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May 2017 Volume 30 Number 5
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LC TROUBLESHOOTING
Increasing resolution by changing selectivity
Drug Target DiscoveryCombining HIC, SEC, and IEX with
fluorescence polarization
GC CONNECTIONS
GC products review
MULTIDIMENSIONAL
MATTERS
Miniaturized LC×LC and HRMS
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Feature240 Hot Topics in Separation Science
Leading separation scientists attending HPLC 2017 in Prague reveal
the latest trends in their areas of expertise, including: Gert Desmet:
(U)HPLC The Shape of Things To Come; Milos Novotny: Advances
in Glycomics in Biology and Medicine; Koen Sandra: Contemporary
Trends in Biopharmaceutical Analysis; E. Michael Thurman and
Imma Ferrer: UHPLC Coupled with Accurate Mass and High
Resolution Mass Spectrometry for Complex Environmental Analyses;
Peter Schoenmakers: The Rising Profile of Comprehensive 2D LC;
Vaclav Kasicka: Affinity Capillary Electrophoresis—A Powerful Tool
to Investigate Biomolecular Interactions; and Gerhard Liebisch: The
Role of LC–MS in Lipidomics.
Columns250 LC TROUBLESHOOTING
Count the Cost, Part 3: Increasing Resolution by Changing
Selectivity
John W. Dolan
Several variables can be used to change selectivity in a liquid
chromatographic (LC) separation. Here we compare the variables in
an effort to prioritize which experiments will be most effective.
256 GC CONNECTIONS
New Gas Chromatography Products for 2016–2017
John V. Hinshaw
The annual review of new developments in the field of gas
chromatography seen at Pittcon and other venues in the past 12
months.
264 MULTIDIMENSIONAL MATTERS
The Benefits of Coupling Miniaturized Comprehensive 2D LC
with Hybrid High-Resolution Mass Spectrometry
Juri Leonhardt, Jakob Haun, Torsten C. Schmidt, and Thorsten
Teutenberg
This month’s “Multidimensional Matters” looks at the benefits of
miniaturization in the first and second dimension.
Departments271 Products
274 Events
COVER STORY232 Combining HIC, SEC, and IEX
with Fluorescence
Polarization for Drug Target
Discovery
Tore Vehus, Jo Waaler, Stefan
Krauss, Elsa Lundanes, and
Steven Ray Wilson
Fluorescence polarization (FP) is
a highly regarded technique for
studying drug–protein interactions,
but has limited value regarding
protein mixtures. As a novel
approach to drug target discovery,
we have explored the possibility
of combining FP with liquid
chromatography (LC).
May | 2017
Volume 30 Number 5
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LCGC ONLINE
Selected highlights of digital content from LCGC Europe and The Column.
NEWSIdentifying Greek Drug
Consumption Using LC–MS/MS
Researchers from the National
and Kapodistrian University
of Athens have used liquid
chromatography with tandem
mass spectrometry (LC–MS/MS)
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illicit drug consumption patterns
following the major socioeconomic changes Greece
has experienced. Read Here: https://goo.gl/rt4tLp
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Drug discovery is essential to combat disease. There
are two main strategies: one is where the target is known
(target based), and the second is phenotype-based
drug discovery (1). The latter variant faces the daunting
task of identifying the target of a prototype drug (drug
deconvolution). This key piece of information allows the
mode of action of the drug to be identified. A central
bottleneck of drug deconvolution is the techniques
available (2,3). Approaches to drug deconvolution
include affinity chromatography, drug affinity responsive
target stability (DARTS), target identification by
chromatographic coelution (4) (TICC), and stability
of proteins from rates of oxidation (SPROX) (5) (the
mentioned approaches are extensively reviewed in
references 3 and 6). However, drug deconvolution is
far from mature, and alternative or complementary
procedures can ease the process. One approach
can be to combine chromatography with detectors
that specifically measure drug–protein binding. There
are a number of approaches that are suited for this
purpose, for example, isothermal titration calorimetry
(ITC) (7), surface plasmon resonance (SPR) (8), nuclear
magnetic resonance (NMR) spectroscopy (9), microscale
thermophoresis (MST) (10), and fluorescence polarization
(FP) (11). Of these, FP is arguably the simplest, and is
quite inexpensive.
FP measurements are based on rotational differences
between an unbound and bound fluorescent
(FLU)-tagged molecule, and can be briefly explained
as follows: a drug rotates freely and rapidly in a
solution, but rotates slower when bound to a much
larger protein; the FP instrument can detect this
rotational difference and hence a binding. Parameters
that can affect the performance of FP measurements
include fluorescence intensity, sample viscosity, and
quenching of drug fluorescence. For more details on
the technique, see Figure 1 and references 12, 13,
and 14. Although FP is frequently used for measuring
binding strength and kinetics (15), it has not been used
in drug deconvolution with biosamples because it has
limitations regarding mixtures. FP should in principle
detect a drug-binding in a protein mixture, but cannot
tell which protein is involved. Therefore, we wanted to
test the approach of chromatographically resolving
proteins in a mixture before FP measurements. FP has
previously been described as a detection device with
capillary electrophoresis (CE) techniques (16–18), but
not with liquid chromatography (LC) (to our knowledge).
We examined a number of chromatographic principles
(size-exclusion chromatography [SEC], hydrophobic
interaction chromatography [HIC], and ion exchange
chromatography [IEX]) that are suited for separating
proteins without perturbing their biological activity and
could be combined with FP. We evaluated LC and FP with
two FLU-tagged drugs that antagonize the Wnt pathway
(“161-FLU” and “XAV-FLU”, see Figure 2[a and b]
and Figure 3), a signaling cascade system strongly
associated with, for example, colon cancer, and a current
focus in drug discovery (19,20). Emphasis was placed
Combining HIC, SEC, and IEX with Fluorescence Polarization for Drug Target DiscoveryTore Vehus1, Jo Waaler2, Stefan Krauss2, Elsa Lundanes1, and Steven Ray Wilson1, 1Department of Chemistry, University
of Oslo, Blindern, Oslo, Norway, 2Unit for Cell Signaling, Oslo University Hospital, Rikshospitalet, Oslo, Norway
Fluorescence polarization (FP) is a highly regarded technique for studying drug–protein interactions, but has limited value regarding protein mixtures. As a novel approach to drug target discovery, the possibility of combining FP with liquid chromatography (LC) was explored. Nondenaturing protein LC principles such as size-exclusion chromatography (SEC), hydrophobic interaction chromatography (HIC), and ion exchange chromatography (IEX) were found to be orthogonal and compatible with FP because the mobile phases used do not negatively affect detection. For simple protein mixtures, the SEC/HIC/IEX–FP approach was able to identify tankyrase as the target of a triazole-based inhibitor of the Wnt signaling pathway, which is heavily associated with colon cancer. However, the total peak capacity of the three LC dimensions was not sufficient to resolve at cell-proteome level, calling for higher resolution of intact proteins to enable stand-alone drug target discovery with LC and FP.
KEY POINTS• SEC, IEX, and HIC are orthogonal.
• Fluorescence polarization is compatible with SEC,
IEX, and HIC.
• SEC/IEX/HIC–FP enables protein–drug interaction
measurement in a mixture.
• The total peak capacity of SEC/IEX/HIC has to be
strengthened.
Ph
oto
Cre
dit: vs1
48
/Sh
utt
ers
toc
k.c
om
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on evaluating the orthogonality of the three separation
principles (crucial for multidimensional separations prior
to FP), optimizing the peak capacity of the LC principles,
and ensuring that LC and FP are compatible.
ExperimentalChemicals: Type 1 water (resistivity [MΩ•cm @ 25 °C]
>18.0) was from a Milli-Q ultrapure water purification
system (Millipore). 10K cut-off 500 μL, 2 mL, or 5 mL
ultracentrifugation filters were also from Millipore.
2-(N -morpholino)ethanesulfonic acid (MES), L-arginine,
ribonuclease B (rib B), γ-globulin, holo-transferrin,
lysozyme, cytochrome C, bovine serum albumin (BSA),
myoglobin, carbonic anhydrase, α-chymotrypsinogen,
trypsinogen, Fluorescein-5-EX N-hydroxysuccinimide
ester (NHS-FLU), sodium phosphate, and ammonium
sulfate were from Sigma-Aldrich. Hemoglobin and NaCl
were from Merck. The SW480 cell line, Leibowitz’s L-15
medium were from ATCC. Fetal bovine serum (FBS),
Penicillin streptomycin (Pen Strep, PS) (Invitrogen),
and trypsin ethylene diamine tetraacetic acid (T-EDTA)
were acquired from Gibco, Invitrogen. Native cell
lysis buffer was from Cell Signaling Technology and
phenylmethylsulfonyl fluoride (PMSF) from Nigu Chemie
GmbH. Compounds OD139, OD198, and G007-LK was
acquired from Prof. Stefan Krauss’s laboratory.
Preparation of Solutions and Samples: Protein
standard mixtures containing different combinations of
rib B, γ-globulin, holo-transferrin, lysozyme, cytochrome
C, BSA, myoglobin, hemoglobin, carbonic anhydrase,
α-chymotrypsinogen, trypsinogen, and TNKS2a were
prepared in appropriate mobile phases. Samples
containing TNKS2a and other standard proteins are
hereafter referred to as TP.
Compound 161 (Figure 2[a] and [b]) was based on
the JW74-molecule (21), where an amine functionalized
polyethylene glycol was added. Labelling of 161 with
FLU was done according to the NHS-FLU manufacturer’s
protocol, and preparation of TNKS2a according to (22).
Cell Culturing: The SW480 wild type cell line was
cultured in Leibowitz’s L-15 medium containing 10%
(v/v) FBS and 1% (v/v) PS. To detach cells, T-EDTA
was used. Cells were harvested when they were ~90%
confluent. Prior to cell lysis, cells were counted using a
hemocytometer. Lysis of cells was performed by adding
400 μL cell lysis buffer per 107 cells with 50 mM PMSF.
Tubes were immersed in a sonication bath at 4 °C for
1 min × 5 with a 20 s delay between sonications. Cell
debris was removed with centrifugation at 13000 × g for
10 min and supernatant was transferred to eppendorf
tubes. Cell lysates were snap-frozen in liquid nitrogen,
and stored at -80 °C until use.
Liquid Chromatography: All protein LC experiments
were performed with a PerkinElmer Series 200 pump and
autosampler. UV absorption was measured at 280 nm
with a SPD-10AV UV detector (Shimadzu). Column
temperature was regulated with a Mistral-column oven
LC•GC Europe May 2017234
Vehus et al.
Polarized light
Drug
Depolarized light
Drug
Polarized light
Polarized light
Fast rotation
Slow rotation
Protein
(a)
(b)
Figure 1: Fluorescence polarization measurement on (a) a freely rotating fluorescent drug leading to depolarized light, and (b) a slowly rotating drug–protein complex maintaining a polarized light.
161-FLU/XAV-FLU/G007-LK
TNKS
Axin2 GSK3
CK1
Cell membrane
ß-catenin
ß-catenin
APC
ß-catenin destruction complex
Nucleus
Transcription factors
Prote
asom
e
Figure 3: Chemical structure of 161-FLU and XAV-FLU molecules.
(a) : 161-FLU
(b) : XAV-FLU
Figure 2: Wnt-pathway antagonism through inhibition of TNKS1/2 and stabilization of beta-catenin destruction complex leading to proteasomal degradation of beta-catenin and suppression of transcription factors.
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(Spark Holland) or a PerkinElmer Series 200 Peltier
column oven. Fraction collection was done with a Gilson
FC204 fraction collector.
IEX separations were performed on a 4.6 × 200 mm
PolyCATWAX mixed-bed ion-exchange column (PolyLC
Inc.) temperature regulated to 20 °C with an injection
volume of 100 μL. The column temperature was set to
4 °C. Mobile phase A (MP A) contained 20 mM MES
and mobile phase B (MP B) contained 20 mM MES and
0.8 M NaCl. For most separations the elution (at 1 mL/
min) started at 10% B for 10 min, then linear gradient
increased to 52.5% B in 20 min and held for 2 min at
100% B. Equilibration was done for 30 min at 10% B
between analyses.
HIC separations were performed on a 4.6 × 100 mm
ProPac HIC-10 column from Dionex (Thermo Fisher
Scientific). The column temperature was 20 °C. Injection
volume was 100 μL. MP A contained 2 M ammonium
sulfate, 0.05 M sodium phoshate pH regulated to 7.0, and
MP B contained 0.05 M sodium phoshate pH = 7.0. Most
gradients started at 0% B for 2 min with a linear gradient
elution to 75% B in 18 min and a hold for 3 min at 100% B.
Equilibration was done for 30 min at 0% B between analyses.
SEC separations were performed with a 4.6 × 35 mm
TSKgel-SuperSW guard column (Tosoh Corp.) coupled
in-line with a 4.6 × 300 mm TSKgel SuperSW3000
SEC column (Tosoh Corp.). Separations were done
isocratically with a flow rate of 0.35 mL/min and a mobile
phase containing 0.05 M sodium phosphate and 0.3 M
NaCl pH adjusted to 7.0 or 0.05 M sodium phosphate
and 0.3 M L-Arginine pH adjusted to 7.0.
The durations of fraction collections are shown in figure
legends and varied for each separation principle and
sample type. After fraction collection, the fractions were
(when needed) concentrated with cut-off filters of various
sizes.
Fluorescence Polarization: Fluorescence polarization
detection was performed with a Tecan F200 Pro
(Tecan Group Ltd.) using either 96- or 384-well plates
(Flat black polysterol, Greiner Bio One GmbH). For
fluorescein-labeled compounds, excitation was
performed at 485 ± 20 nm and emission measured at
535 ± 25 nm with cut-off filters. The number of flashes
was set to 25 and emitted light was integrated for 40 μs.
The settle time before measurements was 60 ms. Four
wells with water were used as instrument blanks. Aliquots
LC•GC Europe May 2017236
Vehus et al.
140
120
100
80
60
40
Tagged drug Tagged drug +binding protein
Tagged drug + non-binding protein
Flu
ore
scen
ce p
ola
riza
tio
n (
mP)
Figure 4: Fluorescence polarization drug binding identification of 200 μL 10 nM 161-FLU with 20 μL 0.2 mg/mL TNKS2a as binding protein and 20 μL 0.15 mg/mL ribonuclease B as non-binding protein added to a 96-well plate (n = 4).
240.0
200.0
160.0
120.0
80.0
40.0
Non-binding protein TNKS2a Cell lysate TNKS2a + Cell lysate
Flu
ore
scen
ce p
ola
riza
tio
n (
mP)
Figure 6: Fluorescence polarization between 161-FLU and non-binding protein (ribonuclease B), binding protein (TNKS2a), and SW480 wt cell lysate. 200 μL 10 nM 161-FLU and 10 μL protein samples in buffer were added. The TNKS2a and cell lysate concentration was 0.3 mg/mL and 21 mg/mL, respectively. In TNKS2a added cell lysate, the TNKS2a concentration was 0.3 mg/mL and the cell lysate concentration 21 mg/mL. Spread bars shown (n = 2).
trypsinogen
cytochrome C
lyzosymeribonuclease B
albumin
holo-transferrin
trypsinogen
cytochrome C
lysozyme
ribonuclease B
albumin
holo-transferrin
γ-globulinsγ-globulins
1.00
0.90
0.80
0.70
0.60
0.50
0.40
0.30
0.20
0.10
0.000.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00
No
rma
lize
d r
ete
nti
on
tim
e (
IIE
X a
nd
HIC
)
Normalized retention time SEC
SEC vs IEX
SEC vs HIC
Figure 7: Orthogonality plot for proteins separated in SEC, IEX, and HIC.
80
P < 0.018 pmole ≈ mLOD
P < 0.011 pmole > mLOD
8 pmole 1 pmole
70
60
50
Flu
ore
sce
nce
po
lari
za
tio
n (
mP
)
Non-binding protein (ribonuclease B)
Mole protein added to 2 pmole 161-FLU
Binding protein (TNKS2a)
Figure 5: Fluorescence polarization between 161-FLU and TNKS2a–ribonuclease B as a function of protein amount. The assay was performed in a 96-well plate with 200-μL 10 nM drug added to each well, and 20 μL protein dissolved in buffer so that the protein:drug ratio was 0.5 and 4. ** Significant difference at P < 0.01.
of 200 μL of the chromatographic fractions solution
were added to the 96-well plates, and 30 μL to the
384-well plates.
LC–MS/MS: LC–tandem mass spectrometry (LC–MS/MS)
experiments were performed as described in reference
23.
237www.chromatographyonline.com
Vehus et al.
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Figure 8: (a) SEC–UV chromatogram of a 100 μL TP sample separated on a 4.6 mm × 300 mm size-exclusion column with flow rate of 0.35 mL/min. Mobile phase consisted of 0.05 M sodium phosphate + 0.3 M NaCl at pH 7. The column was temperature regulated at 20 °C. UV absorbance was measured at 280 nm. Eight fractions were collected from 4.5 min with 2 min in each. *TNKS2a containing fractions. (b) Fluorescence polarization binding study between XAV-FLU and TP fractions from SEC.
*TNKS2a containing fractions. (c) IEX–UV chromatogram of 100 μL TP sample separated at 1 mL/min using gradient elution on a 4.6 mm × 200 mm mixed-bed ion-exchange column. The column was temperature regulated at 20 °C and UV absorbance was measured at 280 nm. MP A and MP B contained 20 mM MES and 20 mM MES + 0.8 M NaCl, both pH regulated to 6. Gradient start was at 10% B for 10 min, with a linear increase up to 52.5% B in 20 min, a wash-out step at 100% B for 2 min, and equilibration at 10% B for 30 min. Seven fractions were collected from 0 to 42 min, with 6 min in each. *TNKS2a containing fractions. (d) Fluorescence polarization binding study between XAV-FLU and TP fractions from IEX. *TNKS2a containing fractions. (e) HIC–UV chromatogram of 100 μL TP sample separated on a 4.6 × 100 mm Propac HIC-10 column. The column was temperature regulated at 20 °C and flow was set to 1 mL/min and the UV absorbance measured at 214 nm. The samples were eluted with 100% A for 2 min followed by a linear decrease to 25% A in 18 min, and a 3 min wash-out at 0% A. Equilibration for 30 min was carried out between analysis at 100% A. MP A contained 2 M ammonium sulfate + 0.05 M sodium phosphate, and MP B contained 0.05 M sodium phosphate, both pH adjusted to 7. A total of 22 fractions was collected in 1.5 min intervals from 0 to 33 min. *TNKS2a containing fractions. (f) Fluorescence polarization binding study between XAV-FLU and TP fractions from HIC. (Fractions with UV-signal from pure TNKS2a sample were collected, thus only three fractions were tested from the other samples to prove that TNKS2a retains its biological activity in a protein standard sample after HIC). *TNKS2a containing fractions.
Results and DiscussionFP as a Tool for Detecting Drug–Protein Interaction:
As a model for this initial investigation, we chose to use
FLU-tagged tankyrase inhibitors. Inhibition of tankyrase
allows axin proteins to engage in a so-called destruction
complex that degrades beta catenin for moderation of Wnt
signaling (Figure 3). TNKS2a (recombinant, binding part of
tankyrase) and ribonuclease B (non-binding) were added
to 161-FLU. For the TNKS2a–161-FLU solution, the FP
signal increased approximately 3.5 times, whereas with the
non-binding protein added, the signal did not significantly
increase (Figure 4). An additional step for verifying a
drug–protein interaction “FP event” is to add a competing,
non-fluorescent drug, which will reduce the FP signal as
a result of the unbinding of the FLU-tagged drug; this was
observed when adding G007-LK (22) (see Supplementary
Figure 1; supplementary information can be found at
http://www.chromatographyonline.com/supplementary-
information-combining-hic-sec-and-iex-fluorescence-
polarization-drug-target-discovery), also performed
in subsequent experiments. Hence, FP was suited for
assessing binding between TNKS2a–161-FLU.
Sensitivity: An approach to detect drug–protein interactions
must be sensitive because biotargets may be present in
minute amounts. Varying amounts of TNKS2a were added
to 2 pmoles 161-FLU (Figure 5). It was found that the
addition of 4 times more moles TNKS2a compared to the
drug gave a significant change in FP value, thus indicating
that the mass limit of detection (mLOD) in this example was
about 8 pmoles; this sensitivity of the TNKS2a–161-FLU FP
experiment was considered satisfactory.
Binding in Complex Samples: To confirm that FP could
detect a binding protein in a complex sample such as a
cell lysate (but not tell which protein is binding), an FP
assay with 161-FLU in SW480 cell lysate (contains human
tankyrase-2) with and without TNKS2a was performed.
Indeed, the FP value increased when a whole cell lysate
was added to 161-FLU, implying that one or more proteins
present in the sample binds to 161-FLU (Figure 6). It is very
likely that this is at least partly a result of tankyrase binding,
because this drug variant is highly selective (21). When the
cell lysate was spiked with TNKS2a, the FP signal increased
further, implying that the drug was initially present at higher
mole ratios compared to the target protein. We concluded
that FP experiments can function for complex samples,
in that a binding can also be detected in the presence of
non-binding proteins.
FP Measurement Robustness in Common Solvents for
Protein LC: To pinpoint which protein in a complex mixture
is causing an FP-event with a drug, they must be separated
prior to measurement. To maintain the tertiary structure of
proteins (typically necessary for interaction with a drug),
nondenaturing chromatographic conditions must be used.
This is achievable with HIC, SEC, and IEX. Substantial
amounts of salts are present in the mobile phase used
for these principles, often varying in composition during
a solvent gradient. It was feared that FP signals could be
affected by varying salt amounts, because this could affect
viscoscity. However, FP-events were largely unaffected by
the mobile phases of HIC, SEC, and IEX (Supplementary
Figure 2). Hence, mobile phases of common nondenaturing
protein LC principles were quite compatible with FP.
Estimating the Orthogonality of HIC, IEX, and
SEC: Cell lysates constitute a typical matrix for drug
deconvolution. However, a single LC separation will not
be able to separate all proteins in a biological sample,
which contains thousands of different proteins. Hence, a
multidimensional approach is called for, which dramatically
increases the resolution if the dimensions have a degree
of orthogonality (24). The orthogonality of HIC, IEX, and
SEC for protein separation was therefore evaluated. Model
proteins chosen featured a range of isoelectric points (pI)
and molar masses (MM). An orthogonality plot (Figure 7)
shows an obvious deviation from linearity in normalized
retention times, indicating that the separation principles are
indeed highly orthogonal. Hence, HIC, IEX, and SEC were
LC•GC Europe May 2017238
Vehus et al.
(a)
(b)
(c)
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Figure 9: (a) SEC–UV chromatogram of 100 μL SW480 wt cell lysate separated on a 4.6 mm × 300 mm size-exclusion column with a flow rate of 0.35 mL/min. Mobile phase consisted of 0.05 M sodium phosphate + 0.3 M NaCl at pH 7. The column was temperature regulated at 20 °C and UV absorbance was measured at 280 nm. Eight fractions were collected from 6 min with 4.25 min in each. (b) Fluorescence polarization binding assay between 161-FLU and SEC fractions collected of SW480 cell lysate. FP assay performed in 384-well format, with 30 μL 1 μM 161-FLU and 10 μL of spin filtrated fractions added to each well (n = 1). (c) Partial amino acid sequence of human tankyrase 2 identified in SEC fraction 2 highlighting peptides (green) identified with LC–MS/MS.
suited for multidimensional LC separation of proteins for FP
measurements.
HIC/IEX/SEC–FP with a Simple Protein Mixture: A
protein mixture was chromatographed in parallel on HIC/
IEX/SEC, and fractions were mixed with 161-FLU and
FP-measured (Figure 8[a–f]). FP-event fractions were
digested with trypsin and a peptide separation and
identification was performed with LC–MS/MS, confirming
the presence of TNKS2a in all the FP-event fractions. None
of the other proteins were identified in all FP-event fractions
(Supplementary Figure 3), and this was in accordance with
the known retention times of the proteins in the various
dimensions. Hence, a simple proof of concept for identifying
a drug target in a mixture, using multidimensional LC and
FP, was demonstrated.
Peak Capacities: The previous experiment showed that
HIC/IEX/SEC–FP could be used to pinpoint drug-binding
proteins in simple mixtures. At this stage, the system can
be functional, for example, as a step following protein
pull-down with immobilized drug columns (25). We
wanted, however, to assess the potential for an LC and
FP-only approach to drug deconvolution of very complex
samples. For maximum resolution, peak capacity was
optimized. Regarding IEX, the peak capacity of a 180-min
long ammonium acetate gradient with the conditions
described was approximately 35 (Supplementary
Figure 4). In HIC, the peak capacity was about 20
(with a 20-min long gradient, and did not substantially
increase with longer gradients) for the 200-mm long
column when applying a decreasing ammonium sulfate
gradient (Supplementary Figure 5). The peak capacity
for SEC was estimated to be 5 with phosphate buffer and
sodium chloride in the MP (Supplementary Figure 6) (in
later experiments NaCl was exchanged with L-arginine
to increase recovery from SEC, but this did not affect the
peak capacity significantly [data not shown]). Combining
the maximum peak capacity from each column with a
generously estimated orthogonality of 0.9, the overall
peak capacity of the system was 20 × 35 × 0.9 × 5 ×
0.9 = 2835. This number is well below the number of
proteins in a biological samples (which can be on the
ten thousand-scale), and an additional separation and
isolation step seems to be necessary, or further peak
capacity enhancements. The approach was, however,
investigated on protein extract from cells, which provided
a strong FP-event signal in fraction number 2 for SEC–FP
(Figure 9).
ConclusionsAs a novel approach to drug deconvolution, FP has
been shown to be compatible with nondenaturing liquid
chromatography. HIC, SEC, and IEX provided orthogonal
separation prior to FP measurements, allowing for drug
targets to be pinpointed in a mixture. For the technique to
be a completely stand-alone technique for very complex
cell samples, the total peak capacity of protein
chromatography must be strengthened; the development
and application of higher resolving columns than
those used here are key. If this level is reached, the
technique can have potential for drug target discovery
for (fluorescent) compounds, even without the need for
immobilization-based approaches.
References(1) G.C. Terstappen, C. Schlupen, R. Raggiaschi, and G.
Gaviraghi, Nature Review Drug Discovery 6, 891–903
(2007).
(2) E. Tashiro and I. Imoto, Bioorganic & Medicinal Chemistry 20,
1910–1921 (2012).
(3) J.N.Y. Chan, C. Nislow, and A. Emili, Trends in
Pharmacological Sciences 31, 82–88 (2010).
(4) J.N.Y. Chan et al., Molecular & Cellular Proteomics 11(7),
M111.016642 (2012).
(5) G.M. West, L. Tang, and M.C. Fitzgerald, Analytical Chemistry
80, 4175–4185 (2008).
(6) J. Lee and M. Bogyo, Current Opinion in Chemical Biology 17,
118–126 (2013).
(7) R. Ghai, R.J. Falconer, and B.M. Collins, Journal of Molecular
Recognition 25, 32–52 (2012).
(8) J.A. Maynard et al., Biotechnology Journal 4, 1542–1558
(2009).
(9) A.L. Skinner and J.S. Laurence, Journal of Pharmaceutical
Sciences 97, 4670–4695 (2008).
(10) S. Patnaik et al., Journal of Medicinal Chemistry 55,
5734–5748 (2012).
(11) F. Perrin, Journal de Physique et le Radium 7, 390–401
(1926).
(12) A.M. Rossi and C.W. Taylor, Nature Protocols 6, 365–387
(2011).
(13) W.A. Lea and A. Simeonov, Expert opinion on drug discovery
6, 17–32 (2011).
(14) N.J. Moerke, Current Protocols in Chemical Biology 1(1), 1–15
(2009).
(15) T.G. Dewey, Ed., Biophysical and Biochemical Aspects of
Fluorescence Spectroscopy (Springer, 1991).
(16) Q.-H. Wan and X.C. Le, Analytical Chemistry 72, 5583–5589
(2000).
(17) E. Ban and E.J. Song, Journal of Chromatography B 929,
180–186 (2013).
(18) L. Ye, X.C. Le, J.Z. Xing, M. Ma, and R. Yatscoff, Journal of
Chromatography B: Biomedical Sciences and Applications 714,
59–67 (1998).
(19) J.N. Anastas and R.T. Moon, Nature Reviews Cancer 13, 11–26
(2013).
(20) B.T. MacDonald, K. Tamai, and X. He, Developmental cell 17,
9–26 (2009).
(21) J. Waaler et al., Cancer Research 71, 197–205 (2011).
(22) A. Voronkov et al., Journal of Medicinal Chemistry 56,
3012–3023 (2013).
(23) M. Rogeberg, S.R. Wilson, H. Malerod, E. Lundanes, N.
Tanaka, and T. Greibrokk, Journal of Chromatography A 1218,
7281–7288 (2011).
(24) M. Gilar, P. Olivova, A.E. Daly, and J.C. Gebler, Analytical
Chemistry 77, 6426–6434 (2005).
(25) S. Sato, A. Murata, T. Shirakawa, and M. Uesugi, Chem. Biol.
17, 616–623 (2010).
Tore Vehus is an Assistant Professor at Department
of Engineering Sciences, University of Agder, Norway.
He graduated with a M.Sc. in analytical chemistry
from University of Oslo (UiO) in 2012 and is currently
finishing a Ph.D. under Ass. Prof. Steven R. Wilson at
UiO. His work is currently focused on the development
of instrumentation and chromatographic columns for
fit-for-all analyses of complex biological samples.
Jo Waaler is a Researcher at Oslo University Hospital.
Stefan Krauss is a Professor at Oslo University
Hospital.
Elsa Lundanes is a Professor at the Department of
Chemistry at the University of Oslo.
Steven Ray Wilson is an Associate Professor at
the Department of Chemistry, University of Oslo.
Research interests have included miniaturization,
multidimensional separations, and on-line
hyphenatations with MS and NMR. Application fields
are in cancer research (diagnostics and drug discovery)
and neuroscience.
239www.chromatographyonline.com
Vehus et al.
LC•GC Europe May 2017240
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(U)HPLC: The Shape of ThingsTo Come
Gert Desmet, Department of Chemical
Engineering, Free University of
Brussels, Belgium
A recent argument was raised in the scientific press that
in pursuit of greater speed and separation resolution,
ultrahigh performance liquid chromatography (UHPLC) is
faced with practical limitations and will struggle with its
own version of Moore’s law (1).
This empirical law was first proposed to describe
the long-term progress made in the micro-electronics
industry. Moore’s law states that speed and memory
storage capacity are roughly doubling every two years.
Progress is occurring by shrinking the distance between
the transistors on the chips to cram even more of them on
the same surface. However, the current spacing between
the transistors is already down to a dazzling small 22 nm,
and most theoretical models predict that the fundamental
laws of physics will prevent the distance being reduced
below 10–7 nm. It is clear that Moore’s law will one day
run into a hard stop and bring a halt to the advances in
speed and data storage if the electronics industry does
not find a new paradigm to store and manipulate data.
A gloomy parallel was drawn with (U)HPLC to
emphasize that this field has been witnessing a Moore’s
law-type of progress in speed and resolution over the
past decade. This progress was essentially realized
by making increasingly smaller particles, and it was
suggested that (U)HPLC is also facing the end of
practical progress with its own version of Moore’s law.
Most specialists agree that with pressure limits entering
a range where the compressibility of the liquid makes
it harder to precisely control the flow rate and where
viscous heating threatens to become unacceptably high,
we have now reached the stage of what can practically
be achieved by particle size reduction.
Slip flow technology has been suggested as a possible
way out of this, but its promises still need to be achieved
in practice (2). Sub-micron particles may also be able
to realize the ultra-rapid separations (in the order of a
few seconds) needed in the final dimension of the best
possible three-dimensional LC (3D LC separations, but
this is likely to remain a very niche application for a
long time.
However, the limits of Moore’s law in (U)HPLC only
relate to packed beds of spherical particles. We should
Hot Topics in Separation ScienceA series of short articles exploring current trends in separation science that will be addressed at the HPLC 2017 conference in Prague, Czech Republic, from 18–22 June 2017.
not forget the sphere is only one of the many shapes
that are possible. Just think of monoliths, perfusion
particles, and pillar arrays. Measured by Golay’s and
Knox’s separation impedance number, these are far
better shapes than the packed bed of spheres and hold
the promise of a 10-fold increase in efficiency (for the
same time) and even a 100-fold reduction of the analysis
time (for the same efficiency). These approaches have
not delivered their promise yet, some because of the
lack of order and some because the size of the individual
elements is still too large to reach their performance limit
in a range of practical times or efficiencies—and some
still suffer from both problems.
However, with new materials engineering possibilities,
such as silicon micromachining and 3D printing, rapidly
gaining widespread availability, it is highly possible
we will one day see a commercially viable production
technology that will be able to produce the perfect
chromatographic column, breaking away from Moore’s
law by trading our spherical particles for supports with a
much more advantageous shape as measured by Golay’s
and Knox’s separation impedance.
Let us not forget how this field recently defeated
Moore’s law already, with the (re-)introduction of
core–shell particles (representing a fundamental
change of the particle design) leading to a large gain
in speed and resolution. So, let us be optimistic and
consider that maybe the next 50 years will be the era
of support shape, rather than of support size. And with
exciting contributions on the possibilities of silicon
micromachining and 3D printing on the programme,
the HPLC 2017 conference could be the start of this
new era.
References(1) M.S. Reisch, C&E News 94(24), 35–36 (2016).
(2) B.A. Rogers, Z. Wu, B. Wei, X. Zhang, X. Cao, O. Alabi, and
M.J. Wirth, Anal. Chem. 87, 2520−2526 (2015).
The Role of LC–MS in Lipidomics
Gerhard Liebisch, Institute of Clinical
Chemistry and Laboratory Medicine,
University of Regensburg, Germany
Lipidomics, the analysis of lipids by mass spectrometric
methods, revolutionized lipid science (1). It provides
detailed quantitative information on hundreds of
0
50
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0 3 6 9 12 15
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LC•GC Europe May 2017242
Hot Topics in Separation Science
LC–MS provides separation of lipids and reduces the
complexity of the matrix. It typically provides a higher
sensitivity than shotgun and offers retention time as an
additional parameter to identify lipid species. Therefore,
low abundant lipid mediator species are typically
analyzed by targeted LC–tandem mass spectrometry
(MS/MS) (5). Lipidomic methods apply both reversed
phase with nonpolar as well as normal phase and
hydrophilic interaction chromatography (HILIC) with polar
selectivity.
Reversed phase separates lipids species based on
their hydrophobic moiety, that is, the hydrocarbon chain
for most lipid classes. This allows the separation of
hundreds of lipid species based on their chain lengths
and double bond number (6,7). The retention behaviour
follows certain rules and increases the confidence of
lipid species identification. This permits the separation
of isomeric lipid species with different acyl chains like
PC 18:1_18:1 (phosphatidylcholine with two acyl chains
containing 18 carbon atoms and 1 double bond) and
PC 18:0_18:2 (18:0 and 18:2 acyl chain). However,
quantification in lipidomics typically relies on lipid
species not present in the samples. In reversed-phase
chromatography most of the lipid species and internal
standards elute at different times, thus experience
different matrix effects and different solvent composition,
which influences their ionization and may result in
inaccurate quantification (8).
The polar selectivity of normal phase and HILIC
provides lipid class-specific separation. This has great
advantages in terms of quantification because analytes
and internal standards show similar retention times.
Moreover, identification of lipid species comprising a lipid
class is straightforward. In contrast to reversed-phase
methods, separation of acyl chain isomers is not usually
possible by normal phase and HILIC methods, but
lipid class-specific separation may resolve isomers like
bis(monoacylglycero)phosphate and phosphatidylglycerol
(9). A promising approach is the application of
polar stationary phases with ultrahigh-performance
supercritical fluid chromatography (UHPSFC), which
offers ultrafast separation for quantitative analysis of
multiple lipid classes (10).
Today, an increasing number of studies are reporting
poor quality lipidomics data with misidentification
and inaccurate or inappropriate quantification of lipid
molecules. These studies primarily use untargeted
metabolomics approaches (11) and the reasons for the
poor data quality include analytical, bioinformatics,
lipid species and opens new possibilities to gain an
insight into lipid biology. This helps not only to explain
the vital role of lipid species as membrane building
blocks, but also to unravel their bioactive functions.
Thus, lipid species can act as signaling molecules and
modulate membrane properties, which are essential for
organelle and membrane protein function. Moreover,
the first examples demonstrated their potential as novel
biomarkers to monitor human health (2).
Lipidomics research is based on two main approaches:
direct infusion mass spectrometry (DIMS) analysis
(shotgun lipidomics) and liquid chromatography (LC)–MS
analysis. In direct infusion analysis a crude lipid extract
is infused into the mass spectrometer and lipid species
identification relies on specific precursor ion, neutral
loss scans (3). The main advantage of infusion-based
analysis is its simplicity and the straightforward way
of quantification. Analytes and internal standards are
present in the same sample matrix and thus experience
the same ion suppression and matrix effects. Shotgun
analysis is therefore able to provide comprehensive,
quantitative lipidomes, as for example demonstrated for
yeast (4). The application of high mass resolution, MSn,
and derivatization–gas phase reactions can provide
detailed lipid structures. However, the application
of shotgun approaches is limited in sensitivity and
separation of isomeric lipid species. Moreover, in-depth
characterization of lipid species in crude lipid extracts
may be complicated by co-isolation of precursor ions.
Lipidomics revolutionized lipid science. It provides detailed quantitative information on hundreds of lipid species and opens new possibilities to gain an insight into lipid biology. This helps not only to explain the vital role of lipid species as membrane building blocks, but also to unravel their bioactive functions.
243www.chromatographyonline.com
Hot Topics in Separation Science
UHPLC Coupled with Accurate Mass and High Resolution Mass Spectrometry for Complex Environmental Analyses
E. Michael Thurman and
Imma Ferrer, Laboratory
of Environmental Mass
Spectrometry, University of
Colorado, Boulder, Colorado,
USA
Environmental analyses of food, soil, and water have
changed dramatically over the last decade. Topics such
as pesticides, food additives, and natural products
have become important as food products are globally
grown and distributed (1). Monitoring their quality is
critical to international business. Pharmaceuticals,
fluorinated surfactants, and endocrine disruptors in
water are major new topics, where not only parent
compounds are unknown but also their metabolites
and degradation products are often more important
or more abundant than the parent compound (2). New
environmental issues, such as hydraulic fracturing
and its wastewater, have captured our attention as the
production of oil and gas has increased exponentially
in the past decade (3). With this technology comes
the problem of wastewater disposal and groundwater
contamination. These environmental issues have greatly
benefited from the combination of ultrahigh-performance
liquid chromatography (UHPLC) mated to high resolution
mass spectrometry (HRMS). Because suppression by
matrices creates challenges in environmental analysis,
both sample preparation, such as solid-phase extraction
(SPE), and UHPLC make important contributions to
eliminating or reducing suppression.
The analysis of environmental samples has challenged
our ability to separate the thousands of compounds
that are present in a food or water extract. Furthermore,
the salts and metal ions associated with these extracts
further complicate the analytical challenges. HRMS,
such as time-of-flight MS and Orbitrap MS, has been
adopted by many laboratories to address these pressing
environmental issues. To gain the most from HRMS,
UHPLC has been rapidly accepted as a separation
method. In particular, the use of sub-2-μm particles in
a variety of packing materials has enabled the mass
spectroscopist to fully appreciate the power of HRMS
and accurate mass by separating compounds of isobaric
mass, as well as isotopes of various compounds that
have the same identical mass. Even the highest resolving
power in mass spectrometry will not separate two isomers
that have the same formula; thus, UHPLC plays a critical
role in separation and identification of environmental
targets. A separation by UHPLC then allows the use of
MS/MS followed by accurate mass for identification.
For example, pharmaceuticals in water may have not
only identical formulas (isomers) but may also have
nearly identical MS/MS spectra. The analysis problem
of tramadol and desvenlafaxine are just that problem
(4). The use of UHPLC using a C-8 column was easily
able to separate these two isomers, such that they
could be identified correctly (4). The importance of
these pharmaceuticals is that they may contribute to
the formation of dimethylnitrosoamine (NDMA), which is
an important new chlorination product created in water
treatment (5).
A valuable mass spectrometry technique, auto
MS/MS, is available from many vendors of high-resolution
mass spectrometers. The peak capacity of the analytical
column used in UHPLC is valuable for this type of
analysis because it gives the auto MS/MS spectra a more
easily interpreted accounting of the unknowns present in
a food or water extract. The MS/MS spectra may then be
correlated to various libraries currently available without
the interference of matrix materials.
Another reason to increase the resolution in
chromatography before mass spectrometry is the
current availability of complex databases. With accurate
mass analysis, it is possible to create a database of
accurate masses for any compounds that one would
like to investigate in an environmental sample, that is,
of course, if that compound will ionize in either positive
or negative ion electrospray. However, the limitation of
HRMS does not stand alone in the conundrum of environmental analysis. The power of separation methods brings us ever closer to fully characterizing the environmental pollutants in food, soil, and water.
and educational aspects. Therefore, it is necessary
to implement reporting standards for lipidomics data
to share with the scientific community (12). These
standards need to cover both shotgun and LC–MS
approaches. Only the application of both approaches
in a complementary and confirmatory way permits a
comprehensive and accurate coverage of the lipidome.
References(1) K. Yang and X. Han, Trends Biochem. Sci. 41(11), 954–969
(2016).
(2) S. Sales et al., Sci. Rep. 6, 27710 (2016).
(3) X. Han, K. Yang, and R.W. Gross, Mass Spectrom. Rev. 31(1),
134–78 (2012).
(4) C.S. Ejsing et al., Proc. Natl. Acad. Sci. U.S.A 106(7),
2136–2141 (2009).
(5) G. Astarita et al., Biochima. et Biophys. Acta 1851(4), 456–68
(2015).
(6) M. Ovcacikova et al., J. Chromatogr. A 1450, 76–85 (2016).
(7) K. Sandra and P. Sandra, Curr. Opin. Chem. Biol. 17(5), 847–53
(2013).
(8) S. Krautbauer, C. Buechler, and G. Liebisch, Analytical
Chemistry 88(22), 10957–10961 (2016).
(9) M. Scherer, G. Schmitz, and G. Liebisch, Analytical Chemistry
82(21), 8794–8799 (2010).
(10) M. Lisa and M. Holcapek, Analytical Chemistry 87(14), 7187–95
(2015).
(11) G. Liebisch, C.S. Ejsing, and K. Ekroos, Clinical Chemistry
61(12), 1542–1544 (2015).
(12) G. Liebisch et al., Biochimica. et Biophysica. Acta (2017).
E. Michael Thurman
Imma Ferrer
LC•GC Europe May 2017244
Hot Topics in Separation Science
the database lies in the fact that sometimes as many as
a thousand isomers may exist for a formula, such as a
simple fungicide of the elements, C, H, N, and O. How
does one tackle this problem? One powerful technique
is slow chromatography with high peak capacity and
reproducibility of retention time. This allows one to use
retention time in the mass spectrometry database to
accurately pull out targeted compounds and radically
decrease the false positives caused by isomeric
compounds.
Another important advance in mass spectrometry is
the use of ion mobility for the separation of surfactants
associated with wastewater from hydraulic fracturing.
The complexity of surfactants adds to the hundreds of
ions associated with a single group of compounds. These
surfactants are used as clay stabilizers and emulsifiers to
move oil and gas from deep underground to the surface.
The combination of a heatmap generated by UHPLC
versus IM drift time is a powerful visual tool to see and
identify new groups of compounds present in wastewater
samples. This is especially important in that these
surfactants may contribute to earthquake occurrence
when these wastewaters are disposed of by deep well
injection, a common technique in the United States (6).
Thus, HRMS does not stand alone in the conundrum
of environmental analysis. The power of separation
methods, including UHPLC and advances in sample
preparation (solid-phase extraction and other sample
preparation tools), brings us ever closer to fully
characterizing the environmental pollutants in food, soil,
and water. HPLC 2017 will highlight and encourage us
in the field of environmental analysis to continue this
interesting journey in high resolution chromatography.
References(1) E.M. Thurman et al., Anal. Chem. 78, 6703–6708 (2006).
(2) M. Strynar et al., Environ. Sci. Technol. 49, 11622–11630 (2015).
(3) Y. Lester et al., STOTEN 512, 637–644 (2015).
(4) I. Ferrer and E.M. Thurman, J. Chromatogr. A 1259, 158–166
(2012).
(5) D. Hanigan et al., Environ. Sci. Technol. Lettrs. 2, 151–157
(2015).
(6) W.L. Ellsworth, Science 341, 142 (2013).
Advances in Glycomics in Biology and Medicine
Milos V. Novotny, Department of Chemistry,
Indiana University, Bloomington, Indiana, USA
and Regional Centre for Applied Molecular
Oncology Masaryk Memorial Oncological
Institute, Brno, Czech Republic
The importance of glycosylated structures in modern
biology and medicine has been beyond dispute for
many years, but there are still gaps in biochemical
understanding. The current realization that virtually
all major human diseases have been associated with
glycosylation changes demands in-depth structural
studies of these highly complex glycobiomolecules.
Glycoscience with its many directions and a broad
scope in both prokaryotic and eukaryotic systems is
currently securing its place at the centre stage of modern
biological research (1).
However, the enormous complexity of glycoconjugate
molecules and the abundance of glycosylated proteins
in biological fluids and tissues present significant
challenges to modern analytical methods and
measurement technologies. As underscored in the
2012 report of the National Research Council to the
U.S. National Academies (2), developing new tools for
glycoscience is one of the highest priorities of the general
scientific inquiry.
The rapidly growing fields of glycomics and
glycoproteomics reflect these on-going efforts.
Biomolecular mass spectrometry (MS) is today the central
identification and measurement technique for glycans
and glycopeptides. The contemporary MS features
state-of-the-art instrumentation in terms of ionization
and fragmentation techniques, highly resolving mass
analyzers, and very sensitive detection. However, the
field of analytical glycobiology also urgently needs
high-performance separation methods to assist MS
measurements because of (a) the sheer complexity
of the mixtures generated during various cleavages
of glycoprotein molecules; and (b) glycan isomerism,
which is both biologically relevant and methodologically
difficult.
The role of separation scientists to develop better
analytical methods and reliable protocols to study
differences in glycosylation (for example, “normal” versus
“aberrant” glycosylation levels) seems secure for a long time
to come. Starting with sample preparation, fractionation, and
preconcentration, and ending with the discrete resolution
of glycoconjugates before MS measurements, many of
these tasks are accomplished through chromatographic
principles. Rapid advances are increasingly seen in
many of these vitally important tasks of glycomics and
glycoproteomics. Carbohydrate derivatization at microscale,
such as permethylation or fluorescent labeling, are often
desirable to enhance identification and measurements.
Recent advanced methods in analytical glycoscience have
been reviewed (3,4).
Glycomic profiling measurements have been
particularly important in a search for disease biomarker
candidates. Additionally, glycosylation analysis of
therapeutic glycoproteins is becoming increasingly
essential to evaluate their bioactivity, safety, immune
response, and solubility. While deglycosylation protocols
for N -linked glycans have been reliably developed
during recent years, the same still cannot be said about
quantitative analysis of O -linked oligosaccharides,
although some progress has recently been made. The
general profiling procedures may involve matrix-assisted
laser desorption–ionization mass spectrometry
(MALDI-MS), HPLC–fluorescence detection, capillary
electrophoresis with laser-induced fluorescence
(CE–LIF) detection, or capillary liquid chromatography
tandem mass spectrometry (LC–MS/MS). Some of the
MS techniques and CE–LIF can be complementary in
yielding the information on glycan isomerism (5,6).
245www.chromatographyonline.com
Hot Topics in Separation Science
Our laboratory has had a long-term interest in the search
for cancer biomarkers through glycomic comparative
measurements (6,7) in the blood serum of patients with
different disease conditions. More recently, we have
extended our studies to glycomic profiling and structural
characterization of urinary exosomes (8). Our current
emphasis has also been on the isomerism concerning
fucosyl substitution and sialyl linkages, in both N - and
O -glycans, in relation to oncological conditions. With
regards to N -glycans, typical profiles reflect high-mannose
and complex-type oligosaccharides resulting from the
major serum glycoproteins. It is now often the case
that trace multiantennary N -glycans represent the very
structures with important disease-related information.
To enhance their detection by MS techniques, we can
use preconcentration based on the hydrophobicity of
permethylated structures or the ion-exchange principles
for multiply-sialylated structures. Certain amidation
reactions can also be helpful in distinguishing sialyl linkage
isomers in both capillary LC–MS/MS and microchip CE (6).
Exosomes originating from different tissues and
encountered in biological fluids have recently received
considerable attention for their diagnostic and therapeutic
potential. Exosomes are extracellular nano-sized vesicles
encapsulating nucleic acids and proteins, both with and
without glycosylation. There is increasing evidence that
certain exosomes are involved in the cancer process,
suppression of the immune system, drug resistance, and
possibly metastasis. As a prelude to studying the exosome
glycoconjugate composition in the genitourinary tract
cancers, we have broadly characterized glycans (8) and
proteins associated with urinary exosomes. The analytical
profiles of N -glycans feature paucimannosidic structures,
high-mannose, and large complex-type structures.
Using long capillaries packed with graphitized carbon
black together with electrospray ionization MS/MS, we
succeeded in partial resolution of some sialylated isomers
of tetra-antennary glycans. This represents one of the most
difficult analytical challenges in glycoscience (1).
References(1) R.D. Cummings and J.M. Pierce, Chem.Biol. 21, 1–15 (2014).
(2) National Research Council of the National Academies,
Transforming Glycoscience: A Roadmap for the Future
(The National Academies Press, Washington, D.C., USA,
2012).
(3) W.R. Alley Jr., B.F. Mann, and M.V. Novotny, Chem. Rev. 113,
2668–2732 (2013).
(4) S. Gaunitz, G. Nagy, N.L.B. Pohl, and M.V. Novotny, Anal.
Chem. 89, 389−413 (2017).
(5) S. Mittermayr, J. Bones, and A. Guttman, Anal. Chem. 85,
4228–4238 (2013).
(6) C.M. Snyder, W.R. Alley Jr., M.I. Campos, M. Svoboda, J.A.
Goetz, J.A. Vasseur, S.C. Jacobson, and M.V. Novotny, Anal.
Chem. 88(19), 9597–9605 (2016).
(7) W.R. Alley Jr., J.A. Vasseur, J.A. Goetz, M. Svoboda, B.F.
Mann, D.E. Matei, N. Menning. A. Hussein, Y. Mechref, and M.V.
Novotny, J. Proteome Res. 11(4), 2282–2300 (2012).
(8) G. Zou, J.D. Benktander, J.T. Gizaw, S. Gaunitz, and
M.V. Novotny, Anal. Chem. 2017. DOI: 10.1021/acs.
analchem.7b000629
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LC•GC Europe May 2017246
Hot Topics in Separation Science
Some members of the separation science community
are still not yet convinced of the value of comprehensive
two-dimensional liquid chromatography (LC×LC). They feel
that the large increase in separation power (that is, in peak
capacity: the number of component peaks that may possibly
be separated) may be compromised by losses in sensitivity
and robustness of the separation. However, the chairmen
of HPLC 2017 will have seen a great number of abstracts
come their way from scientists who want to change this
perception.
Amongst the almost 50 categories in which abstracts could
be submitted, biopharmaceuticals and multidimensional
separations were the most popular and both these
subjects now extend across two sessions. Lectures on
multidimensional chromatography are spread across a
session devoted to LC×LC in the “FUN” (fundamental) track
of the programme and a session devoted to the combination
of LC×LC with mass spectrometry (LC×LC–MS) in the “HYP”
(hyphenation) track.
Contemporary Trends in Biopharmaceutical Analysis
Koen Sandra, Research Institute for
Chromatography, Kortrijk,
Belgium
The HPLC symposium series is recognized as “the forum”
where new developments in liquid phase separations
and their hyphenation to mass spectrometry (MS) for the
analysis of (bio)pharmaceutical compounds and their
metabolites are presented.
At HPLC 2016 in San Francisco, four lecture sessions
were dedicated to biopharmaceutical analysis and one
session to pharmaceutical analysis. The lecture sessions
were accompanied by three extensive poster sessions.
At HPLC 2017 this tradition will continue and dedicated
lecture and poster sessions will be organized under the
umbrella of recent advances in (bio)pharmaceuticals
and pharmaceutical analysis. These topics will also be
discussed in the sessions related to multidimensional
chromatography and MS coupling and to electromigration
techniques.
Biopharmaceuticals are becoming a core aspect of the
pharmaceutical industry and it is therefore logical that the
characterization of the huge and complex structures of
these drugs will be a highlight at HPLC 2017. The sessions
will cover chromatographic, electrophoretic, and mass
spectrometric developments for the characterization of
peptides, recombinant proteins, monoclonal antibodies
and next-generation formats, such as bispecific mAbs
(bsmAbs), antibody–drugs conjugates (ADCs), antibody
mixtures, antibody fragments (nanobodies, Fab), Fc
fusion proteins, and brain penetrant mAbs in addition
to glyco-engineered formats. As a result of the patents
of top-selling mAbs expiring or due to expire soon, an
explosion of biosimilar (generic) activities has been
observed in recent years. The analytical methods have
therefore to embrace comparability assessment for
originators and biosimilars.
For a detailed characterization and comparability
assessment of the complex and heterogeneous protein
biopharmaceuticals (a small drug, such as ibuprofen,
contains 33 atoms, a mAb contains over 20,000 atoms!),
the complete analytical toolbox has to be opened to
determine characteristics, such as amino acid sequence
and composition, molecular weight, N- and O -glycosylation,
N- and C-terminal processing, S–S bridges, free cysteine
residues, deamidation, aspartate isomerization, oxidation,
clipping, and sequence variants. Moreover, for the very
promising ADCs, one also needs to reveal critical quality
attributes such as drug-to-antibody ratio (DAR), drug
distribution, and conjugation sites.
Several lectures and posters will deal with these
determinations and the complete liquid chromatography (LC),
capillary electrophoresis (CE), and MS portfolios are applied,
including the main chromatographic techniques affinity
chromatography (AC), ion exchange chromatography (IEX),
reversed-phase LC, hydrophobic interaction chromatography
(HIC), hydrophilic interaction liquid chromatography
(HILIC), and the electrophoretic techniques capillary zone
electrophoresis (CZE), capillary gel electrophoresis (CGE),
and capillary iso-electrophoretic focusing (CIEF).
One of the more recent innovations in LC, 2D LC, in
the (multiple) heart-cutting mode (LC–LC) or comprehensive
mode (LC×LC) is also being more commonly applied in
practice to characterize biopharmaceuticals at both the
protein and the peptide level. The features of 2D LC will be
discussed and illustrated with detailed analysis of mAbs
and ADCs. It is expected that applications of 2D LC in
the analysis of host-cell proteins and the determination of
pharmacokinetic properties will be demonstrated as well.
For neophytes in the field of 2D LC, a short course on the
fundamentals and applications of 2D LC will be organized
on 18 June 2017 with instructors Peter Schoenmakers and
Dwight Stoll.
In state-of-the-art biopharmaceutical analysis,
hyphenation of the separation tools with mass spectrometry
is indispensable and applications using high resolution (HR)
MS, MS/MS, and ion-mobility spectrometry (IMS) with the
most recently introduced instrumentation will be presented.
Instrument and column manufacturers are also active
participants at the HPLC meetings and new tools to
successfully analyze (bio)pharmaceuticals and drug
metabolites will be shown at the HPLC 2017 exhibition and
discussed in company seminars or the scientific lectures
and poster programmes.
The Rising Profile of Comprehensive2D LC
Peter Schoenmakers,
University of Amsterdam, Amsterdam,
Netherlands
Hot Topics in Separation Science
The latter session features Dwight Stoll, who is this year’s
recipient of the Georges-Guiochon Faculty Fellowship Award.
The award is intended to support an emerging academic
group and to enhance its visibility in the high performance
liquid chromatography (HPLC) community. Despite being
based in a small school (Gustavus Adolfus College, St. Peter,
Minnesota, USA) and despite heading a research group
consisting largely—if not exclusively—of MSc students,
Dwight has done some great research. He will easily be able
to convince LC×LC “sceptics” that the sensitivity of LC×LC
can be much higher than that of conventional high-resolution
one-dimensional (1D) LC. This is because the analytes can
be focused on top of the (small) second-dimension column,
to then be eluted in a short time with minimal dilution. It can
all be achieved with a simple trick from Dwight’s book, just
by adding water prior to a second-dimension gradient-elution
reversed-phase LC separation.
The chairmen of the meeting have done a great job to
attract just about all the leading experts from within the
field of HPLC, as well as a number of great scientists from
a wider circle of separation science. The LC×LC–MS
session will be spearheaded by W.C. Byrdwell from the US
Department of Agriculture (Beltsville, Maryland, USA), who is
a less familiar face in the HPLC community. His contribution
is expected to be unique because he has his own definition
of MSn, namely n parallel mass spectrometers coupled to
a single HPLC system (with n up to at least 4). The majority
of the audience will consist of liquid chromatographers and
such an approach may make them feel mightily important.
If the sceptics find time to attend some of the LC×LC
lectures, they will also be convinced of the reliability and
robustness of LC×LC. Several speakers apply LC×LC
routinely, underlining that the technique has arrived at
a stage where it is applicable in real practice. Andre
de Villiers from Stellenbosch (South Africa) has already
demonstrated a number of excellent separations of
food-related samples and C.J. Venkatramani will describe
how 2D LC–MS of antibody–drug conjugates has
progressed “from research to mainstream pharmaceutical
analysis” in the industrial setting of Genentech. If that does
not suffice, there will be the inimitable Bob Pirok (University
of Amsterdam) from the appropriately named MANIAC
project to explain one of the very many other applications
of LC×LC technology for a completely different industrial
application (comprehensive characterization of polymeric
nanoparticles).
And yes, you may also have to listen to me, but don’t let
that deter you from travelling to Prague and attending the
two conference sessions, as well as the short-course on
Sunday morning devoted to multidimensional LC.
There will be an awful lot more to learn and to enjoy during
the HPLC 2017 Prague meeting. LC×LC only concerns
about 5% of the entire scientific programme. There are many
great speakers and excellent sessions in what promises to
be one of the most exciting and enjoyable meetings in the
HPLC series yet.
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LC•GC Europe May 2017248
Hot Topics in Separation Science
In the biomedical research of molecular bases of both
normal and pathological biological processes, it is
currently necessary not only to detect, identify, and
quantify individual compounds, but also to study their
interactions with endo- and exogenous compounds.
Obviously, for these purposes it is crucial to develop new
advanced high-performance analytical methods providing
high sensitivity, high selectivity, and high throughput.
These challenging requirements are well met by capillary
electromigration (CE) methods. They have developed in
the last three and half decades into high-performance
separation techniques suitable for the analysis of a wide
spectrum of both low- and high-molecular mass bioactive
compounds (1).
Among the variety of modern CE techniques, affinity
capillary electrophoresis (ACE) represents a special
separation mode (2,3). It is based on the monitoring of
migration times and on the measurement of effective
electrophoretic mobilities of the interacting species. It is
used not only for selective analysis of particular types of
compounds (bio)specifically interacting with the affinity
ligands present in the separation medium, but also for
the identification and quantification of these (bio)specific
interactions. Various ACE modes are available that can be
used for the investigation of thermodynamically weak or
strong bindings and kinetically fast or slow interactions both
in homogeneous liquid phase and on the heterogeneous
solid–liquid interface. ACE allows the determination of both
binding (stability, association) constants and stoichiometries
of the biomolecular complexes (3,4).
ACE possesses the advantages of CE methods, that is,
high separation efficiency, ultra-small sample volume with
high mass sensitivity, short analysis time, low consumption
of reagents and solvents, and the capability to separate
a wide range of biologically active compounds, such as
amino acids, peptides, proteins, nucleobases, nucleosides,
nucleotides, nucleic acids, saccharides, steroids,
flavonoids, and other (bio)molecules.
For investigation of biomolecular interactions, the
following ACE methods are available: The first mode is
nonequilibrium ACE of equilibrated mixtures of analyte and
ligand at different analyte–ligand ratios in the background
electrolyte (BGE) free of ligand and analyte can be used
for strong or slowly dissociating complexes. From the peak
areas of the analyte, ligand, and analyte–ligand complex,
their equilibrium concentrations and the stability constant of
the complex can be determined.
The second mode is the dynamic equilibrium ACE of
an analyte in the BGE containing a free ligand at several
distinct concentrations. Electrophoretic migration of the
analyte is retarded as a result of the formation of the
analyte–ligand complex. The advantage of this mode, the
“so-called” mobility shift assay, is that the analyte need not
be perfectly pure (the admixtures can be separated during
the ACE experiment) and concentration of the analyte
need not be exactly known since estimation of the stability
constant is based on measurement of analyte effective
mobility.
Partial filling ACE (PF-ACE) is a special ACE mode in
which only a part of the capillary is filled with ligand solution
in the BGE. This technique has several advantages over
classical ACE. Instead of adding the ligand at several
concentrations to the BGE in the whole capillary and in one
or both electrode vessels, the use of a short ligand zone
means that the consumption of the valuable ligand is very
low. The binding constants can be calculated from a slope
of linear dependence of analyte migration time changes on
the substance amount of ligand in the ligand zone in the
capillary.
In frontal analysis ACE (FA-ACE), a long zone of
equilibrated mixture of analyte–ligand complex at different
analyte–ligand ratios is introduced into the capillary, and
in the applied electric field the complex dissociates and
the zones of free analyte, analyte–ligand complex, and
free ligand are formed. From the heights of the analyte or
ligand zones on the electropherograms, their equilibrium
concentrations, binding constants, and stoichiometry of the
complexes can be determined. These parameters can also
be determined by a special mode of FA-ACE, continuous
FA-ACE, in which the analyte–ligand complex at different
analyte–ligand ratios is electrokinetically introduced in the
capillary.
In ACE with immobilized ligand, the ligand is covalently
or by physical sorption attached to the inner capillary wall
and the analyte is electrophoretically or electroosmotically
transported through this affinity open-tubular column. The
strength of the analyte–ligand interaction is evaluated from
the reduced electrophoretic mobility of the analyte as a
result of its interaction with the ligand.
References(1) R.K. Harstad et al., Anal. Chem. 88, 299–319 (2016).
(2) H.M. Albishri et al., Bioanalysis 6, 3369–3392 (2014).
(3) P. Dubský, M. Dvorák, and M. Ansorge, Anal. Bioanal. Chem.
408, 8623–8641 (2016).
(4) S. Štepánová and V. Kašicka, J. Sep. Sci. 38, 2708–2721
(2015).
The 45th International
Symposium on High Performance
Liquid Phase Separations and Related
Techniques (HPLC 2017) will be
held in Prague, Czech Republic,
from 18–22 June 2017.
Website: www.hplc2017-prague.org
Václav Kašicka, Institute of Organic
Chemistry and Biochemistry of the Czech
Academy of Sciences, Prague, Czech
Republic
Affinity Capillary Electrophoresis—A Powerful Tool to Investigate Biomolecular Interactions
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LC•GC Europe May 2017250
LC TROUBLESHOOTING
This is the third in a series of “LC
Troubleshooting” instalments
that consider how we can use
the fundamental properties of
chromatographic separations to
estimate the impact of different
variables on liquid chromatography
(LC) separations. In the first
instalment (1) we looked at the
column plate number, N, and saw
that it was not as useful a tool as
we might guess, but concluded
that a column with a plate number
of N ≈ 10,000 is a good place to
start. Last month (2), we considered
retention, expressed as the retention
factor, k. It is best to adjust retention
for 2 ≤ k ≤ 10. If the sample won’t fit
in 1 ≤ k ≤ 20, a gradient method is
likely a better choice.
As in the previous two instalments,
we’ll use the fundamental resolution
equation as a guide:
Rs = ¼N0.5 (α-1) [k/(1+k)] [1]
(i ) (ii ) (iii )
This month we’ll focus on
selectivity, α, (or peak spacing) as
expressed in term ii of equation
1, where α is the selectivity factor
between two peaks with k-values of
k1 and k2:
α = k2/k1 [2]
Several variables can be adjusted
to change α. It is not surprising that
some of these variables are more
effective and some are easier to
change than others. By looking at
the effects of different variables
on the separation, we can “count
the cost” of different choices and
choose a balance of the various
costs (effectiveness, time invested,
expense, and so forth) that fit our
requirements.
Orthogonal SeparationsWhen we focus on selectivity during
LC method development, we are
looking for ways to move peaks
relative to each other. This means
changing values of α (equation 2) by
changing the k-value of one or both
peaks under consideration. When
more than two peaks are present, it
may be necessary to move several
peaks relative to each other so that
satisfactory separation is obtained.
As part of the process of improving
selectivity, we’d like to choose a
variable (for example, solvent type
or pH) that has a high probability
of making the desired change.
One way to compare changes in
selectivity is to plot the retention of
the various sample components,
expressed as log k, for two different
variables, as in Figure 1.
In Figure 1(a), the retention
of the sample components
(blue diamonds) falls close to a
straight line, with a coefficient
of determination, r2, of 0.98. If
the slope of the line were 1.0, all
components would have the same
retention with either variable 1
or variable 2. If r2 is close to 1,
but the slope is not, the relative
retention would be approximately
the same, but little or no change
in α would be observed. When
the coefficient of determination is
close to one, we can refer to the
separation conditions as equivalent.
We might see this if the retention
on two different C18 columns were
compared. This approach would be
desirable if we want two columns
that can be used interchangeably,
with one of them designated as
a backup column for a method.
However, this is not a desirable
situation if we want to choose
a variable that will improve the
separation of a hard-to-separate
peak pair.
Figure 1(b) shows more scatter
for the points around the trend
line than Figure 1. This might
occur if we changed the organic
solvent (B-solvent) type in a
reversed-phase method, where
variable 1 is acetonitrile and variable
2 is methanol. Note that when two
adjacent points lie on opposite sides
of the trend line, a retention reversal
has occurred for those two peaks.
For example, the point circled in
red is eluted before the one circled
in green with variable 1, whereas it
comes out second with variable 2.
As the scatter about the trend line
increases r2 drops, the likelihood
of significant changes in relative
retention (selectivity) increases,
also. When the separations under
two conditions are quite different,
we refer to the separation as
orthogonal. (Yes, technically to
be orthogonal, the coefficient of
Count the Cost, Part 3: Increasing Resolution by Changing SelectivityJohn W. Dolan, LC Troubleshooting Editor
Several variables can be used to change selectivity in a liquid chromatography (LC) separation. Here we compare the variables in an effort to prioritize which experiments will be most effective.
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LC•GC Europe May 2017252
LC TROUBLESHOOTING
determination should be zero, but
this is extremely unlikely with two
chromatographic conditions.)
Orthogonal LeverageAlthough we could use r2, we get
a better measure of the scatter of
the points, as in Figure 1(b), as a
function of the standard error of
the curve, which for the present
discussion I’ll call “orthogonal
leverage”. Orthogonal leverage is
the power of a variable to make
significant selectivity changes for
reversed-phase separations.
In Table 1, I’ve summarized
orthogonal leverage data for the
common variables we use to control
reversed-phase separations. This
table is based on a large database
of chemically diverse compounds
under different chromatographic
conditions (see reference 3 for
more discussion). When the
orthogonal leverage for a specified
change in a variable is ≥1, it is a
good choice to try to change α for
hard-to-separate peaks. Yes, this is
an average number that may or may
not apply to a specific sample, but
in our “count the cost” series, we’re
seeking knowledge that will allow us
to choose conditions that increase
the probability of success as well
as learn which conditions are likely
to be a waste of time. In each case,
data are shown for an arbitrary, but
experimentally reasonable, change
in the variable (3). Let me next
briefly interpret the data of Table 1.
%B and tG: %B refers to the
percentage of organic solvent in a
reversed-phase mobile phase, most
commonly methanol or acetonitrile.
In the discussion of k (2), the Rule of
Three was mentioned: We can
expect approximately a threefold
change in k for a 10% change in
the B-solvent. This percentage is
a reasonable amount of change
without being excessive. We can
see that this gives an orthogonal
leverage value of 0.8, which is less
than our target value of ≥1. However,
80% of the way to our goal isn’t bad,
and as we know from experience
and the discussion of k last month
(2), many times a change in %B is
sufficient to pull two peaks apart.
Also, this change is very easy, so
even though it is not as effective as
some other variables, it usually is
worth pursuing. A threefold change
in the gradient time, tG, will have a
similar effect on a gradient method
as a change of 10%B does in an
isocratic one (4). The two changes
also have approximately the same
effect on orthogonal leverage (0.7
versus 0.8). A change in gradient
time is easy and reasonably
powerful, so again we choose easy
over powerful and often implement a
change in gradient time early in the
method development process.
Column Temperature: A change in
the column temperature can change
the selectivity of a separation (5)
because of the affect of temperature
on k and mobile-phase pH. A
change in temperature of 20 °C
gives us an orthogonal leverage of
0.7. Once again, this value is less
than the desired ≥1, but because it
is easy, similar to a change in %B or
tG, it is often a variable we try early
in the method development process.
Another reason that we often
choose to investigate %B or tG
and °C early in our investigations
is that they are easily modelled
based on two experimental values,
much like we saw for k in (2). Thus
Table 1: Comparing “orthogonal leverage” for reversed-phase selectivity
Variable Change Orthogonal Leverage*
%B 10% 0.8
tG 3× 0.7
Temperature 20 °C 0.7
Methanol (acetonitrile) Acetonitrile (methanol) 2.0
Column Fs > 65 1.9
pH 5 units >>7†
[Buffer] 2× 0.2
%B: percent organic solvent in the mobile phase; tG: gradient time; Temperature: column temperature; Methanol (acetonitrile): changing from methanol to acetonitrile as the B-solvent; column: a change in column chemistry; Fs: quantitative comparison of column selectivity described in (7); pH: the pH of the aqueous component of the mobile phase; [buffer]: concentration of the buffer used to control mobile-phase pH.
*Desired to be ≥1.0; here “orthogonal leverage” is defined as 10 × |δlog α|avg or 14 × standard error (SE) as defined in (3)
†Only for ionics
Based on data of reference 3.
(a)
(b)
0.04
0.04
-0.04
-0.04
-0.4
0.04
0.40
0
0.08
0.08
-0.04
log
k,
va
ria
ble
1lo
g k
, va
ria
ble
1
log k, variable 2
log k, variable 2
r2 = 0.98
r2 = 0.25
0
0
Figure 1: Comparing retention between
two conditions. (a) Two variables with
similar (“equivalent”) retention; (b) two
variables with quite different (“orthogonal”)
retention. See text for details.
By looking at the effects
of different variables
on the separation, we
can “count the cost” of
different choices and
choose a balance of the
various costs that fit our
requirements.
253www.chromatographyonline.com
LC TROUBLESHOOTING
versa) as the mobile-phase organic
component usually has a significant
change in the peak spacing of
the chromatogram. This effect is
reflected in the value for orthogonal
leverage in Table 1 of 2.0, twice
the minimum desired target of ≥1.
However, although the solvent
swap is powerful, it can create
such large selectivity changes that
we may have trouble figuring out
which peaks correspond between
experimental runs at two values
of %B, tG, or °C allow prediction
of k and α for other values of that
variable. Counting the cost of the
experiments, these are very high
value, low cost choices.
Methanol–Acetonitrile: We all
know from experience, as well
as other “LC Troubleshooting”
instalments (for example, reference
6) that a change from using
methanol to acetonitrile (or vice
the methanol and acetonitrile
chromatograms. We have to balance
this challenge against the power
of moving the peaks. As a result,
we often delay investigation of the
effect of solvent type changes until
we have checked out the easier, but
less-effective variables noted above.
Column: A change from one column
to another can have a wide range
of results, varying from little or no
change in the separation to large
changes in α. The label on the
column does not necessarily give
us enough information to make
the decision. However, there are
now on-line databases, such as
the one discussed in reference 7,
Table 2: Ranking the variables
VariableChange
in αUniversal Convenient
Low-UV/LC–MS
Robustness Equilibration
%B 0 + + + + +
Temperature 0 (+) + + + + +
Solvent type + + + 0 + 0
Column type 0 (+) + 0 + + +
pH ++ - 0 0 - +
+: effective variable, positive characteristic; 0: less-effective variable, less desirable, still
useful; –: ineffective, poor choice, problems associated.
One way to compare
changes in selectivity
is to plot the retention
of the various sample
components, expressed
as log k, for two
different variables.
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LC•GC Europe May 2017254
LC TROUBLESHOOTING
that can quantify the differences
between columns, making the
choices much easier. Table 1 shows
that the right choice of columns
(Fs > 65 according to reference 7)
can have a very strong influence
on the orthogonal leverage (1.9),
approximately the same as changing
the solvent type (2.0). As with
solvent type changes, however,
the change in the chromatogram
may be too large when the column
is changed. Another drawback of
column changes is that they are
expensive and discrete. That is, two
columns may be compared, but it is
not possible to interpolate between
two columns, as it is with solvent
blends, %B, tG, or °C. For these
reasons, column changes are often
delayed until other variables have
been tried.
pH: Changes in the mobile-phase
pH can have dramatic effects in
changes in selectivity, as reflected
in the orthogonal leverage of 7 or
more for a five-unit change in pH.
However, pH changes work only
for ionizable compounds, so this
variable is not universal and may
have only marginal results on a
sample of unknown compounds if
some or all are neutral. As was seen
in a recent “LC Troubleshooting”
instalment (8), care has to be taken
with pH adjustment to pick an
appropriate buffer for the desired
pH.
Buffer Concentration: Buffer
concentration in the mobile phase
is one of the variables when pH
is used to control selectivity.
Other than generating a minimum
buffering strength (typically
5–10 mM), changes in buffer
concentration have little effect on
the separation, as reflected in the
orthogonal leverage value of 0.2.
This one’s easy in the count the cost
choices—don’t bother. However, if
you are working on an ion-exchange
or hydrophilic interaction
chromatography (HILIC) separation,
buffer concentration can be a very
important variable, so don’t dismiss
it as useless for all LC methods.
Other InfluencesTable 1 summarizes the orthogonal
leverage for several common
variables. This is good information,
but it does not tell the whole story,
because there are other factors
involved in prioritizing which
variable to choose first in the
method development process. One
way of looking at this is expressed in
Table 2.
In Table 2, I’ve chosen the five
important variables for isocratic
separations from Table 1 and added
a few more bits of data. I have
somewhat arbitrarily assigned a
value of + (a good choice, powerful
in changing selectivity), 0 (OK, but
not great), or – (weak or ineffective,
or has major problems). Let’s look
quickly at some highlights.
%B: If we consider acetonitrile
as the B-solvent, we see that it is
OK, but not great, in changing α
(reflected in the orthogonal leverage
of 0.8 in Table 1), but changes
will work for all compound types
(universal), it is easy, works well
down to 200 nm and is compatible
with mass spectrometry (MS)
detectors (for use in LC–MS),
is easily reset (robust), and
equilibrates quickly (an advantage
for gradients and for screening
experiments). So, although it isn’t
the most powerful variable, it has
good marks in most categories and
is easy to change, usually making
it my first choice when exploring
selectivity.
Temperature: It is easy to think of
column temperature as a variable of
marginal power (orthogonal leverage
of 0.7), mainly affecting retention.
However, if ionizable compounds
are present, it can mimic changes
in pH and can be a powerful
variable in adjusting selectivity.
This happens often enough that I
put it near the top of the variables I
investigate. You can see all the other
properties of temperature are good.
Solvent Type: A change in
solvent type shows pluses in most
categories. Care does need to be
taken for work at low wavelengths
in an ultraviolet (UV) detector.
Acetonitrile is good down to 200 nm,
but for gradients below 220 nm,
methanol may have excessive
baseline drift. Changing from one
solvent to another (equilibration) may
take a bit longer than other changes,
but it is not a show stopper.
Column Type: Changing from one
column type to another can be a
powerful way to change selectivity,
but it is usually best when guided
by a quantitative column selectivity
tool (7). Unless you have a
valve-switching setup on your LC
system, exchanging one column
for another is a bit inconvenient,
but other than the cost of buying
another column, this variable has
few drawbacks.
pH: As discussed in the previous
section, pH can be the most
powerful way to change a
separation, but only works for
ionizable compounds. It takes more
effort to make up a new buffer than
to change temperature or solvent.
We have to ensure that the buffer
is compatible with the detector:
Phosphate works well down to
200 nm with a UV detector, but will
cause a snowstorm in an LC–MS
system. Unfortunately, improper
selection of the buffer or pH can
make method robustness a problem.
Two StrategiesWe can use the information
discussed above to approach
selectivity adjustment in method
development in two different ways. If
we want to do some quick screening
experiments to determine if the
different variables will be effective
for our particular samples, we could
pick variables that have large values
Changing from one
column type to another
can be a powerful way to
change selectivity, but
it is usually best when
guided by a quantitative
column selectivity
tool.
pH can be the most
powerful way to change
a separation, but only
works for ionizable
compounds. It takes
more effort to make up
a new buffer than to
change temperature or
solvent.
LC TROUBLESHOOTING
of orthogonal leverage in Table 1.
For example, we could screen two
columns with two solvents at low
and high pH (23 = 8 experiments
total) to choose initial starting
points. On the other hand, if we want
to fine-tune selectivity, it will usually
be easier to start with %B (or tG for
gradient methods) and temperature,
because they are easier to fine-tune
and the results of two experiments
for a variable allow us to predict
retention under other conditions.
SummaryWe might summarize the discussion
of counting the cost in LC method
development with the familiar
statement, “work smarter, not
harder”. Equation 1 helps us evaluate
the impact of N, k, and α on the
separation. A column with N ≈ 10,000
is usually a good place to start;
changes in column length and
particle size are easily calculated
without additional experiments. Next,
adjust retention for 2 ≤ k ≤ 10 or 1 ≤ k
≤ 20 to get “good” chromatography.
Fine-tuning k to adjust α by changes
in %B or temperature will often
give the desired separation. When
choosing which variable to explore
for further changes in α, we have
to balance the cost (both time and
money) against the effectiveness
(orthogonal leverage) and the ease of
making the adjustment; Tables 1 and
2 can help guide these decisions.
Today’s business demands often
do not give us the luxury of taking
as much time as we would like when
developing an LC method. It is more
important than ever to pause before
starting a method development
project and count the cost of a
proposed strategy. We need to take
as little time as possible to develop
fast, robust, and accurate methods.
There are many ways to do this
wrong, but with advanced planning
we can make good choices that will
minimize wasted time.
References
(1) J.W. Dolan, LCGC Europe 30(3),
138–142 (2017).
(2) J.W. Dolan, LCGC Europe 30(4),
190–195 (2017).
(3) J. Pellett, P. Lukulay, Y. Mao, W.
Bowen, R. Reed, M. Ma, R.C. Munger,
J.W. Dolan, L. Wrisley, K. Medwid,
N.P. Toltl, C. Chan, M. Skibic,K.
Biswas, K.A. Wells, and L.R. Snyder, J.
Chromatogr. A 1101, 122–135 (2006).
(4) J.W. Dolan, LCGC Europe 26,
149–154, 210–214, 260–264, 330–336
(2013).
(5) J.W. Dolan, LCGC North Am. 20(6),
524–530 (2002).
(6) J.W. Dolan, LCGC Europe 23(11),
581–584 (2010).
(7) J.W. Dolan, LCGC Europe 24(3),
142–148 (2011).
(8) J.W. Dolan, LCGC Europe 30(1), 30–33
(2017).
“LC Troubleshooting” Editor John
W. Dolan has been writing “LC
Troubleshooting” for LCGC for more
than 30 years. One of the industry’s
most respected professionals, John
is currently a principal instructor
for LC Resources in McMinnville,
Oregon, USA. He is also a member
of LCGC Europe ’s editorial advisory
board. Direct correspondence about
this column via e-mail to LCGCedit@
ubm.com
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LC•GC Europe May 2017256
GC CONNECTIONS
For its 69th session 5–9 March 2017,
the Pittsburgh Conference on Analytical
Chemistry and Applied Spectroscopy
(Pittcon) headed north to McCormick
Place in Chicago, Illinois, USA. In sharp
contrast to the many previous subzero
sessions held in Chicago, the weather
was unusually mild, so much so that I
was able to enjoy a Sunday morning run
along the Lakefront Trail. I passed by
the 17th Annual Chicago Polar Plunge
where hundreds of bewildered swimmers
encountered water colder than the air.
Attendance was up slightly compared to
last year with more than 14,000 registered
participants, 22% of whom came from 89
countries outside the United States. The
exposition hosted 78 exhibitors from 37
countries, 141 of which displayed their
products for the first time.
The technical programme continued
to be a strong part of the conference:
more than 2000 sessions were presented
in 72 symposia, 89 oral sessions, 19
contributed sessions, six workshops, 56
poster sessions, and 15 awards. Among
these were the 2017 LCGC Lifetime
Achievement in Chromatography Award
presented to Professor Pat Sandra
(Research Institute for Chromatography),
and the LCGC Emerging Leader in
Chromatography Award presented to Dr.
Deirdre Cabooter (University of Leuven).
Pittcon 2018 will meet in Orlando,
Florida, 26 February to 1 March at the
Orange County Convention Center, where
participants likely, but not necessarily, will
enjoy even balmier weather. The 2019
session is scheduled for 17–21 March in
Philadelphia, Pennsylvania.
This annual “GC Connections”
instalment reviews gas chromatography
(GC) instrumentation, columns, and
accessories shown at this year’s Pittcon
or introduced during the previous year.
For a review of new products in other
areas of chromatography, columns,
and related accessories, please see
the “Column Watch” and “Perspectives
in Modern HPLC” column instalments
from the April 2017 issue of LCGC
Europe (1,2) as well as the “Sample Prep
Perspectives” column in the May issue of
LCGC North America (3).
The information presented here is
based on manufacturers’ replies to
questionnaires, as well as on additional
information from manufacturers’ press
releases, websites, and product literature
about the past year’s products, and not
upon actual use or experience of the
author. Every effort has been made to
collect accurate information, but because
of the preliminary nature of some of
the material LCGC Europe cannot be
responsible for errors or omissions. This
column instalment cannot be considered
to be a complete record of all new GC
products introduced this year at Pittcon or
elsewhere because not all manufacturers
chose to respond to the questionnaire,
nor is all of the submitted information
necessarily included here because of the
limited available space and the editors’
judgment as to its suitability.
GC: 2016–2017 As the gas–solid variety of GC attains its
70th anniversary in 2017, this past year
had a number of notable advances in
GC technology that clearly demonstrate
its ongoing viability. Far from those
early university experiments, some of
the newest developments help remove
a number of significant obstacles in
routine GC while other advances deliver
even higher performance to the gas
chromatography–mass spectrometry
(GC–MS) realm. The most significant
development in GC instrumentation this
year has to be Agilent’s new Intuvo 9000
GC system, designed for reduced time
spent on routine tasks with minimized
maintenance risks. The core Intuvo
developments are outlined in the following
tables. Laboratories that adopt the system
will deploy columns in instrument-specific
directly heated modules, but this should
prove not to be a significant barrier to the
multiyear cycle of instrument upgrades
and replacements. Ellutia’s 500-Series
GC systems implemented a hybrid
air-oven column heating system, which
supports resistively heated as well as
conventionally heated columns. Qmicro
displayed application-specific versions of
their micro-GC platform, which is based
on microelectromechanical systems
New Gas Chromatography Products for 2016–2017John V. Hinshaw, GC Connections Editor
In this instalment, John Hinshaw presents an annual review of new developments in the field of gas chromatography (GC) seen at Pittcon 2017 and other venues in the past year.
Table 1: Companies introducing new
products in 2016–2017
Company Name
Agilent Technologies
CDS Analytical
Dani Instruments
Ellutia
LECO Corporation
Markes International
Peak Scientific
Phenomenex
Proton OnSite
Qmicro B.V.
Restek Corporation
Scion Instruments
Shimadzu Corporation
The 4S Company
Thermo Fisher Scientific
VICI DBS Ltd.
VUV Analytics
Valves, fittings, detectors, and more for chromatography and liquid handling
�
Valco Instruments Co. Inc.tel: 800 367-8424 fax: 713 688-8106 [email protected]
North America, South America, and Australia/Oceania contact:
VICI AG Internationaltel: Int + 41 41 925-6200 fax: Int + 41 41 925-6201 [email protected]
Europe, Asia, and Africa contact:
Generation of Calibration Gas Standards
Dynacalibrator® Model 505
s�4OUCH�SCREEN�CONTROL
s�4WO�CHAMBERS�WITH�INDEPENDENT� temperature control
s�%XTREMELY�STABLE�TEMPERATURE�CONTROL�n� deviation of <0.002°C at set point / 1°C change in ambient temperature
s�5TILIZES�SAFE��CONVENIENT�$YNACAL®� permeation devices
Dynacal®�0ERMEATION�4UBES
s�00"�TO�HIGH�00-�RANGE
s�!CCURATE��STABLE�CONCENTRATIONS
s�%CONOMICAL��mEXIBLE�ALTERNATIVE�TO � BULKY�BOTTLED�MIXTURES
s�.)34�TRACEABLE
� �
LC•GC Europe May 2017258
GC CONNECTIONS
Table 2: New GC instrument systems
Company Product Description
Agilent
Technologies
GC/MS Arsine
Phosphine
Analyzer
The Agilent GC/MS Arsine Phosphine Analyzer is a robust solution for routine monitoring of arsine
and phosphine impurities in olefin production. It is based on the company’s 5977B High Efficiency
Source (HES) GC–MSD with a 120 m × 0.32-mm, 8-μm film thickness methyl polysiloxane Select
Olefins column. The analyzer allows for fast implementation of precise, stable, and easy to use
methods for single-digit ppb detection of arsine and phosphine.
Intuvo 9000
GC System
The Agilent Intuvo 9000 GC system is designed for contract laboratories that perform routine
environmental and food testing, chemical and energy for raw ingredient and finished product
testing, blood alcohol content analysis, and residual solvent analysis. It features direct column
heating of one or two Intuvo no-trim GC columns at 30–450 °C with programming rates up to
250 °C/min, modular Intuvo Flow Chips, Intuvo disposable Guard Chip retention gap, ferrule-free
click and run connections, a colour touchscreen interface, Intuvo smart ID keys that provide
essential information about critical flow-path components, lower power consumption, and a
small footprint. The Intuvo GC system is compatible with Agilent’s 5977B, 7000D, and 7010B MS
detectors as well as the company’s full suite of GC detectors. According to Agilent, all of its sample
introduction systems can be used with the Intuvo GC system.
SureTarget
GC/MS Water
Pollutants
Screener
Agilent’s GC/MS SureTarget Water Pollutants Screener provides fast data review and reporting of
water pollutants, and the system screens for unknown compounds as well. It is preconfigured with
hardware, consumables, and software, including MassHunter SureTarget deconvolution workflow.
The system allows fast implementation of screening methods for the identification of more than
1000 known water pollutants via the company’s retention time and spectral database, plus many
more unknown pollutants via National Institute of Standards and Technology (NIST) search.
Ellutia
500 Series
GC gas
chromatograph
The 500 Series GC system from Ellutia is a compact single-channel gas chromatograph that, in
addition to being operated as a conventional GC system, can also perform ultrafast chromatography
using directly heated columns. In ultrafast mode, metal capillary columns are directly resistively
heated. This approach produces greatly increased ramp rates and upper temperature limits, and
faster cool down times. Cycle times are reduced by up to 10 times, while using only a fraction of the
energy a conventional GC would require. The 500 Series gas chromatograph comes as standard
with a temperature programmable injector (PTV), which can be used as a conventional split–split-less
injector with operating temperatures up to 400 °C. It also features several other operating modes such
as column oven tracking and large volume injection, plus other PTV modes with temperature ramping
up to 600 °C at ramp rates of up to 750 °C/min. Initially the 500 Series GC system will launch with a
flame ionization detection (FID), to be followed with other detection options such as electron capture
detection (ECD), flame photometric detection (FPD), and heated transfer lines to other detectors such
as a time-of-flight (TOF) mass spectrometer.
LECO
Corporation
Pegasus BT
GC time-of-
flight mass
spectrometer
LECO’s benchtop-sized Pegasus BT GC time-of-flight mass spectrometer incorporates new
software and hardware features, including a StayClean ion source, complete historical records
for data mining, and a deconvolution algorithm. The system has a detection limit of <20 fg
octafluoronapthalene (OFN) and a quantitative dynamic range of up to 105. The open-style electron
ionization (EI) source reportedly virtually eliminates downtime associated with source cleaning.
Markes
International
XR series
thermal
desorption
instruments
Markes International introduced its new xr series of thermal desorption (TD) instruments: The
TD100-xr 100-tube automated thermal desorber; the UNITY-xr single-tube thermal desorber;
the ULTRA-xr 100-tube autosampler for UNITY-xr; and the Air Server-xr on-line volatile organic
chemical (VOC) monitoring system. The samplers are designed for use with GC–MS analyzers.
A key new feature is an extended capability for automated sample splitting and re-collection
that allows valuable samples to be reanalyzed for method development and compliance with
standard methods. The samplers can recover compounds from C2 to C44, and they incorporate
a new water-management module for on-line monitoring of humid air streams. Markes also has
redesigned the instrument control software.
Qmicro B.V.
Explosion
proof micro GC
analyzer
This explosion proof (IECEx/ATEX certified) gas analyzer from Qmicro is based on the company’s
micro GC platform. Several custom, specifically configured applications are enabled by the
four-channel GC cartridge. The transmitter performs on-line gas analysis and heating value
computations and has external dimensions of 29 × 26 × 12 cm3. The replaceable GC cartridge
encompasses the injector and sample loop, MEMS micro thermal conductivity detectors with a
500 ppb detection limit, GC column, and zone heating up to 180 °C at a maximum ramp rate
of 5 °C/s. The micro GC platform is designed for fast reliable gas mixture analysis in on-line
monitoring. It allows fully autonomous gas analysis and can run unattended preconfigured
analysis methods, including peak identification, integration, and result communication via industry
standards. Other features include automated calibration, low consumption of consumables, and
less maintenance. The micro GC platform can be configured for a variety of applications, such as
variable natural gas or biogas applications, as well as others.
259www.chromatographyonline.com
GC CONNECTIONS
the past year. Continuing their
advancements in vacuum ultraviolet
spectroscopic detection for GC,
VUV Analytics expanded the
wavelength range of its VGA-101
detector as well as enabling series
coupling with some conventional GC
detectors. From Dani Instruments,
the DiscovIR–GC Solid Phase GC–
FTIR detector produces solid-phase
transmission infrared (IR) spectra of
eluted GC components.
A number of sampling accessories
round out the newest instrumental
offerings, including the XR Series of
thermal desorption instruments and
HiSorb extraction probes from Markes,
the 6000 Series Pyroprobe pyrolyzers
from CDS Analytical, and the Master
MTAS robotic autosampler platform
from Dani, all of which advance their
respective sampling tasks with new or
improved capabilities.
(MEMS) for the inlets and detectors plus
direct column heating. All three of these
systems make use of customized column
formats.
For GC–MS, the Exactive GC Orbitrap
GC-MS system from Thermo-Fisher
Scientific, the GCMS-QP2020
GC–MS system and GCMS-TQ8050
triple-quadrupole GC–MS system
from Shimadzu, and the Pegasus BT
GC–time-of-flight MS system from
LECO represent the latest advances
in GC–MS technologies. Two new
application-specific offerings from Agilent
in this area, the SureTarget GC/MS Water
Pollutants Screener and the GC/MS Arsine
Phosphine Analyzer, plus the EPA 8270D
analyzer kit from Thermo Fisher Scientific,
all emphasize the ongoing march towards
routine application of high-performance
GC–MS for standardized methods.
Two optical spectroscopy GC
detectors were announced during
The zone of general GC accessories
was not left out this year, either. The
ADM flowmeter from Agilent has
some new calibration capabilities that
minimize downtime. New Thermolite
septa and Topaz inlet liners from Restek
are designed to extend trace-level
analysis capabilities. Restek also now
offers an electron ionization (EI) filament
replacement for a number of Agilent MS
detectors. There was a large crop of new
gas generators, too: VICI DBS entered
this segment with several offerings for GC
systems, Proton OnSite showed a new
G-Series family of hydrogen generators,
and Peak Scientific has a new nitrogen
generator.
In GC columns, Agilent and
Phenomenex introduced some new polar
columns that offer increased stability
and upper temperature limits as well as
application-specific selectivity tailoring.
Agilent’s new Intuvo System modular
Table 2: New GC instrument systems Contd....
Company Product Description
Scion Instruments
Scion Analytical GC Analyzer Solutions
Scion Instruments announced GC analyzers that tailor the capabilities of GC systems to meet specific analytical requirements. Based on the company’s 436 and 456 gas chromatography platforms, these systems can be configured with multiple columns, switching valves, and temperature-controlled ovens. The Scion SPT (sample preconcentration trap) helps chromatographers perform low level determinations from environmental to gas purity analysis. Available configurations include Simulated Distillation (SIMDIST), Detailed Hydrocarbon Analysis (DHA), Refinery Gases (RGA), Oxygenates, Natural Gas Analyzers (NGA), and Transformer Oil Gas Analysis (TOGA).
Shimadzu
Corporation
GCMS-
QP2020
GC–MS
system
The Shimadzu GCMS-QP2020 GC–MS system offers Advanced Scanning Speed
Protocol, which allows scans up to 20,000 μ/s. The system features a new large-capacity
turbomolecular pump with heightened exhaust efficiency for all carrier gases, including
nitrogen, and the system enables simultaneous scan and single-ion monitoring (SIM) for
qualitative and quantitative data in a single run. The new Smart SIM creation function
automatically creates a program that enables a staggered SIM of multiple components,
resulting in higher SIM sensitivity, while the Quick-CI function allows users to introduce
reagent gas while using the EI source to look for the molecular ion. The ion source is
accessible from the front of the instrument.
GCMS-
TQ8050
triple-
quadrupole
GC–MS
system
The Shimadzu GCMS-TQ8050 MS detector enables detection of femtogram-level concentrations.
It utilizes a new turbomolecular pump that is designed to achieve a higher vacuum and yield
higher sensitivity, accuracy, and stability. Shimadzu’s UFsweeper technology helps to conduct
multiple reaction monitoring (MRM) analysis speeds up to 800 transitions/s, while the company’s
Smart MRM technology helps accurately create methods for ultra-trace analysis and ensures
high sensitivity for MRM measurements. A high-efficiency ion source generates and transmits
ions directly to the detector for higher sensitivity and improved repeatability. Alongside the mass
spectrometer, Shimadzu’s LabSolutions Insight software provides analysts with multianalyte data
review, colour-coded quantitative flags, and a status review function. The system has a stated
instrument detection limit (IDL) of 0.36 fg OFN.
Thermo
Fisher
Scientific
Exactive GC
Orbitrap
GC–MS
system
The Thermo Scientific Exactive GC Orbitrap GC–MS system is designed to provide sensitive,
routine-grade performance for both targeted and nontargeted analyses. The system reportedly
offers the quantitative power of a GC triple-quadrupole mass spectrometer combined with the
advantages of Orbitrap high-resolution accurate mass technology. The new system is designed
for scientists working in routine environments who are looking to increase their reach beyond
targeted quantitation in analysis. The GC–MS system has a resolving power of up to 50,000
(FWHM) at m/z 272, routine sub-part-per-million mass accuracy, and an instrument detection
limit of less than 6 fg OFN. The electron ionization/chemical ionization (EI/CI) ExtractaBrite
ion source is removable under vacuum through a vacuum interlock. The system is capable of
vent-free column exchange with a source plug.
LC•GC Europe May 2017260
GC CONNECTIONS
Table 3: New GC accessories
Company Product Description
Agilent Technologies
ADM flowmeter The Agilent ADM flowmeter provides an external reference for verifying flows and is intended for use when troubleshooting detectors or other GC problems. The flowmeter measures flow volumetrically, which eliminates the need to select a gas type and allows for composite gas streams. The flowmeter incorporates a removable calibrated cartridge. Instead of returning the meter to a third party for recalibration, the cartridge can be replaced regularly, once a year, to keep the meter compliant. Range: 0.5–750 mL/min; accuracy: ±2% of reading or 0.2 mL/min—whichever is greater. The meter has a USB port and can record up to four flows on screen.
Intuvo Flow Chips
Agilent’s Intuvo Flow Chips are modular components that enable flexible configuration of the Intuvo 9000 GC system flow path. These application-specific chips provide simplified connections between the inlet and column (via a guard chip), and column to detector without the need for ferrules. The chips are fitted with smart keys that plug directly into the instrument and automatically configure it for backflush, flow splitting, or MS detection.
Intuvo gasket
Agilent’s Intuvo 9000 GC system uses ferrule-free face seals called gaskets for all fittings within the sample gas flow path. These gaskets take the place of ferrules throughout the GC system, providing a face seal between components of the flow path. These connections are reportedly easily replaced, provide leak-free connections, and enable click-and-run column changes. They are available in three types: polyimide, nickel, and as a plug. The polyimide gasket is designed for standard use up to 350 °C, and the nickel gasket provides a solution for applications at temperatures as high as 450 °C. The plug gasket can be used to check for leaks and for troubleshooting the flow path.
Intuvo Guard Chip
The Intuvo Guard Chip is a simple disposable chip that contains flow channels that connect the inlet of the Intuvo 9000 GC system to the Intuvo column via an inlet flow chip. The guard chip acts as a guard column within a single, disposable chip to prevent unwanted material from depositing on and damaging the head of the column and is designed to be easily installed and replaced. It provides almost 1 m of sample flow path just before the Intuvo GC column. This protection eliminates the need for retention time adjustment and the need to trim a column.
CDS Analytical
6000 Series Pyroprobe pyrolyzers
CDS Analytical introduced its 6000-series Pyroprobe pyrolyzers in two models: the 6150 base model and the 6200 with analytical trap, reactant gas, and sorbent tube capability. Both pyrolyzers can heat to 1400 °C at up to 20 °C/ms using up to 10 stored temperature profiles. The interface temperature is settable as well, up to 400 °C, and is programmable up to 100 °C/min. A heated sample line and valve oven maintain temperatures over the entire gas flow path. The 6200 trap can be heated at up to 1000 °C/min up to 400 °C. An available dynamic headspace option for the 6200 Pyroprobe can sample from 25-mL test tubes or from an 800 mL vessel, at up to 300 °C. A liquid nitrogen cryotrap is also available. The 6000-Series instruments use a LCD touchscreen and a Windows-based software package.
Dani Instruments
DiscovIR–GC Solid Phase GC–FTIR detector
Dani Instruments’ DiscovIR–GC Solid Phase GC–FTIR detector couples a gas chromatograph to Fourier-transform infrared (FT-IR) spectroscopic detection for the identification of GC eluants by depositing them in a spiral track onto a -40 °C cryogenically cooled rotating sample collection disk. Trapped components are then spectrally scanned by a FT-IR interferometer to acquire searchable solid-phase transmission spectra for identification. The spectrometer operates at 4 cm-1 resolution. The FT-IR detector is compatible with the company’s Master GC gas chromatograph and Master AS autosampler.
Master MTAS autosampler
Dani Instruments’ Master MTAS robotic autosampler platform can be configured for conventional autosampling, dual injection, or automated solid-phase microextraction (SPME). The Master Dual-AS autosampler performs simultaneous injection into two GC inlets, allowing analyses on two columns or detectors at the same time. Beyond decreasing overall analysis times for otherwise serially performed dual-column GC, the dual injection mode can provide increased selectivity and sensitivity through the use of two different columns or detectors. The autosampler can hold up to two trays of 80 vials apiece and has a liquid injection volume range of 0.1–500 μL.
Master SHS Robotic autosampler with standard addition
Dani Instruments’ Master SHS autosampler for static headspace can now be used for standard, surrogate, and reagent addition. In one example, in which the autosampler was configured with the company’s Master GC and Master TOF-MS Plus, detection limits of 1 μg/L of formaldehyde in cosmetic products via standard addition and derivatization with PFBHA were reported, with a linear dynamic range to 100 μg/L.
Markes International
HiSorb extraction probes
Markes’ HiSorb extraction probes are a sampling system for the analysis of volatile and semivolatile organic compounds (VOCs and SVOCs) that can be used for immersive or headspace sampling of liquids and solid samples. They are compatible with thermal desorption (TD)GC–MS analysis using industry-standard tubes on all leading commercial systems. The probes feature detection limits lower than for SPME because of the larger capacity of their polydimethylsiloxane (PDMS) sorbent. Cryogen-free preconcentration by TD before automated GC–MS analysis reportedly improves sensitivity. Markes’ HiSorb Agitator heats and agitates HiSorb probes in standard 10- or 20-mL sample vials. The probes are then washed, dried, and inserted into a conventional thermal desorption tube for automated TD-GC–MS analysis.
Peak ScientificSolaris nitrogen generator
Peak Scientific's Solaris nitrogen generator has been engineered and designed as a gas delivery solution that can reduce downtime and increase workflow efficiencies for compact mass spectrometer instruments or for evaporative light scattering detection (ELSD). Built in the company’s ISO-9001 manufacturing facility, the Solaris nitrogen generator can provide up to 10 L/min of high purity nitrogen (up to 99.5%). Developed with a space-saving design, Solaris can be placed on a benchtop and paired with an additional air compressor unit to provide air supply for laboratories without an in-house air supply or who wish to contain their gas supply in a single system.
261www.chromatographyonline.com
GC CONNECTIONS
Table 3: New GC accessories Contd...
Company Product Description
Proton OnSite
Hydrogen generators: G200, G400, G600, G600-HP, G4800
Proton OnSite’s G Series benchtop hydrogen generators utilize proton-exchange membrane (PEM) technology to produce ultra-high purity hydrogen on-site. The generators sense demand and adjust gas production accordingly. The G-series family is available with flow rates of 200, 400, and 600 mL/min and produce 99.9995% pure hydrogen at output pressures from 43–119 psig (3–8 barg). The G600-HP model scrubs the hydrogen to a 99.99999% purity level. The G4800 model provides 99.9999+% purity hydrogen at up to 4.8 L/min at pressures up to 200 psig (13.8 barg).
Restek Corporation
EI filament replacement part for Agilent MS detector
Restek’s EI filament replacement part is designed for Agilent 5972, 5973, 5975, and 5977 GC–MS systems. The filaments meet or exceed original manufacturer’s performance and are subjected to quality control (QC) tests including heat, electrical current, and resistance. In addition, samples from each filament manufacturing lot are installed in a mass-selective detector for in situ testing.
Restek Methanizer
Restek’s Methanizer is an aftermarket add-on for Agilent 5890, 6890, and 7890 GC FID systems. A methanizer allows parts-per-billion-level determination of CO and CO2 by converting them to methane upstream of an FID system. The system incorporates temperature control to ensure complete conversion of CO and CO2 to CH4. A separate installation kit includes all parts needed for installation into any Agilent GC system.
Thermolite Plus Septa for GC inlets
Restek’s Thermolite Plus septa are usable with inlet temperatures as high as 350 °C, and reportedly have ultra-low bleed levels. The septum incorporates a new plasma coating that eliminates sticking in the injection port. Some of the septa have a CenterGuide design to minimize coring, and the 5-mm septa are partially predrilled for improved puncturability. The septa come preconditioned and ready to use, packaged in ultraclean blister packs. Each batch is reportedly GC–FID tested.
Topaz inlet liners
Restek’s Topaz inlet liners feature an improved deactivation designed to help push detection limits downward for reactive compounds, which also yields better reproducibility and enables longer liner lifetime. Topaz liners are available in clean blister packs for most laboratory GC inlet systems.
The 4S Company
GC-SOS gas chromatography simulation and optimization software
The 4S Company’s GC-SOS simulation and optimization software is an effective tool for developing highly efficient GC methods that reportedly can reduce development time from hours to minutes and can produce more efficient methods. This new version features flexible input with one to three training runs and up to five temperature segments. In many cases an existing method can be used as a training run. The software uses an auto-optimization proprietary numerical algorithm to provide a highly optimized method in seconds, and it has an animation viewer that provides visualization of separations and can be used for teaching as well.
Thermo Fisher Scientific
EPA 8270D analyzer kit
Thermo Scientific’s EPA 8270D kit is designed for use with Thermo Scientific ISQ single-quadrupole GC–MS systems coupled with the Thermo Scientific TRACE 1300 Series gas chromatographs. The kit allows laboratories updating their current GC–MS system to take advantage of key features, including a single-column method and modular GC injectors and detectors, as well as a removable ion source under vacuum. The EPA 8270D analyzer kit includes column, liners, septa, and ferrules specifically designed for EPA semivolatile analysis and a CD with specific instrument and data processing methods, e-workflow, compound retention time database, environmental method specific reports, and an instructional user guide. A video tutorial for method setup ensures that the instrument is up and running with EPA Method 8270D immediately following system installation. Both the Thermo Scientific Dionex Chromeleon chromatography data system (version 7.2 SR4 MUB or newer) and the Thermo Scientific TraceFinder software (EFS version 4.1 or newer) are compatible. The kit also features a dynamic range of 0.2–200 ppm with a single column and liner, plus reduced helium usage with Thermo Scientific’s Instant Connect Helium Saver module.
VICI DBS Ltd.
FID Plus Hydrogen Gas and Zero Air Generator
The VICI DBS range of FID gas generators combines hydrogen and zero air generators into one system. Available in high and ultrahigh purity for all GC detector and carrier gas applications, the generator has software control via USB and alarm capability. It is available in two styles: the FID Station Plus is flat for placement under a GC system, and the FID Tower Plus is a tower configuration for benchtop placement next to instruments. The FID gas generators are available with H2 flow ranges up to 1 L/min and 150 psig (10.5 barg) and air output flow at a maximum of 1.5 L/min.
N2 TOWER Plus nitrogen gas generator
The VICI DBS N2 Tower Plus is a high-purity nitrogen gas generator that produces up to 99.999% pure N2 from a range of models at flow rates up to 4 L/min. An optional catalytic furnace reportedly reduces total hydrocarbon levels to below 0.1 ppm. An external air compressor is required.
NM-H2 Plus Hydrogen Gas Generator
The VICI DBS NM-H2 Plus is a high-purity hydrogen generator that produces 99.999999% pure hydrogen gas for GC carrier gas and flame ionization detectors. The device uses a proton-exchange membrane (PEM) purifier that does not employ palladium membranes. A cascading configuration allows multiple generators to be connected for scalable laboratory expansion. The generators are available in various models with output flow rates ranging from 100 to 1000 mL/min, and the outlet pressure is adjustable from 1 to 160 psig (0.1–11 barg).
VUV AnalyticsVGA-101 gas chromatography detector
The VGA-101 vacuum ultraviolet (VUV) benchtop spectrometer from VUV Analytics is designed to meet the needs of scientists with advanced GC applications. The VGA-101 features a wavelength spectrum of ~120–430 nm, which provides selectivity for complex structures such as polyaromatic hydrocarbons (PAHs). The operating temperature of up to 450 °C allows the deconvolution and analysis of high boiling-point compounds. The detector can be placed in series with MS and other GC detectors. The detector reportedly provides sampling rates up to 100 Hz. Applications include the analysis of high-boiling-point fuel samples containing complex hydrocarbon mixtures, and characterization of isomeric compounds with extensive branching or ring structure that are difficult to distinguish with alternative methodologies.
LC•GC Europe May 2017262
GC CONNECTIONS
columns are being made available in
a variety of common dimensions and
stationary phases; the modules feature a
quick-connect planar cage that does not
use conventional ferrules.
2016–2017 was another very active
year in GC that again emphasized the
pivotal role that small-molecule and
volatile component analyses fulfil in
the fields of separation science. As we
spin around to Pittcon 2018, I expect to
be pleasantly surprised by more new
developments and innovations.
Acknowledgements I would like to thank the manufacturers
and distributors that kindly furnished
the requested information, which
allowed a timely report on new product
introductions over the past year. For
those manufacturers who did not
receive a “New Products” questionnaire
this year and would like to receive one
and be considered for early inclusion
into the 2018 new GC and related
product introductions review, as well as
the other related review articles to be
published in LCGC, please send the
name of the primary company contact
plus the mailing and e-mail addresses
to Laura Bush, Editorial Director, LCGC
and Spectroscopy, UBM Americas,
485 Rte. 1 South, Bldg. F, Suite 210,
Iselin, NJ 08830, USA, Attn: 2018
New Chromatography Products. The
questionnaire will be sent out later in
2017.
References(1) D.S. Bell, LCGC Europe 30(4), 196–207
(2017).
(2) M.W. Dong, LCGC Europe 30(4), 208–218
(2017).
(3) D.E. Raynie, LCGC North Am. 35(5),
296–305 (2017).
“GC Connections” editor John V.
Hinshaw is a Senior Scientist at
Serveron Corporation in Beaverton,
Oregon, USA, and a member of
LCGC Europe’s editorial advisory
board. Direct correspondence about
this column to the author via e-mail:
Table 4: New GC columns
Company Product Description
Agilent
Technologies
Intuvo no-trim
GC column
modules
Agilent’s modular wall-coated open-tubular (WCOT)
GC columns for their Intuvo GC are offered in
lengths of 5, 15, 20, 30, and 60 m, inner diameters
of 0.18 mm, 0.25 mm, and 0.32 mm, and with film
thicknesses up to 1.8 μm; not all stationary phase and
column dimension combinations are available. The
columns are held in a quick-connect planar cage that
requires no ferrules and includes an electronic ID key
with column information and usage tracking.
J&W CP-Wax
52 CB column
J&W have improved their CP-Wax CB columns with
greater inertness lifetime over repeated temperature
cycling to the columns’ upper temperature limits
of 250 °C isothermal and 275 °C maximum ramp
excursion. The columns are available in standard
lengths from 5 to 100 m, inner diameters from 0.10 to
0.53 mm, and film thicknesses from 0.10 to 1.20 μm.
J&W
HP-INNOWax
column
J&W’s HP-INNOWax columns deliver improved
inertness lifetime over repeated temperature cycling
to the columns’ upper temperature limits of 260 °C
isothermal and 270 °C maximum ramp excursion for
film thicknesses up to 0.50 μm and 240–250 °C for
thicker films. The columns are available in standard
lengths from 5 to 60 m, inner diameters from 0.18 to
0.53 mm, and film thicknesses from 0.10 to 1.0 μm.
Phenomenex
Zebron GC
FAME Testing
Trio columns
Phenomenex is making available a trio of fatty
acid methyl ester (FAME) testing columns that are
optimized for different FAME applications. The
Zebron ZB-FAME has optimized selectivity that is
compliant with compendial fatty-acid GC methods
while delivering run times as short as 11 min. The
Zebron ZB-23 is an alternative column selectivity
optimized for marine oils and omega fatty acids. The
Zebron ZB-88 is well suited for separation of olive oil
and other hydrogenated oil analysis.
Introduction
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Da
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Expertise in all aspects of GC
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PLENARY SPEAKERS
SYMPOSIUM CHAIRMEN Michal Holčapek and František Foret
Four parallel program tracks
Alberto Cavazzini, University of Ferrara, Ferrara (Italy)
Thermodynamic and kinetic considerations on fully
porous and core-shell particles for ultrafast
enantioseparations via liquid chromatography
with Pirkle-type chiral stationary phases
Doo Soo Chung, Chung-Ang University, Seoul (South Korea)
Sample preconcentration techniques in-line coupled
with capillary electrophoresis
Gert Desmet, Vrije Universiteit Brussel (Belgium)
Current state and future directions in liquid
chromatography
Norman J. Dovichi, University of Notre Dame, Indiana (USA)
Capillary zone electrophoresis as a tool for eukarotic
proteomics
Pat Sandra, Research Institute for Chromatography, Kortrijk (Belgium)
The LC toolbox for biopharmaceutical characterization
Zoltán Takáts, Imperial College, London (UK)
Ambient and LC-MS lipidomic profi ling of clinical
samples – new era in cancer diagnostics
Peter A. Willis, NASA, Pasadena (USA)
Development of liquid phase separation systems
for spacefl ight missions of exploration
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LC•GC Europe May 2017264
MULTIDIMENSIONAL MATTERS
The development of separation
systems on the basis of on-line,
comprehensive two-dimensional
liquid chromatography (LC×LC)
is a highly complex task, not only
because of the high number of
variable operation parameters, but
also because of the high demands
on the instruments. Recently, the
commercial availability of new high
performance liquid chromatography
(HPLC) systems specifically
designed for LC×LC operation
has attracted much interest in
the academic and industrial
community. The latest innovations
in multidimensional separations
were collected in a special issue
of a journal dedicated to this topic
(1). Although LC×LC appears to
have matured, there are some
specific problems still present that
hamper the widespread use of this
technology. One key aspect is the
coupling of an on-line LC×LC system
to a mass spectrometer. Generally,
on-line LC×LC is based on a very
fast second dimension separation
to achieve low cycle times (2). This
often results in flow rates that are far
above the optimum for electrospray
ionization mass spectrometry
(ESI-MS). In order to circumvent
the necessity for flow-splitting, a
miniaturized LC×LC system with
nano-LC in the first dimension and
micro-LC in the second dimension
was described previously (3). This
month’s “Multidimensional Matters”
explores the benefits of coupling
miniaturized comprehensive 2D LC
to a hybrid high-resolution mass
spectrometer (HRMS) with a focus
on its application in environmental
(water) analysis.
The Selection of Suitable
Stationary Phases—The First
Dimension: The selection of a
suitable stationary phase in on-line
LC×LC not only encompasses
the need for a high orthogonality,
but also the appropriate column
dimensions. Although there are
numerous combinations of different
column chemistries to enhance
orthogonality (4), it has become
common practice for the selection
of two reversed-phase stationary
phases in on-line LC×LC to use
a less retentive column in the
first dimension (1D) to avoid the
necessity of a very strong eluent
in the first dimension that would
be a strong eluent in the second
dimension (2D) as well (2). However,
this practice is inconsistent with the
need for good sensitivity because
on-column focusing increases with
the retentivity of the stationary phase
material (5). This is especially true
for a nano-LC column where the
sample volume has to be adapted
in conjunction with the internal
diameter. Moreover, polar analytes
that experience no retention on
a classical silica-based reversed
phase stationary phase cannot
be trapped. It is these analytes,
however, that play a pivotal role in
environmental analysis (6). Porous
graphitic carbon (PGC) is ideally
suited to trap very polar compounds
that would elute at the void volume
on a silica-based reversed stationary
phase. Leonhardt et al. recently
demonstrated that 5-fluorouracil,
which has almost no retention
on a “classical” reversed-phase
stationary phase, could be eluted
with a retention factor of 146 on
a PGC stationary phase with an
internal diameter of 75 μm (5). The
authors noted that small fluctuations
The Benefits of Coupling Miniaturized Comprehensive 2D LC with Hybrid High-Resolution Mass SpectrometryJuri Leonhardt1, Jakob Haun1, Torsten C. Schmidt2, and Thorsten Teutenberg1, 1Institut für Energie- und Umwelttechnik
e. V., Duisburg, Germany, 2University Duisburg-Essen and Centre for Water and Environmental Research (ZWU), Essen, Germany
Comprehensive two-dimensional liquid chromatography (LC×LC) is evolving and becoming more commonly used in practice, but there are some specific problems still present that hamper the widespread use of this technology. One key aspect is the coupling of an on-line LC×LC system to a mass spectrometer. Generally, on-line LC×LC is based on a very fast second dimension separation to achieve low cycle times. This often results in flow rates that are far above the optimum for electrospray ionization mass spectrometry (ESI-MS). This month’s “Multidimensional Matters” looks at the benefits of miniaturization in the first and second dimension for coupling with a high-resolution mass spectrometer (HRMS) and describes an environmental analysis application.
265www.chromatographyonline.com
MULTIDIMENSIONAL MATTERS
in the composition of the injection
solvent could lead to fronting
effects. This means that the method
is most suited for a large-volume
direct injection of an aqueous
sample without the need for further
preconcentration. This strategy is
currently applied in most laboratories
dealing with water analysis, because
the availability of very sensitive mass
spectrometers allows for a direct
injection of large sample volumes
(for example, 1000 μL injected onto
a 4.6 mm i.d. column [7]). Leonhardt
et al. successfully increased the
absolute injection volume to 5 μL
on a 12 mm × 0.075 mm PGC
nano-LC column coupled to a
50 mm × 0.1 mm reversed-phase
C18 core–shell stationary phase.
Moreover, West et al. described
essential differences in retention
behaviour of compounds separated
on PGC phases compared to
common reversed-phase C18
materials (8). At that time this
contradicted the popular opinion
that a PGC phase is simply a
hydrophobic phase comparable
to C18 phases. From this, it
was decided to use PGC as the
stationary phase with an internal
diameter of 100 μm for the first
dimension that would include a
large volume injection to counteract
analyte dilution—a major problem in
on-line LC×LC approaches (9).
The Selection of Suitable
Stationary Phases—The Second
Dimension: With regard to the
differences in retention behaviour to
PGC phases, a reversed-phase C18
Small 2dc
Large 2d
c
Reduction ofextra-column delayand band broadeningin 2D(due to higher 2Dflow rates)
Higher 2D linearflow velocity(at constant volumeflow)
Reduction ofanalyte dilution
Better on-columndilution of transferred1D sample(reduced solventeffects / better peakfocusing)
Figure 1: Advantages and disadvantages of using a smaller or larger internal diameter second dimension column in on-line LC×LC techniques.
100
10
1
0.1
Flow Rate (μL/min)
0 50 100 150 200 250
V_GD: 1 μL V_GD: 3 μL V_GD: 5 μL2 s gradient delayV_GD: 50 μLV_GD: 10 μL
Gra
die
nt
Dela
y T
ime (
s)
Figure 2: Dependence of gradient delay time on the flow rate for different delay volumes (V_GD).
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LC•GC Europe May 2017266
MULTIDIMENSIONAL MATTERS
material was chosen for the second
dimension separation. In order to
obtain a fast second dimension
separation, the use of elevated
temperatures is recommended to
operate a column at high efficiency
even if the flow rate is far above the
van Deemter minimum (10). In terms
of ultimate temperature stability,
bridged ethyl hybrid particles have
proven to be extremely temperature
resistant, even if the temperature is
increased to 150 °C (11). However,
it is an essential requirement that
the column hardware in which the
stationary phase is packed is also
stable at the applied temperatures.
Unfortunately, this point turned
out to be the Achilles heel for the
application of very high-temperature
LC in the 2D. The main problem was
that the available capillary column
hardware was either based on
packed fused-silica capillaries that
needed plastic parts for the fittings
or even based on packed PEEK
capillaries. Often, steel sheathings
covered the packed capillaries to
mimic the outer appearance of a
standard HPLC column. In both
cases, high temperatures cause
deformation and carry a high
potential of hardware failure. For this
reason, the temperature was set to
60 °C. In order to further increase
the separation speed in the 2D, a
core–shell stationary phase with
a particle diameter of 2.6 μm was
chosen instead of a fully porous
sub-2-μm particle packed column.
Since the 2D column has to fulfill
significantly more requirements
compared to the 1D column, the
main arguments for choosing either
a small or a large 2D column internal
diameter are visualized in Figure 1.
As can be seen, the preselection
of the 2D column internal diameter
is dominated by questions of speed
and dilution effects. A large diameter
increases analyte dilution simply by
additional dispersion that occurs
within the large column volume. On
for increased flow rates. It can be
concluded that a smaller column can
enhance overall speed by increasing
the speed of the separation itself,
whereas the use of higher flow rates
in combination with a larger column
internal diameter decreases the
contribution of extra-column volumes
to the analysis time. This means that
for an optimum speed, the stationary
phase in combination with the
column internal diameter should be
chosen so that the optimum flow rate
is high enough to keep the influence
of the extra-column volumes low,
but low enough to allow the use of
a small internal diameter column to
optimize towards linear velocity. An
essential requirement for a linear
velocity optimization is that the
latter does not result in significant
efficiency losses, which depend
on the stationary phase and the
the other hand, the transferred 1D
solvent will be far better diluted as
well, so the analyte bands potentially
can be better refocused on the 2D
stationary phase. The latter process
counteracts analyte dilution (12).
The question of speed, however,
is of greater importance when fast
LC is applied. As demonstrated in
the theory, chromatographic speed
is proportional to the average
linear mobile phase velocity (u).
This means linear velocity can
be increased by decreasing the
column diameter at a constant flow
rate. An increase of the column
diameter implies that significantly
higher flow rates are necessary
to keep u constant, so solvent
consumption increases as well. On
the other hand, extra-column delay
times, such as for example the
gradient delay time, are reduced
(a) (b)
Figure 3: The use of PEEK tubing sleeves when mounting a 50-μm i.d. 360-μm o.d. fused-silica capillary to a column end fitting for 1/16” capillaries. (a) Ideal arrangement before tightening the nut. (b) Void volume formation: dislocated inner sleeve and fused-silica capillary after tightening the nut. Grey: connection bore of the column end fitting. Brown: fused-silica capillary. Orange: PEEK sleeve 1/32” A 360 μm o.d. Blue: PEEK sleeve 1/16” A 1/32” o.d. Arrows: approximate grip of the ferrule.
0.6 P 7
P 6
P 55
4
2
3
1
6
P 4
P 3
P 2
P 8
0.5
0.4
0.3
0.2
0.1
2D
Rete
nti
on
Tim
e (
min
)
1D Fraction Number
0 10 20 30 40 50 60 70 80
Figure 4: The areas used for the calculation of the surface coverage. Adapted with permission from reference 3.
Although LC×LC appears to have matured, there are some specific problems still present that hamper the widespread use of this technology.
267www.chromatographyonline.com
MULTIDIMENSIONAL MATTERS
experimental conditions. However,
in the case of a sub-3-μm core–
shell stationary phase at elevated
temperature, this requirement is
fulfilled (13).
The influence of the extra-column
volume on analysis time will be
discussed by using the example of
the gradient delay volume (Vdwell).
This is defined as the volume from
the point of gradient mixing to the
column head. The gradient delay
time (tdwell) needed to flush this
volume can be calculated by using
the fundamental equation that
defines the flow rate as volume per
time, thus:
[1]tdwell =
Vdwell
F
where F is the volumetric flow
rate. If Vdwell is held constant as
in Figure 2, functions of the type
f(x) = b/x are obtained on the basis
of equation 1.
The maximum value of the time
scale in Figure 2 has been chosen
as 60 s. This is the intended time
for a complete 2D cycle in this
example. As can be seen from
Figure 2, the gradient delay time
drastically increases at low flow
rates. A delay time of 2 s (3.3%)
was set as a significance level. A
delay below this tolerable value
guarantees that the gradient delay
time is not a significant part of
the cycle time, keeping in mind
that the extra-column volume from
the column end to the detector
will also be added to the analysis
time. As shown in Figure 2, this
significance level can only be
reached by gradient delay volumes
much less than 10 μL within the
given flow rate range. Moreover,
it can be seen that the gradient
delay volumes have to be reduced
together with the flow rate at
an equal rate to keep the delay
constant. A gradient delay volume of
50 μL is a typical value for modern
ultrahigh-pressure LC (UHPLC)
instruments with small standard
mixers (usually ~ 35–45 μL).
Miniaturized mixers (1–25 μL) are
available for capillary-UHPLC
systems and columns. Accordingly,
smaller gradient delay volumes
below 10 μL can be obtained if the
tubing dimensions are selected
appropriately. The lowest delay
volumes of around 1 μL can be
obtained if no mixer is used.
However, this potentially results in
a baseline ripple for conventional
piston-based pumps that affects
the sensitivity of the detector. The
pump systems used for this study
are pneumatic pumps that do not
need a separate mixer post to the
tee-connector that unifies and mixes
the flow of both gradient channels.
Thus, gradient delay volume is easily
reduced to 1 μL for micro-LC. From
Figure 2 it can be deduced that the 2D column internal diameter should
allow a flow rate of at least 30 to
40 μL/min to avoid a too strong
influence of the gradient delay in a
60 s cycle time.
It was therefore decided to
use a stationary phase for the
second dimension with an internal
diameter of 300 μm and a length
of 50 mm. The application of even
smaller internal diameters was not
considered with respect to the
already high pressure drop and the
need for high loading capabilities as
a result of the transfer volume from
the 1D. It can therefore be concluded
that the final diameters were 0.1 mm
and 0.3 mm for the first and second
dimension, respectively. In order
to compare these values to that of
typical conventional on-line LC×LC
setups, which usually use 1.0 mm or
2.1 mm in the 1D and 4.6 mm in the 2D, the relative difference between
the column internal diameters of the
two coupled LC dimensions (Δ i.d.)
was calculated using equation 2:
[2]i.d. =
—2dc
2dc
1dc𝚫
where 2dc is the internal diameter
of the second dimension, and 1dc
is that of the first dimension. The
results are listed in Table 1.
As can be seen from Table 1, the Δ
i.d. used for the miniaturized on-line
LC×LC system lies in between the
Δ i.d. of typical conventional on-line
LC×LC systems. This indicates that
the contribution to analyte dilution
caused by the difference in internal
diameter is not expected to be
significantly higher or lower than in
conventional LC×LC systems.
Note on Column Fittings: Usually,
there is no discussion on column
fittings as the capillary outer
diameter (o.d.) is standardized to
1/16” in conventional HPLC. Several
column manufacturers, however,
still pack micro-LC columns with
the corresponding fittings for
standard capillaries, which are very
large in comparison to the internal
Table 1: Comparison of the Δ i.d. between non-miniaturized on-line LC×LC and the miniaturized approach described in this
article. Conventional setups 1 and 2 show typical inner diameters of non-miniaturized on-line LC×LC systems that do not use a
flow split between the dimensions.
1D column i.d. 2D column i.d. ∆ i.d.
Conventional setup 1 1.0 4.6 0.78
Conventional setup 2 2.1 4.6 0.54
Miniaturized LC×LC system 0.1 0.3 0.66
The selection of a suitable stationary phase in on-line LC×LC not only encompasses the need for a high orthogonality, but also the appropriate column dimensions.
Chromatographic speed is proportional to the average linear mobile phase velocity (u). This means linear velocity can be increased by decreasing the column diameter at a constant flow rate.
LC•GC Europe May 2017268
MULTIDIMENSIONAL MATTERS
diameter of the column and the
capillaries that are usually used in
micro-LC (o.d.: 1/32” or 360 μm).
Consequently, PEEK sleeves—for
360-μm o.d. capillaries there are
often two—are frequently used to
bridge the gap between the outer
diameter of the capillary and the
connection bore size of the column
end fitting (see Figure 3).
Significant extra-column band
broadening can result from unwanted
void volumes at the column end
fittings. The void volumes can be
formed as shown in Figure 3, or
between the stacked sleeves or the
sleeve and the capillary up to the
grip point. Accordingly, columns with
end fittings for 1/32” o.d. capillaries
should be used to avoid peak
broadening. Additionally, the use of
zero dead-volume fitting assemblies
is recommended where possible.
The Simplified Heating
Concept: To keep the setup of the
multidimensional system as simple
as possible, it was decided to heat
both LC dimensions equally by using
the air-bath oven of the column
compartment. A detrimental point,
however, is the stability of the valves
used for the modulation, which
were also included in the heating
compartment. Since both extended
pressure and temperature might
lead to a continuous abrasion of the
rotor, 60 °C was chosen as the oven
temperature for isothermal heating of
both LC dimensions to increase the
lifetime of the valves as well as the
stationary phases.
Selection of Mobile Phases: For
a further optimization of selectivity,
different mobile phases should
be used in the first and second
dimension. Therefore, methanol was
selected as protic solvent in the 1D
and acetonitrile was selected as
aprotic solvent in the 2D. The reason
for the chosen order is the different
viscosity maxima of binary solvent
systems consisting of
water–acetonitrile and water–
methanol. A mixture of water–
methanol exhibits a much higher
pressure maximum when a solvent
gradient is applied than a mixture
of water–acetonitrile (14) and is
therefore more suitable to be used
in the 1D. In the 2D, a very high
linear velocity has to be achieved
to reduce the cycle time. Hence,
a mixture of water–acetonitrile has
been used in the 2D.
Gradient Programming: If an
unknown sample has to be analyzed
or the information about the sample
is limited, a generic gradient should
be used. This means that for both
dimensions, a linear gradient
that covers the full range from,
for example, 5% to 95% B should
be applied. Many studies have
described more advanced gradient
programming for LC×LC methods
including so-called shift-gradients
(15). A shift-gradient usually refers to
a change of the starting conditions
of the second dimension separation
during the linear gradient of the first
dimension separation. The potential
benefit of shift-gradients is that the
gradient window for consecutive 2D runs can be adapted so that the
resolution of compounds eluting
during these gradients is higher
when compared to full-gradients.
Unfortunately, this concept does
not consider that the approach
is no longer generic. Many users
would prefer easy-to-use generic
methods without changing the
gradient parameters. The technical
feasibility to apply shift-gradients
is an advantage if fine-tuning of
a separation needs to be done
to optimize a two-dimensional
separation. For screening methods,
the application of full-gradients
seems to be a better way because it
is difficult or impossible to anticipate
all theoretical combinations of a
complex sample. The gradient
programming has therefore been
kept as simple as possible for
a generic approach. For the 1D
separation, a linear gradient was
programmed with an isocratic
plateau at the end of the gradient.
The overall analysis time for each
injection is about 110 min. In the 2D
separation, the cycle time was 1 min.
Hyphenation to Mass
Spectrometry: The hyphenation of
a miniaturized LC×LC system to a
mass spectrometer is very critical
in terms of the extra-column dead
volume. This refers to the transfer
line connecting the column outlet
with the ion source of the mass
spectrometer as well as the emitter
tip of the ion source itself. Whereas
a short connection between the LC
99
4844
16
99
65 64
31
0A B DC
50
100
Nu
mb
er
of
dete
cted
targ
ets
1D LC2D LC
Figure 5: Overview of the identified analytes by 1D HPLC–MS and 2D nLC×μLC–MS. Detailed list of detected targets is given in reference 18. A: Detected targets in reference standard by < 5 ppm; B: detected targets in wastewater sample by < 5 ppm; C: detected targets in wastewater sample by < 5 ppm and retention time < 2.5%; D: detected targets in wastewater sample by < 5 ppm, retention time < 2.5% and MS/MS hit. Adapted with permission from reference 18.
A gradient delay volume of 50 μL is a typical value for modern ultrahigh-pressure LC (UHPLC) instruments with small standard mixers (usually ~ 35–45 μL).
269www.chromatographyonline.com
MULTIDIMENSIONAL MATTERS
system and the mass spectrometer is
not the main obstacle, an unsuitable
internal diameter of the emitter tip
can be devastating in terms of the
observed separation efficiency.
To reduce the band broadening
behind the column, an emitter tip
with an internal diameter of 50 μm
was installed instead of the classical
100-μm i.d. emitter tip. While the
classical tip is made of stainless
steel, the modified miniaturized
emitter tip is based on a PEEKSil
capillary. To ensure ionization, at
the top of the PEEKSil capillary a
stainless steel tip with the respective
internal diameter was installed. With
regards to the connection technique
it should be noted that the PEEKSil
emitter is designed for 1/32” fittings.
High pressure-resistant fittings are
screwed to a 1/32” union. This union
offers the advantage of being able
to be used as the grounding point.
The change of the emitter tip can be
easily accomplished in a few minutes.
In the next section, we describe
the application of a miniaturized,
on-line dual-gradient LC×LC
system coupled to hybrid HRMS
detection. A 99-component standard
mixture and a complex wastewater
sample were used to demonstrate
the performance of this approach.
Moreover, a comparison was made
between the miniaturized 2D LC
approach and a conventional 1D LC
approach that is usually used for
suspected target screening of
environmental samples.
Results and DiscussionCalculation of the Surface
Coverage as a Measure of
Orthogonality: First of all, the
surface coverage for the LC×LC
separation of the 99-component
standard mixture was calculated
using the convex hull that includes
all analyte spots (see Figure 4).
In this case, the convex hull is
an eight-sided irregular polygon
described by the points P1 to P8.
The area of this convex hull was
calculated by a vector method that
Dück et al. used in their work (16).
Accordingly, the area of the convex
hull can be divided in six scalene
triangles that are numbered in
Figure 4. Each of these triangles
can be described by two vectors
that have the same corner point as
origin and the other corner points as
heads. The vectors that were used to
determine the six areas are marked
red in Figure 4. P1 was chosen
as the origin of all vectors. The
complete calculation can be found
in reference 3. The surface coverage
can now be approximated by the
area ratio of the convex hull and the
rectangular area, which is ~0.61 (or
61%). This result clearly underlines
that both dimensions are only weakly
correlated. According to Gilar et al.,
a coverage of 60% of the available
separation space can be considered
very high (4). The authors even state
that for most practical applications
a surface coverage higher than 63%
cannot be achieved.
Comparison of 2D LC with 1D
LC: A higher peak capacity is
usually obtained in two-dimensional
liquid chromatography compared
with one-dimensional separations
resulting in a higher number
of compounds that can be
chromatographically resolved.
The user, however, is not primarily
interested in whether it is
possible to obtain a higher peak
capacity. For practical purposes
it is more important whether a
two-dimensional system will also
lead to a significantly higher
number of detected or identified
compounds when compared to
a one-dimensional separation.
Therefore, the sample itself and
not the theoretical peak capacity
should be the basis for this
evaluation. A direct comparison
between a one-dimensional and a
two-dimensional separation would be
useful for an objective judgement of
the performance of two-dimensional
separation approaches. However,
there is only a very limited number
of dedicated studies dealing
with this issue (17). Leonhardt et
al. recently made a comparison
between a one-dimensional and a
two-dimensional chromatographic
approach coupled to hybrid
high-resolution mass spectrometry
(18). The comparison was based
on a screening approach in
environmental analysis, where three
criteria for compound identification
were employed. First, the accurate
mass with a deviation of less than
5 ppm was used for identification.
If the first criterion was fulfilled, the
retention time should not deviate
more than 2.5% from the retention
time measured in the reference
standard. If this criterion was also
fulfilled, MS/MS spectra of the
reference standard were compared
with that of the sample. Figure 5
summarizes the results of this
comparison.
All chosen compounds could
be detected with both approaches
on the basis of the accurate mass
in the reference standard. When
a real sample was analyzed,
48 compounds were found with
the 1D LC approach, while 65
compounds could be detected
with the miniaturized 2D LC
approach. Using the retention
time as an additional filter for the
elimination of false positive hits,
four compounds had to be removed
in the 1D LC approach, while only
one compound had to be eliminated
for the 2D LC approach. It should
be emphasized that retention time
is a powerful parameter to ensure
the identification of a compound.
Of course, a further criterion for an
unambiguous identification should
be considered. MS/MS information
can help to distinguish even isobaric
compounds that have the same
mass-to-charge ratio, m/z. The
possibility of acquiring additional
MS/MS information also depends
on the peak width. As can be seen
from Figure 5, MS/MS information is
only obtained for a small number of
analytes that have been identified
on the basis of the first and second
criterion. The reason is that the
For a further optimization of selectivity, different mobile phases should be used in the first and second dimension.
A direct comparison between a one-dimensional and a two-dimensional separation would be useful for an objective judgement of the performance of two-dimensional separation approaches.
LC•GC Europe May 2017270
MULTIDIMENSIONAL MATTERS
algorithm for acquiring product ion
spectra looks for the most intense
peak in the full scan MS spectrum,
which is then fragmented. Peaks with
a lower intensity are not selected.
Even if 10 MS/MS experiments
are performed during one cycle,
the compounds of interest might
be missed because they possess
an intensity too low compared to
high abundant ions of the matrix.
This could be circumvented if the
algorithm is programmed that
only ions of known substances
are selected for fragmentation
experiments. Nevertheless, the
number of compounds that can
be confirmed by applying all three
criteria is significantly higher
for the 2D LC approach. This
demonstrates that the miniaturized
LC×LC system performs well for
screening purposes, although the
absolute injection volume is reduced
by a factor of 13 and a further
dilution cannot be avoided by the
modulation.
Solvent ConsumptionThe overall 2D solvent consumption
of this miniaturized on-line LC×LC
approach was compared to that of
conventional fast 2D systems that
use flow rates between 1 mL/min
and 5 mL/min. While conventional
fast 2D systems consume 1.44 L
to 7.2 L of solvent over 24 h,
only 0.0576 L are needed by a
miniaturized 2D system operated
at 40 μL/min. Keeping the rising
solvent costs in mind, there are also
reduced solvent costs when using
miniaturized, on-line LC×LC systems
compared with non-miniaturized
versions.
References(1) T.C. Schmidt, O.J. Schmitz, and T.
Teutenberg, Analytical and Bioanalytical
Chemistry 407, 117 (2015).
(2) D.R. Stoll, J.D. Cohen, and P.W. Carr,
Journal of Chromatography A 1122, 123
(2006).
(3) J. Haun, J. Leonhardt, C. Portner, T.
Hetzel, J. Tuerk, T. Teutenberg, and
T.C. Schmidt, Analytical Chemistry 85,
10083 (2013).
(4) M. Gilar, P. Olivova, A.E. Daly, and J.C.
Gebler, Analytical Chemistry 77, 6426
(2005).
(5) J. Leonhardt, T. Hetzel, T. Teutenberg,
and T.C. Schmidt, Chromatographia 78,
31 (2015).
(6) E.L. Schymanski, H.P. Singer, P.
Longree, M. Loos, M. Ruff, M.A.
Stravs, C.R. Vidal, and J. Hollender,
Environmental Science & Technology
48, 1811 (2014).
(7) Y.T. Li, J.S. Whitaker, and C.L. McCarty,
Journal of Chromatography A 1245, 75
(2012).
(8) C. West, C. Elfakir, and M. Lafosse,
Journal of Chromatography A 1217,
3201 (2010).
(9) S.Y. Wang, L.Z. Qiao, X.Z. Shi, C.X. Hu,
H.W. Kong, and G.W. Xu, Analytical and
Bioanalytical Chemistry 407, 331 (2015).
(10) G. Vanhoenacker and P. Sandra,
Journal of Separation Science 29, 1822
(2006).
(11) T. Teutenberg, K. Hollebekkers, S.
Wiese, and A. Boergers, Journal of
Separation Science 32, 1262 (2009).
(12) D.R. Stoll, X.P. Li, X.O. Wang, P.W. Carr,
S.E.G. Porter, and S.C. Rutan, Journal
of Chromatography A 1168, 3 (2007).
(13) F. Gritti and G. Guiochon, LCGC North
America 30, 586 (2012).
(14) T. Teutenberg, S. Wiese, P. Wagner, and
J. Gmehling, Journal of Chromatography
A 1216, 8470 (2009).
(15) D.X. Li and O.J. Schmitz, Analytical
and Bioanalytical Chemistry 405, 6511
(2013).
(16) R. Duck, H. Sonderfeld, and O.J.
Schmitz, Journal of Chromatography A
1246, 69 (2012).
(17) Y. Wagner, A. Sickmann, H.E. Meyer,
and G. Daum, J. Am. Soc. Mass
Spectrom. 14, 1003 (2003).
(18) J. Leonhardt, T. Teutenberg, J. Tuerk,
M.P. Schlusener, T.A. Ternes, and T.C.
Schmidt, Analytical Methods 7, 7697
(2015).
Juri Leonhardt studied instrumental
analytical chemistry and laboratory
management and received his Ph.D.
from the faculty of “Instrumental
Analytical Chemistry” at the
University Duisburg-Essen in 2016.
Since 2011 he has been scientific
coworker at the Institut für Energie-
und Umwelttechnik e. V. (Institute
of Energy and Environmental
Technology) in Duisburg, Germany.
His research is primarily focused
on the development of miniaturized
comprehensive multidimensional
liquid chromatography systems
based on nano- and micro-liquid
chromatography and their
hyphenation to different detection
techniques.
Jakob Haun studied water science
at the University of Duisburg-Essen,
Germany. From 2008 to 2012 he was
a scientific coworker at the Institut
für Energie- und Umwelttechnik
e. V. in Duisburg. In 2014, he was
awarded the Eberhard Gerstel Prize
by the German Chemical Society
(GDCh) and received his Ph.D.
from the Faculty of Chemistry of the
University of Duisburg-Essen. His
related research was focused on the
development of a multidimensional
separation system for the qualitative
screening analysis of complex
samples (for example, wastewater).
It is based on miniaturized on-line
LC×LC hyphenated to quadrupole–
time-of-flight mass spectrometry.
Torsten C. Schmidt is head of
the Department of Instrumental
Analytical Chemistry and the
Center for Water and Environmental
Research (ZWU) at the University
of Duisburg-Essen, and scientific
director at the IWW Water Centre in
Mülheim an der Ruhr, Germany. He
is currently president of the German
Water Chemistry Society. In 2013,
he received the Fresenius Award
of the German Chemical Society.
His main research interests include
the development and application
of analytical methods with a
focus on separation techniques
(GC, LC), sample preparation
and compound-specific stable
isotope analysis, process-oriented
environmental chemistry, and
oxidation processes in water
technology.
Thorsten Teutenberg studied
chemistry at Ruhr University
Bochum, Germany. He studied for a
doctorate in analytical chemistry at
this institution, submitting a thesis
on high-temperature HPLC. In 2004
his career took him to the Institut
für Energie- und Umwelttechnik
e. V. in Duisburg as a research
associate. Since 2012 he has been
in charge of the Research Analysis
Department, mainly working on the
various aspects of high-temperature
HPLC, miniaturized separation
and detection techniques, and
multidimensional chromatography
processes.
“Multidimensional Matters” editor
Robert Shellie has extensive
experience in hyphenated
techniques. He joined Australian
Centre for Research on Separation
Science (ACROSS), University of
Tasmania, Hobart, Australia, in 2005.
He is currently Chromatography
Market Manager at Trajan Scientific
and Medical.
It should be emphasized that retention time is a powerful parameter to ensure the identification of a compound.
271www.chromatographyonline.com
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seals up to 19,000 psi (-1310 bar) and can be connected
and disconnected more than 100 times.
www.biotech.se/products/marvelxact/
Biotech AB, Onsala, Sweden.
Nitrogen generator
Peak Scientific’s Solaris
nitrogen generator has been
engineered as a gas delivery
solution for ELSD or compact
mass spectrometer instruments.
Delivering up to 10 L/min of
ultrahigh purity nitrogen (up to
99.5%), Solaris is a benchtop
solution with a space-saving
design, according to the
company. A compatible air compressor unit is available as
an optional extra.
www.peakscientific.com/elsd
Peak Scientific Instruments Ltd, Glasgow,
Scotland, UK.
SEC-MALS detector
The miniDawn Treos II detector for
SEC-MALS measures absolute
molar mass and size, eliminating
uncertainty in macromolecular
characterization, according to
the company. Covering ranges
of 200–10,000,000 g/mol (MW)
and 10–50 nm (Rg), the detector
can measure as little as 25 ng of 100 kDa polystyrene. An
optional DLS module adds size measurements down to 0.5 nm.
Productivity enhancements include on-site serviceability,
“one-click-MW”, and upgradeability to UHPLC–SEC-MALS.
www.wyatt.com
Wyatt Technology Corporation, Santa Barbara, California,
USA.
LC•GC Europe May 2017272
PRODUCTS
VOC analysis
The PTR-TOF 6000 X2 from Ionicon
is reportedly the premium PTR-TOF
trace VOC analyzer. The system is
comprised of a novel high-resolution
TOF and Ionicon’s PTR technology
with the new “X2” features, for the
ultimate PTR-TOF-MS experience.
According to the company, the analyzer
combines the latest generation of
performance-enhancing tools, including the Ion-Booster funnel
as well as the hexapole Ion-Guide. The ion funnel focuses the
ions into the hexapole ion guide, which results in nearly lossless
transmission of an extremely focused ion beam into the TOF
mass spectrometer. This increases the sensitivity dramatically
and also improves the instrument’s mass resolving power.
www.ionicon.com
Ionicon Analytik GmbH, Innsbruck, Austria.
MALS detector
The Postnova PN3621
Maximum Angle MALS
detector provides precise
multi-angle light scattering
detection for size-exclusion
chromatography (SEC) and
field-flow fractionation (FFF),
according to the company.
The detector simultaneously measures the scattering
intensity at a maximum of 21 angles, which enables
superior determination of absolute molecular weight and
size of proteins, polymers, and nanoparticles.
www.postnova.com
Postnova Analytics GmbH, Headquarter Landsberg,
Germany.
Sample preparation
The latest addition to UCT’s
QuEChERS line puts a new
“spin” on dSPE, according
to the company. SpinFiltr
combines the practice of
conventional dSPE with the
added benefit of ultrafiltration.
UCT’s new format of dSPE
sorbents paired with a 0.2-μm
filtration device, simultaneously removes unwanted matrix
compounds and filters samples without the need for any
additional steps.
https://sampleprep.unitedchem.com/products/
quechers/dispersive-clean-up/spinfiltr
UCT LLC, Bristol, Pennsylvania, USA.
HPLC columns
Phenomenex Inc. has introduced a 5-μm
particle size to its family of Kinetex F5
pentafluorophenyl propyl (PFP)
core–shell columns. The Kinetex F5 is a
robust PFP core–shell phase that offers
excellent reproducibility and performance
and significantly reduces method
development time with its dynamic
and responsive chemical functionality,
according to the company. With five
retention mechanisms and five separation
modes, it is reportedly an effective
orthogonal alternative to the widely used
C18 and C8 phases.
www.phenomenex.com
Phenomenex Inc., California, USA.
Hydrogen gas for GC
The VICI DBS range of FID gas
stations with sophisticated software
control and alarm capability present
the GC user with the opportunity
to reap all the benefits offered
by hydrogen carrier gas, whilst
overcoming the safety concerns.
According to the company, these
unique instruments combine the
reliability of the VICI DBS hydrogen
and zero air generators into one compact and convenient
FID package.
http://www.dbsinstruments.com/en/prodotti/fid_
tower_plus/
VICI AG International, Schenkon, Switzerland.
Mobile benches
Manufacturer of benches for
mass spectrometry and elevating
UHPLC benches. Mass Spec
IonBench products integrate MS
peripherals, a built-in vacuum pump
noise reduction enclosure, and
protect turbomolecular pumps by
reducing vibration by 99%. There
is reportedly up to a 30% saving
in laboratory space allocation.
Solidly built lockable casters simplify moving the system. The
integrated vacuum pump enclosure reduces noise emissions by
80% in perception. LC Elevating IonBench can be easily lifted
up or down, for a convenient and safe access to the top of your
UHPLC.
www.ionbench.com
IonBench, Joigny, France.
273www.chromatographyonline.com
PRODUCTS
Microchip-based column
The μPAC from Pharmafluidics
is a microchip-based
chromatography column with a
perfectly ordered pillar array as
separation bed. μPAC columns
facilitate a significant increase
in peak capacity and sensitivity
at moderate column pressure.
According to the company, they enhance the detection of
molecules in tiny, complex biological samples in the field of
biomarker discovery and development of biopharmaceuticals.
Furthermore, μPAC columns are compatible with any third-party
nano LC–MS system.
www.pharmafluidics.com
Pharmafluidics, Ghent, Belgium.
Preparative system
Quattro countercurrent and centrifugal
partition chromatographs and extractors
are designed to work with, and
complement, standard flash and HPLC
laboratory and process instrumentation.
When appropriate, replacing the solid–
liquid columns with unique liquid–liquid
instrumentation allows preparations
from milligram to tonnes every year. No on-column adsorption
or degradation will occur, according to the company. A
mass-balance is the norm for CCC/CPC. Typically a 50–80%
solvent saving occurs. Standard biphasic solvents, ionic
liquids, liquid chiral selectors, and ion exchangers may all be
used. According to the company, crude material that would
poison standard columns can be injected without causing
contamination.
www.quattroprep.com
AECS-QuikPrep Ltd., London, UK.
LC/SFC system
Shimadzu’s Nexera UC/s
(SFC/UHPLC switching
system) allows measurements
by liquid chromatography
(LC) and supercritical fluid
chromatography (SFC) on
a single system. Switching
between SFC and LC enables
rapid screening for optimum separation conditions
resulting in improved analytical efficiency. Through a
newly released upgrade kit, UHPLC units already installed
can be upgraded to the Nexera UC/s to decrease the
investment cost for an additional SFC system.
www.shimadzu.eu
Shimadzu Europa GmbH, Duisburg, Germany.
LC system
Agilent has launched its
new InfinityLab product
family, including the Agilent
1260 Infinity II liquid
chromatography system.
Integrating the high-end
technology of Agilent’s
flagship 1290 LC system
into the company’s core platform, the 1260 Infinity system
offers an ergonomic design that aims to increase customer
efficiency, reduce costs, and improve overall usability.
According to the company, the InfinityLab family provides
the end-to-end solution laboratories need to ensure
analytical excellence.
www.agilent.com
Agilent Technologies, Inc., California, USA.
HILIC columns
iHILIC-Fusion, iHILIC-Fusion(+),
and iHILIC-Fusion(P) columns
are based on spherical silica
or polymer particles. According
to the company, their unique
surface-bonding technologies
provides customized selectivity,
high separation efficiency, and
ultra-low column bleeding.
They are suitable for the
separation of polar compounds
in metabolomics, proteomics, glycomics, lipidomics, and ion
analysis.
www.hilicon.com
Hilicon AB, Umeå, Sweden.
Thermal desorption tubes
Providing optimum
sampling and analytical
performance for
VOC and SVOC
analysis from air
and solids, Markes’s
range of sorbent
tubes are reportedly
manufactured to the highest quality, with each individually
checked for total quality assurance. Available in metal,
inert-coated, or glass with a range of packing materials
suitable for the widest range of applications.
www.markes.com
Markes International Ltd., Llantrisant, UK.
LC•GC Europe May 2017274
EVENT NEWS
11–14 June 2017
30th International Symposium
on Polymer Analysis and
Characterization (ISPAC)
Linz, Austria
E-mail: [email protected]
Website: www.ispac-conferences.org
18–22 June 2017
HPLC 2017
Prague, Czech Republic
E-mail: [email protected]
Website: www.hplc2017-prague.org
3–6 July 2017
Sample Preparation Summer
Course
Chania, Crete, Greece
E-mail: sampleprep2017@
enveng.tuc.gr
Website: www.sampleprep2017.tuc.gr
16–19 July 2017
International Symposium, Exhibit,
& Workshops on Preparative and
Process Chromatography
Philadelphia, Pennsylvania, USA
Website: www.prepsymposium.org
19–21 July 2017
37th International Symposium
and Exhibit on the Separation and
Characterization of Biologically
Important Molecules (ISPPP 2017)
Philadelphia, Pennsylvania, USA
E-mail: [email protected]
Website: www.ISPPP.org
18–19 September 2017
4th Stir-Bar Sorptive Extraction
Technical Meeting
Novotel Paris Sud, France
E-mail: [email protected]
Website: www.sbsetechnicalmeeting.com
9–12 October 2017
17th International Nutrition &
Diagnostics Conference
Hotel Duo, Prague, Czech Republic
E-mail: [email protected]
Website: www.indc.cz
Please send any upcoming event
information to Lewis Botcherby:
International Symposium for High-Performance Thin-Layer Chromatography (HPTLC 2017)
The International Symposium for High-Performance
Thin-Layer Chromatography (HPTLC 2017) will
take place in Berlin, Germany, 4–8 July 2017.
Progress and innovation are rarely found on the
well-trodden path of a known scientific routine,
according to the organizers, and inspiration comes
from the cross-pollination of ideas from multiple
fields and institutions. It is this spirit of science that the organizers seek to
evoke at HPTLC 2017. Visitors can see firsthand the continuing evolution of
high-performance thin-layer chromatography into a modern and powerful tool
with broad applicability throughout laboratory science.
If you are interested in what HPTLC could do for your laboratory, or simply want
to update your understanding of methods using this technique, then HPTLC 2017
is the venue for you. The symposium showcases the most up-to-date research
methods with the latest proven applications, highlighting recent instrumental
advances as well as advances in automation and data analysis. A series of
advanced training courses in cutting-edge techniques are also available, along
with a social programme to promote networking and contact with thought leaders.
Food chemists can obtain 30 ZFL credit points when visiting HPTLC 2017.
The AK Separation Science of the GDCh division Analytical Chemistry offers
funding for poster presenters. Last minute poster presentations will still be
accepted and included in the book of abstracts.
E-mail: [email protected]
Website: www.hptlc.com
ChromSoc’s Spring Symposium 2017: Supercritical Fluid and Ultra-High Performance Chromatography
ChromSoc’s Spring Symposium will take
place in the Discovery Event Centre,
Discovery Park, Sandwich, Kent, UK,
15–17 May 2017. The symposium will feature
oral presentations by leading practitioners of
SFC and UHPLC. The latest innovations and applications will be described and
the lectures will be augmented and supported by a comprehensive table-top
exhibition of instrumentation and consumables.
Held over three days at the Discovery Park, the event will feature 30 lectures
with plenty of time allowed for discussions and questions both at exhibitor
stands and in the lecture theatre. Additional scientific, technical, practical, and
computational demonstrations from our sponsoring companies will augment the
programme, with 16 exhibition table-top displays available throughout the event.
Exhibition setup and delegate registration will be on Monday 15 May 2017
and a buffet lunch will be provided around midday. The scientific sessions will
start on Monday afternoon with a specialized workshop on CHROMacademy
software by Crawford Scientific followed by a “How to do it tips and tricks”
demonstration at Jaytee’s laboratory located close by on the Discovery Park site.
The meeting is supported by scientists from across Europe and USA,
including Roman Szucs of Pfizer UK, who will be hosting sessions and helping in
the coordination of the event at The Discovery Centre.
ChromSoc bursaries are available for this event: www.chromsoc.com/
academic-support.aspx
For further information on the meeting’s registration, table-top exhibiting, the
social event, sponsorship, or payment details please visit: www.chromsoc.com/
resources/1/meetings/2017/Spring%20flier.pdf
Telephone: +44 (0)141 945 6880
E-Mail: [email protected]
EVENT NEWS
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