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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
ZZZ�KLFKURP�FR�XN VDOHV#KLFKURP�FR�XN
Chr
omat
ogra
phy
Find what you are looking for!
MACHEREY-NAGEL
HPLC columnspacked by the sorbent manufacturer!
www.mn-net.com
NUCLEOSIL® – the originalNUCLEODUR® – professional solutionsNUCLEOSHELL®�¶�OPNOLZ[�Lᄗ��JPLUJ`
3
Editorial Policy:
All articles submitted to -$t($�"TJB�1BDJGJD
are subject to a peer-review process in association
with the magazine’s Editorial Advisory Board.
Cover:
Original materials: Hluboki Dzianis/
shutterstock.com
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
www.chromatographyonline.com
4 -$r($�"TJB�1BDJGJD November 2016
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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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Antec Leyden, Zoeterwoude, The
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Hernan J. Cortes
H.J. Cortes Consulting,
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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”,
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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�$IFNJTUSZ �
University of Vienna, Austria
Henk Lingeman
Faculteit der Scheikunde, Free University,
Amsterdam, The Netherlands
Tom Lynch
BP Technology Centre, Pangbourne, UK
Ronald E. Majors
Analytical consultant, West Chester,
Pennsylvania, USA
Debby Mangelings
Department of Analytical Chemistry
and Pharmaceutical Technology, Vrije
Universiteit, Brussels, Belgium
Phillip Marriot
Monash University, School of Chemistry,
Victoria, Australia
David McCalley
Department of Applied Sciences,
University of West of England, Bristol, UK
Robert D. McDowall
McDowall Consulting, Bromley, Kent, UK
Mary Ellen McNally
DuPont Crop Protection,Newark,
Delaware, USA
Imre Molnár
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Luigi Mondello
Dipartimento Farmaco-chimico, Facoltà
di Farmacia, Università di Messina,
.FTTJOB �*UBMZ
Peter Myers
Department of Chemistry,
University of Liverpool, Liverpool, UK
Janusz Pawliszyn
Department of Chemistry, University of
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Colin Poole
Wayne State University, Detroit,
Michigan, USA
Fred E. Regnier
Department of Biochemistry, Purdue
6OJWFSTJUZ �8FTU�-BGBZFUUF �*OEJBOB �64"
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Trajan Scientific and Medical, Milton
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Kortrijk, Belgium
Pat Sandra
3FTFBSDI�*OTUJUVUF�GPS�$ISPNBUPHSBQIZ �
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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-$r($�"TJB�1BDJGJD November 20166
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�
Ph
oto
Cre
dit: L
eo
nid
An
dro
nov/S
hu
tte
rsto
ck.c
om
,&:�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
Gilson PLC Purification Systems + CPC columns
Ultimate Performance inSilica-free Purification.
Gilson Glider Prep Software
simplifies system control
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isolates the maximum yield at the highest purity, in minimum
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• No columns to replace or silica to recycle
• Five times less solvent consumption and zero sample loss
• High performance: Purity > 99% and Recovery > 90%
-$r($�"TJB�1BDJàD November 20168
3FVCTBFU�BOE�)BMWPSTFO�
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
3FVCTBFU�BOE�)BMWPSTFO�
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��
3FVCTBFU�BOE�)BMWPSTFO�
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
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IS t
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43.4
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GR
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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.
Joanna Simpson
comfortable with on the
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• Maximum sensitivity
• Minimal sample transport and storage costs
• Ease of sample collection
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offering easy sample collection, transport and storage. Our revolutionary DBS Autosampler™ maintains the
integrity of the sample through automation, offering time and cost savings. Innovative patented Flow-through
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workflow for DBS analysis in minutes, providing maximum sensitivity without any manual intervention.
BE
TT
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RE
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P. +31 591 631 700F. +31 591 630 035E. [email protected]. www.sparkholland.com
Head Offi ce:P. de Keyserstraat 87825 VE EmmenThe Netherlands
*US 8586382 B2
-$r($�"TJB�1BDJàD November 2016��
3FVCTBFU�BOE�)BMWPSTFO�
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
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6
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'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
MRGRELPLVLLALVLCLAPRGRAVPLPAGGGTVLTKMYPRGNHWAVGHLMGKKSTGESSSVS
ERGSLKQQLREYIRWEEAARNLLGLIEAKENRNHQPPQPKALGNQQPSWDSEDSSNFKDVG
SKGKVGRLSAPGSQREGRNPQLNQQ
Isoform 3
MRGRELPLVLLALVLCLAPRGRAVPLPAGGGTVLTKMYPRGNHWAVGHLMGKKSTGESSSVS
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
3FVCTBFU�BOE�)BMWPSTFO�
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.
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www.ludger.com
Are you seeking regulatory approval for your glycosylated biopharmaceutical?
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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.
3FVCTBFU�BOE�)BMWPSTFO�
(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�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.
5IFSF�BSF�UXP�DPNNPO�
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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
8F�IBWF�TFFO�UIBU�B�
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JOKFDUJPO�PS�FYDMVTJPO�
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-$r($�"TJB�1BDJàD November 2016��
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
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.
-$r($�"TJB�1BDJGJD November 201622
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
The inventors of the Valco gas-tight valve for chromatography
45 years of experience in valves for chromatography
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-$r($�"TJB�1BDJGJD November 201624
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.
25www.chromatographyonline.com
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.
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
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WE MAKE THEM JUGGLE...
THEN WE SAVE SPACE
AND REPLACE
“UGLY-LOOKING”
INSTRUMENTS
BY FASHIONABLE DESIGN
ZERO AIR+H2
ZERO AIR+N2
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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.
-$r($�"TJB�1BDJàD November 201628
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
Retention = 9.8 minWidth (50%) = 0.075 minTailing factor = 1.22
Retention = 13.4 minWidth (50%) = 0.084 minTailing factor = 1.05
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
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��
(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
-$r($�"TJB�1BDJàD November 2016��
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.
$PODMVTJPOTHPLC 2016 was again an eventful
symposium bringing together
researchers interested in separation
science from around the globe. The
2015 meeting was described as
evolutionary rather than revolutionary
(16). The same can be said of
the 2016 meeting as most of the
presentations provided information
on steps forward for existing
technologies and the coupling
of multiple existing techniques to
meet the challenges of complex
separations. There were, however, a
few exceptions. The idea that HPLC
columns could be 3D printed is an
exciting new reality. The adoption of
traditionally academic techniques
by industry such as 2D-LC further
indicates significant changes in the
practice of HPLC. As with HPLC 2015,
there was an evident trend towards a
focus on large-molecule separations.
LCGC’s global digital magazine provides unique and
timely applications, news, and interviews especially
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Sign up for FREE at chromatographyonline.com/column-subscribe
-$r($�"TJB�1BDJàD November 2016��
COLUMN�8"5$)
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
44th International Symposium of High
Performance Liquid Phase Separations
and Related Techniques (HPLC 2016),
San Francisco, California, USA, 2016.
(14) M.F. Wahab, C.A. Lucy, and
M.K. Pappoe, “Peaks Behaving
Badly: Overload Behaviour in Ion
Chromatography”, presented at the
44th International Symposium of High
Performance Liquid Phase Separations
and Related Techniques (HPLC 2016),
San Francisco, California, USA, 2016.
(15) D.S. Bell, “Understanding Separations
in HILIC Chromatography: Theory
to Practice”, presented at the 44th
International Symposium of High
Performance Liquid Phase Separations
and Related Techniques (HPLC 2016),
San Francisco, California, USA, 2016.
(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
of High Performance Liquid Phase
Separations and Related Techniques
(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
at the 44th International Symposium
of High Performance Liquid Phase
Separations and Related Techniques
(HPLC 2016), San Francisco, California,
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
at the 44th International Symposium
of High Performance Liquid Phase
Separations and Related Techniques
(HPLC 2016), San Francisco, California,
USA, 2016.
(10) N. Lambert, N. Tanaka, and A. Felinger,
“The Performance of Columns for Fast
Liquid Chromatography”, presented
at the 44th International Symposium
of High Performance Liquid Phase
Separations and Related Techniques
(HPLC 2016), San Francisco, California,
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
of High Performance Liquid Phase
Separations and Related Techniques
will be chaired by Michal Holcapek
and Frantisek Foret and held
18–22 June 2017, in Prague, Czech
Republic.
The 46th International Symposium
of High Performance Liquid Phase
Separations and Related Techniques
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.
33www.chromatographyonline.com
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.
-$r($�"TJB�1BDJGJD November 201634
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