create an explosive concentration

in moments. In its favour, hydrogen

rapidly diffuses in air so that venting

the flows encountered in flame

detectors or when used for carrier

gas present no significant hazard

under normal conditions. However,

hydrogen can accumulate in a closed

GC oven in the event of a broken

column. Most electronic pressure

control (EPC) systems incorporate

flow-monitoring safety features that will

detect this condition and shut down

the carrier-gas flow.

The same evacuation procedure

is required with other flammable

gases like propane and acetylene or

reactive gases and oxidizers such as

oxygen and nitrous oxide. Breathing

air contains about 20% oxygen, but

high oxidizer concentrations will

accelerate combustion dangerously

and can cause serious burn injuries.

Remember that clothing, paper,

paint, and plastic can all burn rapidly

in the presence of high oxidizer

concentrations.

If a gas fire starts and the gas

leak cannot be stopped safely and

positively, don’t try to extinguish

the flame. Unburned gas may

How fast will a cylinder go?Let’s assume that helium is allowed to vent unobstructed through a 1.1-cm (0.5 in.) orifice, such as the cylinder valve stem,

over a 10-s interval. That’s just my guess at the time frame that seems reasonable. The thrust or force exerted on the

cylinder at any moment will be the sum of two terms: the mass flow of the helium times its exit velocity through the orifice,

and the pressure differential across the orifice times its area, as delineated in equation 1:

F = q × Ve + (pe − pa) × Ae [1]

where q is the rate of helium mass flow, Ae is the orifice cross-sectional area, Ve is the exit velocity through the orifice, and

pe and pa are the cylinder and ambient pressures, respectively.

The helium will need to expand through the orifice — which has a diameter of 0.95 cm2 — into an 8.3 m3 volume in 10 s,

which gives an average exit velocity over the duration of the release of 87 m/s. That’s approximately 314 km/h, 200 miles

per hour, or 25% of the speed of sound, and these numbers certainly accentuate the hazards of rapid decompression. The

exit velocity will be higher at first and then slow as the tank pressure decreases. This reaction mass of the helium will impart

an average force of about 12 kg-m/s2. Acting for 10 s against the mass of the cylinder — we’ll ignore the loss of the helium’s

mass — this average force will impart a velocity change of around 4.8 km/h or 3 mph. That number is not very impressive,

but it seems right for a relatively small mass of helium acting against a heavy cylinder.

The rocket effect primarily comes from the second term of equation 1, which involves the high pressure drop from the cylinder

to the atmosphere. At the first instant of decompression from a full cylinder at 18.1 MPa, there will be a force of 1710 kg-m/

s2 exerted by the pressurized gas across the orifice. This is so much larger than the first term that we can ignore the helium

reaction mass effect, as equation 2 shows below. As the remaining gas pressure drops off the force will decrease as well

and reach zero after 10 s, for all practical purposes. Recalling that F = ma (force equals mass times acceleration) and then

integrating the decreasing acceleration across time, equation 2 describes the situation for an exponential decay in pressure:

ν = (pe−pa)Ae ÷ m \�0∞0 e−ktdt = (pe−pa)Ae ÷ k\m [2]

where ν is the cylinder velocity after the gas has escaped, k is the pressure decay constant, and t is the time interval. A

pressure decay rate of 50% per second, where k = 1 – 1/e = 0.632, gets the pressure down to less than 0.2% after 10 s.

With a 91-kg cylinder mass, the terminal velocity is approximately 30 m/s, 108 km/h, or 66 mph. Even if the pressure drop

decreased more rapidly and approached zero after 5 s, the velocity would still be as high as 19 m/s, 68 km/h, or 42 mph.


GC CONNECTIONS

fluoride, carbon monoxide, and

various highly reactive silanes —

not all in the same laboratory,

fortunately — carelessly stored

on shelves above floor level with

unprotected valves. No analytical

or chemical laboratory can justify

operation with such hazards present.

Improperly stored or deployed toxic

gas cylinders have no place in

anyone’s workplace. If any are found,

it’s good procedure to evacuate

the area and call in a hazardous

materials team to remove the danger.

In any case, never try to move or

dispose of hazardous or unknown

chemicals in gaseous, liquid, or solid

form yourself — it’s not worth the

risk.

Heavy LiftingNo one should try to lift a cylinder

that weighs more than about 12 kg

(26 lb). Heavy cylinders belong on

the floor, restrained to a bench or a

wall. Always use a cylinder cart to

move cylinders around, even from

one part of the laboratory to another.

The practice of rolling a cylinder on

its bottom edge, while prevalent,

risks injury to feet — and the risk of

the cylinder becoming unbalanced

and falling over. Never place a

cylinder on its side and roll it: the

sidewalls are the thinnest parts and

aren’t designed to take any weight.

You could be creating a dangerously

weak cylinder that may explode the

next time it’s filled with gas.

Liquid carbon dioxide cylinders,

used for cryogenically cooling GC

ovens, weigh much more when full

because of liquid carbon dioxide’s

density, and they can be deceptively

heavy. Always pay special attention

to these cylinders. In all cases, it’s

good practice to wear protective

eyewear, shoes, gloves, and clothing

when manipulating large gas

cylinders.

CryocoolingCryogenic liquefied gases such as

liquid nitrogen or carbon dioxide

present additional hazards in the

laboratory. Carbon dioxide, a liquid

when stored under pressure at

room temperature, cools to subzero

temperatures when decompressed

because of both expansive and

evaporative cooling. Liquid nitrogen is

stored under low positive pressure in

special Dewar tanks at around -195 °C.

Both cryogenic gases can cause

immediate frost burns on exposed

skin. Liquid nitrogen also presents

a cryogenic freezing hazard that

embrittles almost any object it contacts

in bulk, including fingers. Connecting

tubing that conducts cryogenic liquids

also presents a freezing hazard — the

tubing should always be insulated

or shielded to prevent accidental

contact. Again, appropriate protective

measures such as thermal gloves,

eyewear, and skin-covering clothing

help prevent accidents.

High PressureThe hapless lab rat in the video liked

to crack open the high-pressure

valve with no regulator attached.

I suppose the idea is to blow out

any dust particles as well as to see

if the tank is pressurized, but this

behaviour is never a good idea. The

force exerted by gas decompressing

from high pressures is tremendous. If

he happened to have part of his hand

or arm in front of the cylinder fitting

he could suffer a serious abrasion,

deep cut, or worse. A much better

way to clear the dust out is to spray

the area with clean, dry compressed

air from a good air source. Never

spray a halocarbon-based material

onto the cylinder fitting — the gas

can get into the lines and cause

problems with electron-capture and

mass spectrometry detectors.

ConclusionI’ve addressed many of the hazards

associated with compressed and

liquefied gases in this month’s

“GC Connections”. The four most

important considerations when

dealing with compressed gas

cylinders are proper physical

restraint, personal protection,

knowledge of potential hazards, and

appropriate emergency procedures.

After a cylinder is in place in the

laboratory, the next step is to hook it

up and put it in service. In the next

instalment I’ll present some good

procedures to follow when installing,

using, and replacing gas cylinders

and pressure regulators.

References(1) C. Hallenbeck and D.F. Gill, LCGC

North Am. 25(1), 40–47 (2007).

(2) J.V. Hinshaw, LCGC Europe 27(3),

144–148 (2014).

(3) Code of Federal Regulations (CFR),

29 CFR 1910.101, “Compressed

Gases” (U.S. Government Printing

Office, Washington, D.C., USA).

Available at https://www.osha.gov/pls/

oshaweb/owadisp.show_document?p_

table=standards&p_id=9747.

(4) “Compressed Gas Safety Guide,” at

http://www.stonybrook.edu/facilities/

ehs/occupational/cg.shtml (SUNY

Stony Brook University, USA,

September, 2016).

“GC Connections” editor John

V. Hinshaw is a senior scientist

at Serveron Corporation in

Beaverton, Oregon, USA, and a

member of LCGC Asia Pacific’s

editorial advisory board. Direct

correspondence about this column

should be addressed to “GC

Connections”, LCGC Asia Pacific,

Hinderton Point, Lloyd Drive,

Ellesmere Port, Cheshire, CH65 9HQ,

UK, or e-mail the editor-in-chief,

Alasdair Matheson, at alasdair.

[email protected]

Omission

In our June review of new gas

chromatography products (J.V.

Hinshaw, LCGC Asia Pacific 19[2],

20–26 [2016]), the following product

was inadvertently omitted:

Product

G908 GC system

Company

908 Devices

Product Description

The G908 GC system from 908

Devices is designed to be an

all-in-one, multicolumn, plug-and-go

system for in-line, at-line, laboratory,

and remote field qualitative and

quantitative analysis. The 28-lb

system includes a microscale ion

trap mass spectrometer and flame

ionization and thermal conductivity

detectors. Applications include

detection of benzene, ethylbenzene,

toluene, and xylene (BTEX) in

crude oil and seawater; extended

analysis of natural gas composition;

determination of ethers, alcohols,

aldehydes, and ketones in

hydrocarbon process streams, and

speciation of sulphur compounds

in natural, refinery, landfill, sewage

digester, and other fuel gases.

-$r($�"TJB�1BDJà�D November 201626

The 44th International Symposium

of High Performance Liquid Phase

Separations and Related Techniques

(HPLC 2016), convened 19–24 June

in San Francisco, California, USA, at

the Marriott San Francisco Marquis

Hotel and Conference Center. This

was the fourth time the conference

was held in San Francisco (also in

2006, 1996, and 1986 — notice

a trend?). HPLC 2016, which

has grown into the premier event

bringing together leading scientists

in the field of liquid chromatography

and related techniques, attracted

1007 delegates from numerous

countries. The attendance was a

considerable increase compared

to HPLC 2014 in New Orleans,

Louisiana, USA, but similar in

numbers to previous European

events. HPLC 2016 was chaired by

Professor Robert Kennedy of the

University of Michigan. As noted

by Kennedy, the programme was

grouped into three main areas:

“Improving Separations”, which

was devoted to new technology

and theory; “Making Discoveries”,

which was concerned with

applications of separation science;

and “Harnessing the Power”, which

centred on hyphenation of liquid

separations with mass spectrometry

(MS). Professor Kennedy also

noted that because of inspiration

regarding advances in personalized

medicine, aspects of proteomics,

metabolomics, and pharmaceutical

analyses were emphasized.

About 180 oral presentations,

including several plenary lectures,

were featured within the conference.

There were also numerous poster

presentations and several short

courses, vendor seminars, and

tutorials within the programme.

This instalment of “Column Watch”

reports on highlights and trends

observed at the conference.

)JHIMJHIUT�BOE�5SFOETOver the past few years the articles

highlighting topics at the HPLC

symposium have listed the main

topics covered and contrasted these

with previous years. This year several

colleagues present at the symposium

were asked what struck them as

most interesting. The following is a

synopsis of their responses along

with some personal views.

5ISFF�%JNFOTJPOBM�1SJOUJOH�"QQMJFE�UP�4FQBSBUJPOT�5FDIOPMPHZFalling under the label of

“improving separations”, the most

cited of the exciting advances

noted at the conference centred

on three-dimensional (3D)

printing of high performance

liquid chromatography (HPLC)

columns. Simone Dimartino,

from the University of Edinburgh,

presented a talk entitled “3D

Printing of Chromatography Media:

Closing the Loop between Real

World Experiments and Computer

Simulations” (1). On the basis that

the column walls are the ultimate

problem in column packing, the

idea presented was to 3D print half

spheres of particles into the walls of

the columns to eliminate, or at least

minimize, wall effects. Dimartino was

awarded with the Csaba Horváth

Young Scientist Award. As another

sign of the excitement surrounding

the 3D concept, Vipal Gupta of the

University of Tasmania was awarded

first prize in the “Best Poster

Award” contest for his effort entitled

“3D Metal Printed Miniaturized

Chromatographic Columns” (2). A

quick survey of recent literature

indicates that 3D printing of

microdevices for liquid handling,

interfacing various devices, and, in

some cases, separations is gaining

momentum. It will be interesting to

see where this concept can take

separations in the future.

"EWBODFT�JO�4VQFSà�DJBMMZ�1PSPVT�1BSUJDMF�5FDIOPMPHZ��"QQMJDBUJPOT�UP�-BSHF�.PMFDVMF�4FQBSBUJPOTAnother common observation among

our colleagues was the continuing

focus and advances in superficially

porous particle (SPP) technologies.

Wu Chen of Agilent Technologies

presented a paper entitled,

“Comparison of Optimized Wide Pore

Superficially Porous Particles (SPPs)

Synthesized by One-Step Coating

Process with Other Wide Pore SPPs

for Fast and Efficient Separation

of Large Biomolecules” (3). Chen

described several processes

for the manufacture of SPPs,

including “layer-by-layer” as well as

“coacervation” (Figure 1). Chen went

on to compare the impact of pore

size, shell thickness, and particle

size on resolution in large-molecule

separations and determined that

pore size provides the largest impact

on chromatographic performance.

Chen then described application

results comparing several differing

Highlights from )1-$��%BWJE�4��#FMM �Column Watch Editor

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COLUMN WATCH


COLUMN�8"5$)

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BY FASHIONABLE DESIGN

ZERO AIR+H2

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POSSIBLE COMBINATIONS

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GAS GENERATORS FOR GC INSTRUMENTS !

surface chemistries built on

3.5-μm, 450-Å SPPs exhibiting

a 3-μm core diameter and a

0.25-μm shell thickness. Figure 2

shows a comparison of retention

and resolution for a set of protein

standards on several different

chemical modifications of the

3.5-μm, 450-Å SPPs. More details

can be found in reference 4.

In a similar manner, Barry Boyes

of Advanced Materials Technologies

described the development of an

SPP with a 2.7-μm particle size and

a pore diameter of 1000 Å (5). Using

several experiments comparing

the large-pore SPP to both fully

porous particles (FPPs) and other

SPPs with smaller pore diameters,

Boyes demonstrated significant

advantages of the larger pore size

for large-molecule analyses. He

went on to conclude that SPPs

with enlarged pore sizes (400

and 1000 Å) have particular utility

for protein analyses, are highly

robust, and allow faster protein

separations with higher efficiency.

He also posed the question, “Is

there any disadvantage to the use

of the largest feasible pores for

(a)

Raise pH

Add cationicpolymer, rinse

Add silicasol, rinse

(b)

Solid silica cores made bymodified Stöber process

Add morecationicpolymer, rinse

Repeatcoating

steps

Burn offpolymer,

sinter

Burn offpolymer, sinter

Superficially porous particles

Superficially porous particles

Nearly monodisperse solid silica cores made by modified Stöber process

Urea, formaldehyde polymerization coats sol and core,and coated sol then adsorbs to coated core.

Add moresilica sol,

rinse

'JHVSF�� Processes for the preparation of superficially porous particles: (a) layer

by layer approach, (b) coacervation. Adapted with permission from reference 3.


COLUMN�8"5$)

generic protein separations?” In

addition to the discussion on the

advantages of large pore structures

for large-molecule separations,

Boyes also presented compelling

data for the use of difluoroacetic

acid in place of the more common

trifluoroacetic acid and formic acid

in large-molecule separations.

Trifluoroacetic acid generally

provides improved chromatography,

but has the drawback of causing

ion suppression when using MS

for detection. Formic acid, on

the other hand, is MS friendly,

but often produces poorer peak

shapes for large molecules. Boyes

pointed out through comparisons

that difluoroacetic acid provides

a suitable compromise between

the two extremes. Figure 3 shows

a comparison of chromatographic

results obtained for a monoclonal

antibody (mAb) using formic

acid, difluoroacetic acid, and

trifluoroacetic acid as mobile-phase

modifiers. As shown, the

difluoroacetic acid provides peak

shapes that rival those obtained

using trifluoroacetic acid.

Hydrophobic interaction

chromatography (HIC) is often

a valuable tool used to separate

polar variants of proteins. HIC,

however, uses high concentrations

of nonvolatile buffers, rendering it

incompatible with MS detection. In a

paper presented by Andrew Alpert

of PolyLC Inc., he described efforts

to render HIC MS friendly (6). Using

more-hydrophobic analogs of the

commercially available poly(propyl

aspartamide) stationary phase,

Alpert demonstrated that proteins

could be eluted in MS-friendly

0.0 1.0 2.0 3.0 4.0

4035302520151050

4035302520151050

4035302520151050

5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0

Ab

sorb

an

ce (

280 n

m)

0.1% Formic acid

0.1% Difluoroacetic acid

0.1% Trifluoroacetic acid

Retention = 6.5 minWidth (50%) = 0.34 minTailing factor = 3.01



Time (min)

'JHVSF�� Mobile phases for improved mAb LC. Column: 150 mm × 2.1 mm Halo Protein 400 C4; gradient: 28–38% acetonitrile

–0.1% acid as indicated over 15 min; flow rate: 0.3 mL/min; temperature: 80 °C; sample volume: 2 μL of Intact SILu Lite

SigmaMAb (0.5 μg/μL in water). Adapted with permission from reference 5.

SPP, 3.5 μm, 450 Å, C4

SPP, 3.5 μm, 450 Å, SB-C8

SPP, 3.5 μm, 450 Å, Diphenyl

1 2 2

4

4 6 8 10 12 14

5 6

Ab

sorb

an

ce (

mA

U)

Time (min)

'JHVSF�� Different selectivity of a protein standard. Column dimensions:

100 mm × 2.1 mm; gradient: A: 0.1% trifluoroacetic acid in water, B: 0.1% trifluoroacetic

acid in acetonitrile, 20–50% B in 15 min, 3-min wash at 95% B, 2-min re-equilibration at

20% B; flow rate: 0.3 mL/min; temperature: 60 °C; detection: UV absorbance at 220 nm;

injection volume: 5 μL. Peaks: 1 = ribonuclease A (14 kDa), 2 = cytochrome C (12 kDa),

3 = holo-transferrin (80 kDa), 4 = α-lactalbumin (14 kDa), 5 = catalase (240 kDa),

6 = carbonic anhydrase (30 kDa). Adapted with permission from reference 3.

XXX�DISPNBUPHSBQIZPOMJOF�DPN

COLUMN�8"5$)

��

(or friendlier) mobile phases that

may lead to a possible marriage

of the two techniques. Alpert

went on to show that the resulting

chromatography is not exactly

HIC or reversed-phase liquid

chromatography, but a useful hybrid

of the two modes. Initial sample

loading is accomplished at 0.7–1 M

ammonium acetate conditions and

elution takes place by decreasing the

salt concentration and increasing the

concentration of acetonitrile.

)JHI�4QFFE�$ISPNBUPHSBQIZProfessor Dan Armstrong of the

University of Texas at Arlington

presented data on very fast chiral

and achiral separations in his talk

entitled “Practice and Consequences

of Ultra-Fast, High Efficiency Achiral

and Chiral Separations” (7). Armstrong

reported routinely completing

separations in 30–60 s using some

traditional and novel phases built

on modern superficially porous and

highly efficient sub-2-μm fully porous

particles. An example of achiral

separations is presented in Figure 4

where difficult-to-separate desfluoro

analogs are separated from their

respective parent molecules in under

60 s. Armstrong described this as

an important achievement because

many pharmaceutical drugs now

contain labile fluorine atoms within their

structures. Upon comparing brush-type

phases bonded to 2.7-μm SPPs against

commercially available 5-μm columns

of the same chemistry, Armstrong

obtained 20–40% higher resolution

under the same mobile phase

conditions with 40–70% faster elution

times and 2–5 times more efficiency.

Under isoelutropic conditions the SPP

phases generate nearly 70% greater

resolution. Armstrong went on to say

that the effects are even greater at

higher linear velocities. Similar results

were reported for chiral stationary

phases (CSPs) built on efficient sub-2-

μm phases. Armstrong also pointed

out in his lecture that instruments,

even modern ultrahigh-pressure liquid

chromatography (UHPLC) systems, still

need to be modified to produce the

best results.

5BCMF�� Best poster award winners

Award Poster Title Authors

First PrizeMetal Printed Miniaturized Chromatographic

Columns

Vipul Gupta, Mohammad Talebi, Stephen Beirne,

Pavel Nesterenko, Gordon Wallace, and Brett

Paull

Second PrizeMinimizing Dispersion During Single-Cell

ElectrophoresisQiong Pan, Kevin Yamauchi, and Amy Herr

Third PrizeUHPSFC-MS/MS as a Viable Option in Doping

Control Analysis

Lucie Novakova, Vincent Desfontaine, Federico

Ponzetto, Raul Nicoli, Martial Saugy, Jean-Luc

Veuthey, and Davy Guillarme

Honourable MentionApplications of Deep Eutectic Solvents as Green

Solvent Media in Extraction ProcessesKyung Min Jeong and Jeongmi Lee

Honourable Mention

A Widely Targeted Metabolomic Method for

Neurochemicals Using Benzoyl Chloride

Derivatization and Liquid Chromatography–Mass

Spectrometry

Paige Malec, Jenny-Marie Wong, Omar Mabrouk,

and Robert Kennedy

Honourable MentionHydrazine Functionalized Zwitterionic Organic

Polymer for Specific Enrichment of GlycopeptidesZhongshan Liu, Junjie Ou, and Hanfa Zou

Honourable Mention

Improving the Temperature Control and the Heat

Transfer in CE-ESI-MS and CE-C4D-ESI-MS by

Using a 3D Printed Cartridge

Claudimir do Lago and Kelliton Francisco

Honourable Mention

Clinical Mass Spectrometry: Introduction to

Kinetic Study of Acidosis in Patients with Severe

Malaria

Natthida Sriboonvorakul, Sasithon

Pukrittayakamee, Kesinee Chotivanich, Yaowalark

Sukthana, Nicholas Day, Niklas P.J. Lindegardh,

Nicholas J. White, Arjen Dondorp, and Joel

Tarning

Honourable Mention

Studies of Drug Interactions with Alpha1-Acid

Glycoprotein by Using On-Line Immunoextraction

and High-Performance Affinity Chromatography

Cong Bi, Ryan Matsuda, Chenhua Zhang, Zitha

Isingizwe, and David Hage

Honourable Mention

Capillary Liquid Chromatography for Ultra Trace

Neuropeptide Measurement and Application to

Neurochemical Changes in Parkinson’s Disease

Jenny-Marie T. Wong, Omar S. Mabrouk, and

Robert T. Kennedy


COLUMN�8"5$)

both small molecule and conjugated

antibody drugs (12). From these talks

it is apparent that the technique is

making its way into the real world.

Multidimensional separations will

likely continue to grow within the

pharmaceutical industry, especially in

complex large molecule separations.

Another approach towards

multidimensional separations was

discussed by Professor Gerard

Hopfgartner of the University of

Geneva (13). Hopfgartner, who is well

known in the MS world, discussed the

coupling of liquid chromatography

with differential ion mobility (DMS)

mass spectrometry. In ion mobility

MS, ions can be separated based

on their mobility in a carrier gas. Ion

mobility is dependent on applied field

strengths that may be varied. This

property is taken advantage of in

DMS. Hopfgartner presented several

examples of improved separations

of small molecules and investigated

the use of organic additives to the

transport gas. He also noted that

gas-phase separations take place on

a faster time scale than liquid-phase

separations and therefore provide

a more perfect marriage than,

perhaps, 2D-LC. There have been

many improvements in ion mobility

analyzers over the past decade that

may stimulate additional growth of

the technique within the separation

science community.

*PO�$ISPNBUPHSBQIZAnother talk that was cited several

times by our colleagues was presented

by Farooq Wahab of the University of

Texas at Arlington (14). The main idea

of Wahab’s talk was that it is easy to

predict whether a given analyte will

front or tail under column overload

conditions on ion chromatography

columns as well as for most modes

of liquid chromatography. The

rule he described is simple: If the

mobile-phase components have

higher affinity towards the stationary

phase than the analyte, the peaks

will front under overload conditions. If

the mobile-phase components have

lower affinity towards the stationary

phase than the analyte, the peaks

will always tail. Wahab also pointed

out that an ion-exchange column can

have more than one kind of charged

site, each with different kinetics for

ion exchange.

There were several other

interesting talks related to

high-speed separations, including

“New Developments in Fast

Chromatography for Supporting

Pharmaceutical Process Research”,

where Erik Regalado of Merck

Research Laboratories presented

a compelling case for the need for

speed in pharmaceutical research

(8). John Engen of Northeastern

University demonstrated fast UHPLC

separation of peptides in his talk

entitled “Peptide Separations in

Short Times and Low Temperature at

15,000 psi” (9). In another interesting

talk, Attila Felinger of the University

of Pecs used monolithic column

constructs to examine the impact

of frits on column efficiencies (10).

Felinger demonstrated that for

early eluted compounds, monolithic

columns demonstrated significantly

better performance than packed bed

columns; however, when frits were

added to the monolithic columns,

the performance was similar. The

study provides evidence that the

frits used to maintain particles

within the column may contribute to

inefficiencies.

.VMUJEJNFOTJPOBM�4FQBSBUJPOTSimilar to last year’s meeting,

there was extensive interest in

developments in multidimensional

chromatography (two-dimensional

[2D] liquid chromatography [LC]).

With three separate sessions

dedicated to the technique, attention

seemed even greater this year. The

most apparent difference to past

years was that industry is beginning

to utilize the technology. For example,

Samuel Yang of Genentech presented

an interesting lecture entitled “Method

Validation of a Two-Dimensional

Liquid Chromatography Quality

Control Method for Pharmaceutical

Materials: A Focus on Special

Considerations Unique to 2D-LC

Method Qualification” (11). Likewise,

Kelly Zhang, also of Genentech,

spoke about the use of 2D-LC in

0 20 40 60

0 20 40

Time (s)

Time (s)

R = F (Ofloxacin)

R = F (Des-F-ofloxacin)

R = F (Ciprofloxacin)

R = F (Des-F-Ciprofloxacin)

R = F (Ezetimibe)

OH

OH

OH

O O

O

ON

NN

NN

HN

OH

FR

R

R

R = F (Des-F-ezetimibe)

Time (s)

60

0 20

(a)

(b)

(c)

40 60

'JHVSF�� Ultrafast separation of ezetimibe, ciprofloxacin, ofloxacin, and their

desfluoro analogues. (a) Column: 5 cm × 0.46 cm Hydroxylpropyl-β-cyclodextrin

SPP; mobile phase: 50:50 5 mM ammonium acetate (pH 4.0)–methanol; flow rate:

2.0 mL/min. (b) Column: 15 cm × 0.46 cm CF6 SPP; mobile phase: 90:10:0.3:0.2

acetonitrile–methanol–trifluoroacetic acid–trimethylamine; flow rate: 4.5 mL/min.

(c) Column: 15 cm × 0.46 cm CF6 SPP; mobile phase: 90:10:0.3:0.2 acetonitrile–

methanol–trifluoroacetic acid–trimethylamine, flow rate: 4.5 mL/min. Adapted with

permission from reference 7.

��XXX�DISPNBUPHSBQIZPOMJOF�DPN

COLUMN�8"5$)

)ZESPQIJMJD�*OUFSBDUJPO�$ISPNBUPHSBQIZHydrophilic-interaction

chromatography (HILIC) was once

again a highly discussed topic at the

HPLC meeting. In a quick perusal

of the conference proceedings,

31 separate talks, tutorials, and

posters were observed relating to

developments and applications in

HILIC. The desire to understand

more about HILIC was obvious

from the number of colleagues that

attended the tutorial on Tuesday

entitled “Understanding Separations

in HILIC Chromatography: Theory

to Practice” (15). Ron Orlando of the

University of Georgia presented a

talk he titled “Predicting the HILIC

Retention Behaviour of Glycans,

Glycopepetides, and other Modified

Peptides” (15). Orlando pointed out

that there is now enough information

to be able to predict retention of

glycans based on their structure.

Furthermore, retention characteristics

of unknown glycans can be used

to predict structure. He went on to

describe the same for glycopeptides

and that the ability to determine

glycans, while attached to peptide

backbones, enables site-specific

glycan determination.

1PTUFS�1SFTFOUBUJPOT�Poster presentations are a vital

component of the HPLC symposia.

This fact is evidenced by the

numerous posters that were presented

at HPLC 2016. The tradition of Best

Poster Award, sponsored by Agilent

Technologies, at HPLC conferences

continued in San Francisco. There

were 273 poster entrants in 24 session

topics, and 43 of our colleagues

devoted a great deal of their time to

review the posters. Each poster was

evaluated for inspiration (novelty and

originality), scope (amount of work,

technical quality, and execution of

the experiments) and presentation

(clarity, readability, and author’s

explanation). The nominations were

eventually whittled down to 10, and

eight reviewers evaluated these to

select the final awardees. These 10

finalists, who were presented with

their awards by Monika Dittmann

of Agilent Technologies on the last

day of the symposium, are listed in

Table 1. Congratulations to all those

nominated.

$PODMVTJPOTHPLC 2016 was again an eventful

symposium bringing together

researchers interested in separation

science from around the globe. The

2015 meeting was described as

evolutionary rather than revolutionary

(16). The same can be said of

the 2016 meeting as most of the

presentations provided information

on steps forward for existing

technologies and the coupling

of multiple existing techniques to

meet the challenges of complex

separations. There were, however, a

few exceptions. The idea that HPLC

columns could be 3D printed is an

exciting new reality. The adoption of

traditionally academic techniques

by industry such as 2D-LC further

indicates significant changes in the

practice of HPLC. As with HPLC 2015,

there was an evident trend towards a

focus on large-molecule separations.

LCGC’s global digital magazine provides unique and

timely applications, news, and interviews especially

relevant to separation scientists across industry sectors.

Sign up for FREE at chromatographyonline.com/column-subscribe


COLUMN�8"5$)

Francisco, California, USA, 2016.

(12) K. Zhang, “Multi-Dimensional LC:

Navigating the Chromatographic Space

from Small Molecules to Conjugated

Antibody Drugs”, presented at the

44th International Symposium of High

Performance Liquid Phase Separations

and Related Techniques (HPLC 2016),

San Francisco, California, USA, 2016.

(13) M. Raetz, R. Picenoni, G. Boehm,

and G. Hopfgartner, “LC-SWATH/

MS Metabolomics Platform with

Hyphenation of Extraction and Analysis

of Polar and Non-Polar Metabolites in

Plasma and Urine”, presented at the





(14) M.F. Wahab, C.A. Lucy, and

M.K. Pappoe, “Peaks Behaving

Badly: Overload Behaviour in Ion

Chromatography”, presented at the





(15) D.S. Bell, “Understanding Separations

in HILIC Chromatography: Theory

to Practice”, presented at the 44th

International Symposium of High




(16) R. Orlando, M. Badgett, and B. Boyes,

“Predicting the HILIC Retention

Behavior of Glycan, Glycopeptides and

Other Modified Peptides”, presented

at the 44th International Symposium



(HPLC 2016), San Francisco, California,

USA,2016.

(17) D.S. Bell and X. Wang, LCGC Europe

28(9), 506–518 (2015).

%BWJE�4��#FMM is a manager in

pharmaceutical and bioanalytical

research at MilliporeSigma (formerly

Sigma-Aldrich/Supelco). With a B.S.

degree from SUNY Plattsburgh and a

PhD in Analytical Chemistry from The

Pennsylvania State University, Dave

spent the first decade of his career

within the pharmaceutical industry

performing analytical method

development using various forms of

chromatography and electrophoresis.

During the past 15 years, working

directly in the chromatography

industry, Dave has focused his

efforts on the design, development,

and application of stationary phases

for use in HPLC and hyphenated

techniques. In his current role at

MilliporeSigma, Dr. Bell’s main focus

has been to research, publish, and

present on the topic of molecular

interactions that contribute to

retention and selectivity in an array of

chromatographic processes. Direct

correspondence to: LCGCedit@ubm.

com

Nesterenko, G. Wallace, and B.

Paull, “3D Metal Printed Miniaturized

Chromatographic Columns”, presented





USA, 2016.(3) W. Chen, A. Mack, and X. Wang,

“Comparison of Optimized Wide Pore Superficially Porous Particles (SPPs) Synthesized by One-Step Coating Process with Other Wide Pore SPPs for Fast and Efficient Separation of Large Biomolecules”, presented at the 44th International Symposium of High performance Liquid Phase Separations and Related Techniques (HPLC 2016), San Francisco, California, USA, 2016.

(4) W. Chen, K. Jiang, A. Mack, B. Sachok, X. Zhu, W.E. Barber, and X. Wang, J.

Chromatogr. A. ��, 147–157 (2015).(5) B. Boyes, B. Wagner, S. Schuster,

W. Miles, and J. Kirkland, “Improving Superficially Porous Particles for Larger Protein Separations”, presented at the 44th International Symposium of High Performance Liquid Phase Separations and Related Techniques (HPLC 2016), San Francisco, California, USA, 2016.

(6) A. Alpert, “A Series of New Materials for Direct HIC-MS Analysis of Proteins in Top-Down Proteomics”, presented at the 44th International Symposium of High Performance Liquid Phase Separations and Related Techniques (HPLC 2016), San Francisco, California, USA, 2016.

(7) D. Armstrong, “Practice and Consequences of Ultra-Fast, High Efficiency Achiral and Chiral Separations”, presented at the 44th International Symposium of High Performance Liquid Phase Separations and Related Techniques (HPLC 2016), San Francisco, California, USA, 2016.

(8) E. Regalado, K. Zawatzky, and C. Welch, “New Developments in Fast Chromatography for Supporting Pharmaceutical Process Research”, presented at the 44th International Symposium of High Performance Liquid Phase Separations and Related Techniques (HPLC 2016), San Francisco, California, USA, 2016.

(9) T. Wales and J. Engen, “Peptide

Separations in Short Times and Low

Temperature at 15,000 psi”, presented





USA, 2016.

(10) N. Lambert, N. Tanaka, and A. Felinger,

“The Performance of Columns for Fast

Liquid Chromatography”, presented





USA, 2016.

(11) S. Yang, J. Wang, B. Scott, and

K. Zhang, “Method Validation

of a Two-Dimensional Liquid

Chromatography Quality Control

Method for Pharmaceutical Materials:

A Focus on Special Considerations

Unique to 2D-LC Method Qualification”,

presented at the 44th International

Symposium of High Performance

Liquid Phase Separations and

Related Techniques (HPLC 2016), San

It is expected that this trend will

continue and result in many new

developments in liquid separation

technologies.

6QDPNJOH�.FFUJOHTThe 45th International Symposium



will be chaired by Michal Holcapek

and Frantisek Foret and held

18–22 June 2017, in Prague, Czech

Republic.

The 46th International Symposium



will be chaired by Doo Soo Chung

and held 5–9 November 2017, in

Jeju, South Korea.

"DLOPXMFEHFNFOUTCoverage of such a large symposium

is impossible without a great amount

of assistance. The author would

like to acknowledge the invaluable

assistance from Dr. Wu Chen and Dr.

Xiaoli Wang (Agilent Technologies),

Professor Dan Armstrong, and Dr.

Barry Boyes (Advanced Materials

Technology) for their kind permission

to print representations from

their talks. In addition, the author

would also like to thank Professor

Dwight Stoll (Gustavus Aldophus

College), Dr. Andrew Alpert,

Professor Ron Orlando, Professor

James Jorgenson (University

of North Carolina), Professor

Gerard Hopfgartner, Professor

Attila Felinger, Professor Oliver

Trapp (Ruprecht-Karls-University

Heidelberg), Dr. Farooq Wahab,

Professor David McCalley (University

of the West of England), Mr. Timothy

Langlois (Advanced Materials

Technology), Professor Deirdre

Cabooter (University of Leuven), Dr.

Monika Dittmann, and Professor Gert

Desmet (Vrije Universiteit Brussel)

for providing notes, insights, and

discussions regarding the content of

various sessions.

3FGFSFODFT(1) S. Nawada, F. Dolamore, C. Fee,

and S. Dimartino, “3D Printing of Chromatography Media: Closing the Loop between Real World Experiments and Computer Simulations”, presented at the 44th International Symposium of High performance Liquid Phase Separations and Related Techniques (HPLC 2016), San Francisco, California, USA, 2016.

(2) V. Gupta, M. Talebi, S. Beirne, P.


PRODUCTS

HPIC system

The Thermo Scientific Dionex Integrion

High-Pressure Ion Chromatography

(HPIC) system is the newest addition

to the Thermo Fisher Scientific ion

chromatography portfolio. According to

the company, it is intuitive and easy-to-use

and capable of addressing challenging

laboratory workflows. The system delivers

features previously available only on

Thermo Scientific high-end systems, including high-pressure

capability and optional electrochemical detection. With a simple,

flow-based plumbing layout and integrated performance features,

including whole-system smart monitoring, the system offers fast

run times in a robust and reliable format.

www.thermoscientific.com/chromatography

Thermo Fisher Scientific, California, USA.

Purification system

PLC Purification Systems

with the Verity 1900

MS Detector reportedly

conveniently provides

the capability to perform

mass-directed purification

by flash and preparative

chromatography on one

system. According to the company, efficiency can be maximized

and expenses minimized by confirming the contents of the

collected fractions.

www.gilson.com

Gilson, Middleton, Wisconsin, USA.

LC and LC–MS application guide

Advanced Chromatography Technologies

has published a comprehensive LC and

LC–MS applications guide bringing together

over 340 of the latest applications from

a wide range of fields, including clinical,

forensic & bioanalysis, environmental, food

& beverage, and pharmaceutical. More than

1600 analytes are included, from vitamins,

steroids, catecholamines, and metanephrines to organic

acids, amino acids, and sugars, along with extensive

screens for drugs of abuse and pesticides.

www.ace-hplc.com

Advanced Chromatography Technologies Ltd,

Aberdeen, Scotland.

Thermal desorption

The Thermal Desorption Unit (TDU

2) from Gerstel offers flexible,

multi-technique sample introduction.

According to the company, the MPS

robotic adds efficient automation

with barcode reading, tube

spiking options, and 240 sample

capacity. Sample introduction techniques include liquid,

headspace, solid-phase microextraction (SPME), stir bar

sorptive extraction (SBSE), thermal desorption, dynamic

headspace, and pyrolysis. The company reports that

switching between these techniques is easy.

www.gerstel.com/en/thermal-desorption-unit.htm

Gerstel GmbH & Co. KG, Mülheim an der Ruhr,

Germany.

MALS detector

The μDAWN is, according

to the company, the

world’s first multi-angle

light scattering (MALS)

detector that can be

coupled to any UHPLC

system to determine

absolute molecular

weights and sizes of polymers, peptides, and proteins or other

biopolymers directly, without resorting to column calibration

or reference standards. The WyattQELS Dynamic Light

Scattering (DLS) module, which measures hydrodynamic radii

“on-the-fly”, reportedly expands the versatility of the μDAWN.

www.wyatt.com

Wyatt Technology, Santa Barbara, California, USA.

HPLC columns

Macherey-Nagel,

a manufacturer of

chromatography sorbents

such as Nucleosil and

Nucleodur, introduced

Nucleoshell as core–shell

silica for the highest

efficiency. HPLC columns packed with the C18 modified

core–shell phases RP 18 and RP 18plus as well as the

phases phenyl-hexyl, PFP, and HILIC are available.

According to the company, the columns fulfill the demands

for HPLC separations with respect to separation efficiency,

detection limits, and time requirements for each analysis.

www.mn-net.com

Macherey-Nagel GmbH & Co. KG, Düren, Germany.


PRODUCTS

New manufacturer forLC columns

Hichrom Limited,

a manufacturer

of UHPLC/HPLC

columns with

manufacturing

facilities accredited

to ISO9001 (Quality)

and ISO14001 (Environmental) standards, has acquired the

exclusive rights to manufacture Vydac, Alltima, Alltima HP,

Prevail, Apollo, Allsep, Apex, and Genesis analytical HPLC

column ranges from Grace. Manufacture is to the identical

protocols and specifications previously used by Grace/Alltech.

Part numbers also remain unchanged.

www.hichrom.co.uk

Hichrom Ltd, Theale, Reading, Berkshire, UK.

Mass spectrometer

The new Xevo TQ-XS

mass spectrometer is a

highly sensitive benchtop

tandem quadrupole

instrument. Enabled by the

newly designed StepWave

XS ion guide, this mass

spectrometry system

combines ion optics,

detection, and ionization

technologies resulting in

high levels of sensitivity. The system also features UniSpray,

a novel, ionization source capable of ionizing a wider range of

analytes in a single chromatographic run.

www.waters.com

Waters Corporation, Milford, Massachusetts, USA.

Mobile MS benches

Manufacturer of

innovative mobile

benches for LC/GC/

MS. IonBench for mass

spectrometry reportedly

removes 75% of the

noise, eliminates 99%

of the vibration, and

saves up to 30% of floor

space. According to the company, IonBench for HPLC

improves system performance, enhances laboratory

safety, and contributes to productivity.

www.ionbench.com

IonBench, Joigny, Burgundy, France.

Gas generator

The Precision Hydrogen

Trace 250 generator

from Peak Scientific is

designed primarily for GC

carrier gas use and can

also be used for detectors

requiring hydrogen fuel

gas such as FID and FPD.

The company reports that

one generator is capable

of supplying multiple

GC instruments. The generator also comes with robust

safety features as standard.

www.peakscientific.com

Peak Scientific, Inchinnan, Scotland, UK.

UHPLC valves

The VICI Cheminert UHPLC

valve portfolio offers a wide

range of injection valves,

as well as solvent and

column-selection valves for

all kinds of applications, for

example, detector selection

or loop sampling/backflush

to detector. With a range of

actuator types that can control

UHPLC systems from almost

every supplier, every laboratory can now automate their liquid

handling requirements, according to the company.

www.vici.com

VICI AG International, Schenkon, Switzerland.

Field-flow fractionation

The Postnova AF2000

MultiFlow is a high

performance flow field-flow

fractionation (FFF) platform for

the high-resolution separation

of proteins, polymers, and

nanoparticles. According to

the company, it uses unique

crossflow field technology to

separate by dynamic diffusion

on the basis of molar mass or

particle size. Detection is made by UV, RI, and MALS and

can be easily interfaced to ICP-MS or DLS.

www.postnova.com

Postnova Analytics GmbH, Landsberg, Germany.

MethodsNow is a breakthrough solution from CAS that allows

researchers to quickly compare analytical and synthetic methods

side-by-side. With access to the largest collection of methods

and preparations from top journals and patents, MethodsNow

displays experimental details, including materials, instrumentation

and conditions.

Visit www.cas.org/MethodsNow to learn more.

COMPARE. DECIDE. SOLVE.

WHEN COMPARING METHODS,

HOW DO YOU PICK?

www.gerstel.com

Performance- Enhancing Automation

Headspace and Dynamic Headspace (DHS)

SPE, Online SPE and Dispersive SPE

Derivatization and Addition of Standards

Agitate, Heat, Mix

Twister (SBSE), SPME and Thermal Desorption

MAESTRO PrepAhead for enhanced productivity

Efficiently automated Solutions for Pharmaceutical Analysis: Extractables & Leachables (E&L) Residual Solvents (USP<467> / OVI) Fatty Acid Methyl Esters (FAMEs) Genotoxic Impurities (GTI/PGI) Metabolomics Automated Sample Preparation

GERSTEL Solutions for GC/MS and LC/MS –For enhanced performance and highest quality of results.

A

Headspace and Dynamic Headspace (DHS)

SPE, Online SPE andDispersive SPE

Documents

ZZZ KLFKURP FR XN VDOHV#KLFKURP FR XNfiles.alfresco.mjh.group/alfresco_images/pharma/2018/12/... · 2019. 10. 8. · November 2016 Volume 19 Number 4 PEER REVIEW Detecting low-abundance