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TRANSCRIPT
THE
APPLICATIONSBOOK
September 2015
www.chromatographyonline.com
ES665957_LCESUPP0915_CV1.pgs 08.31.2015 14:21 ADV blackyellowmagentacyan
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ES667258_LCESUPP0915_CV2_FP.pgs 09.01.2015 21:14 ADV blackyellowmagentacyan
THE APPLICATIONS
BOOKEnvironmental5 Analysis of VOC and FOG Emissions from Molded Components for Automobiles According to VDA 278 Dr Rebecca Kelting, Shimadzu Europa GmbH
8 Analysis of Organochlorine Pesticides (OCPs) on SGE BP5MS Trajan Scientific and Medical
Food and Beverage9 Determining Aroma Compounds in Edible Oils by Direct Thermal Desorption GC–MS Using Microvials Gerstel
10 Rapid Aroma Prof ling of Cheese Using a Micro-Chamber/Thermal Extractor with Thermal Desorption–GC–MS AnalysisPaul Morris, Caroline Widdowson, and David Barden, Markes International
12 Vitamin D2 and D
3 Separation by New Highly Hydrophobic UHPLC/
HPLC Phase YMC Europe GmbH
General14 GC–TEA Detection of Nitrosamines within Toys and Rubber/Latex Products Donna Kinder, Ellutia
Medical/Biological15 Blood Alcohol Content Analysis Using Nitrogen Carrier Gas Ed Connor1 and Greg Dooley2, 1Peak Scientific, 2Colorado State University
17 Membrane Proteins Wyatt Technology Corporation
18 Molecular Weight Determination of LMWH SEC–MALS vs. SEC–UV–RI Wyatt Technology Corporation
Pharmaceutical/Drug Discovery20 Authentication of Traditional Chinese Prescriptions Using Comprehensive 2D-LC Sonja Krieger, Agilent Technologies Inc
21 Investigation of Iron Polysaccharide Complexes by GPC/SEC Using RI- and UV-Detection PSS Polymer Standards Service GmbH
22 Accurate Pain Management Analysis in Under 5 Min on Raptor™ Biphenyl Superf cially Porous Particle LC Columns Sharon Lupo, Ty Kahler, and Paul Connolly, Restek Corporation
24 UHPLC Analysis of Immunoglobulins with TSKgel® UP-SW3000 SEC Columns Tosoh Bioscience GmbH
26 Chiral Separation of Beta Blocker Pindolol Enantiomers YMC Europe GmbH
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3 THE APPLICATIONS BOOK – SEPTEMBER 2015
CONTENTS
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4 THE APPLICATIONS BOOK – SEPTEMBER 2015
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The Publishers of LC•GC Europe would like to thank the members of the Editorial Advisory Board for
their continuing support and expert advice. The high standards and editorial quality associated with
LC•GC Europe are maintained largely through the tireless efforts of these individuals.
LCGC Europe provides troubleshooting information and application solutions on all aspects of
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readers with the tools necessary to deal with real-world analysis issues, thereby increasing their
efficiency, productivity and value to their employer.
Kevin AltriaGlaxoSmithKline, Harlow, Essex, UK
Daniel W. ArmstrongUniversity of Texas, Arlington, Texas, USA
Michael P. BaloghWaters Corp., Milford, Massachusetts, USA
Brian A. BidlingmeyerAgilent Technologies, Wilmington,
Delaware, USA
Günther K. BonnInstitute of Analytical Chemistry and
Radiochemistry, University of Innsbruck,
Austria
Peter CarrDepartment of Chemistry, University
of Minnesota, Minneapolis, Minnesota, USA
Jean-Pierre ChervetAntec Leyden, Zoeterwoude, The
Netherlands
Jan H. ChristensenDepartment of Plant and Environmental
Sciences, University of Copenhagen,
Copenhagen, Denmark
Danilo CorradiniIstituto di Cromatografia del CNR, Rome,
Italy
Hernan J. CortesH.J. Cortes Consulting,
Midland, Michigan, USA
Gert DesmetTransport Modelling and Analytical
Separation Science, Vrije Universiteit,
Brussels, Belgium
John W. DolanLC Resources, Walnut Creek, California,
USA
Roy EksteenSigma-Aldrich/Supelco, Bellefonte,
Pennsylvania, USA
Anthony F. FellPharmaceutical Chemistry,
University of Bradford, Bradford, UK
Attila FelingerProfessor of Chemistry, Department of
Analytical and Environmental Chemistry,
University of Pécs, Pécs, Hungary
Francesco GasparriniDipartimento di Studi di Chimica e
Tecnologia delle Sostanze Biologica-
mente Attive, Università “La Sapienza”,
Rome, Italy
Joseph L. GlajchMomenta Pharmaceuticals, Cambridge,
Massachusetts, USA
Jun HaginakaSchool of Pharmacy and Pharmaceutical
Sciences, Mukogawa Women’s
University, Nishinomiya, Japan
Javier Hernández-BorgesDepartment of Analytical Chemistry,
Nutrition and Food Science University of
Laguna, Canary Islands, Spain
John V. HinshawServeron Corp., Hillsboro, Oregon, USA
Tuulia HyötyläinenVVT Technical Research of Finland,
Finland
Hans-Gerd JanssenVan’t Hoff Institute for the Molecular
Sciences, Amsterdam, The Netherlands
Kiyokatsu JinnoSchool of Materials Sciences, Toyohasi
University of Technology, Japan
Huba KalászSemmelweis University of Medicine,
Budapest, Hungary
Hian Kee LeeNational University of Singapore,
Singapore
Wolfgang LindnerInstitute of Analytical Chemistry,
University of Vienna, Austria
Henk LingemanFaculteit der Scheikunde, Free University,
Amsterdam, The Netherlands
Tom LynchBP Technology Centre, Pangbourne, UK
Ronald E. MajorsAgilent Technologies,
Wilmington, Delaware, USA
Phillip MarriotMonash University, School of Chemistry,
Victoria, Australia
David McCalleyDepartment of Applied Sciences,
University of West of England, Bristol, UK
Robert D. McDowallMcDowall Consulting, Bromley, Kent, UK
Mary Ellen McNallyDuPont Crop Protection,Newark,
Delaware, USA
Imre MolnárMolnar Research Institute, Berlin, Germany
Luigi MondelloDipartimento Farmaco-chimico, Facoltà
di Farmacia, Università di Messina,
Messina, Italy
Peter MyersDepartment of Chemistry,
University of Liverpool, Liverpool, UK
Janusz PawliszynDepartment of Chemistry, University of
Waterloo, Ontario, Canada
Colin PooleWayne State University, Detroit,
Michigan, USA
Fred E. RegnierDepartment of Biochemistry, Purdue
University, West Lafayette, Indiana, USA
Harald RitchieTrajan Scientific and Medical, Milton
Keynes, UK
Koen SandraResearch Institute for Chromatography,
Kortrijk, Belgium
Pat SandraResearch Institute for Chromatography,
Kortrijk, Belgium
Peter SchoenmakersDepartment of Chemical Engineering,
Universiteit van Amsterdam, Amsterdam,
The Netherlands
Robert ShellieAustralian Centre for Research on
Separation Science (ACROSS), University
of Tasmania, Hobart, Australia
Yvan Vander HeydenVrije Universiteit Brussel,
Brussels, Belgium
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10% Post
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Waste
ES666795_LCESUPP0915_004.pgs 09.01.2015 00:46 ADV blackyellowmagentacyan
ENVIRONMENTAL
THE APPLICATIONS BOOK – SEPTEMBER 2015 5
Millions of new cars are produced and registered in Germany
every year. In the supply chain of a vehicle, control standards
have to be followed to ensure a high quality final product. The
VDA regulations, for example, address the organic emissions
from automotive components. Based on thermodesorption
techniques, VDA 278 regulates the test procedure for
non-metallic materials used for molded components in
automobiles. Using this method, two classes of compounds are
distinguished: highly and medium volatile substances (VOC)
up to C25 and those of low volatility (FOG) in the range C14
up to C32. In this application note the upper layer material of
fairing parts used in automobiles has been analyzed according
to VDA 278 regarding its organic emission. The influence of
open storage time on the VOC and FOG content has also been
investigated.
Workflow and Experimental Conditions
According to VDA 278 the workflow shown in Figure 1 has to be
followed. A control standard solution of 18 compounds has to
be checked to ensure instrument performance followed by the
measurement of two standards as calibration substances. The
VOC content is calibrated using toluene; for the FOG calibration
hexadecane is used to determine the respective response factor.
Each sample has to be measured twice: one sample is used for
a VOC analysis only, while the second one is measured under
VOC conditions followed by FOG analysis.
The measurements were carried out using a GCMS-QP2010 SE
combined with the TD-20 thermodesorption unit. Following VDA
278, desorption temperatures for the tubes were set to 90 °C
(VOC) and 120 °C (FOG), and desorption time was 30 min. The
chromatographic separation was performed using a 50 m × 0.32
mm, 0.5-µm Optima5MS column. The oven temperature was
programmed and began at 50 °C, held for 2 min, ramped with
25 °C/min to 160 °C followed by a second ramp of 10 °C/min
up to 280 °C final temperature, and held for 30 min. Compound
detection was done by an MS full scan over the expected mass
range. As a result of the high concentrations of both standards
and samples, a split of 100:1 was used to prevent detector
saturation. In addition, the amount of standards injected into the
TD tubes could be decreased by a factor of 4 to 0.5 µg absolute.
The sample was cut into small pieces of approximately 10 mg
and placed into empty TD tubes as shown in Figure 2. For the
standard solution, tubes filled with Tenax were used, and the
solvent was evaporated after injection under a continuous flow
of nitrogen gas (5 min at 100 mL/min).
Results
The chromatogram of the control standard solution is shown in
Figure 3. As required by VDA 278, o-xylene and n-nonane are
baseline separated. While undecane and 2,5-dimethylphenol
coelute (see insert in Figure 3), both can be identified using the
library search. Recovery rates for the compounds checked were
well within the limits of 60–140%. For toluene, the recovery was
98%. Response factors calculated for VOC and FOG were 0.08
and 0.06, respectively. With these factors, emissions from the
upper layer material of fairing parts were measured directly after
opening the package. The chromatograms for the VOC and FOG
run are shown in Figure 4. For emission calculations all peaks
have been summed. An analogous measurement was repeated
after 7 days of open sample storage in a neutral environment.
Analysis of VOC and FOG Emissions from Molded Components for Automobiles According to VDA 278Dr Rebecca Kelting, Shimadzu Europa GmbH
Figure 2: Sample placed into the TD tube.
Control standard solution System performance check
Response factor VOC
Response factor FOG
Emission VOC / FOG
Toluene standard
Hexadecane standard
Sample
Figure 1: Workf ow according to VDA 278.
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ENVIRONMENTAL
6 THE APPLICATIONS BOOK – SEPTEMBER 2015
Shimadzu Europa GmbHAlbert-Hahn-Str. 6–10, D-47269 Duisburg, Germany
Tel: +49 203 76 87 0 fax: +49 203 76 66 25
E-mail: [email protected]
Website: www.shimadzu.eu
The emission values for all measurements before and after
storage are summarized in Table 1. As would be expected,
the emission decreased significantly after longer storage time.
Reduction in VOC value was much stronger than FOG content
as these compounds have higher volatilities.
Conclusion
In this application note the upper layer material of fairing
parts used in automobiles was analyzed for VOC and FOG
compounds, according to VDA 278. The GCMS-QP2010 SE in
combination with TD-20 proved to be fully sufficient for this
type of analysis. Furthermore, a high influence of the storage
time on the emission values was determined, indicating that
defined storage times are extremely important for reliable and
reproducible detection of the emission values.
Figure 3: Chromatogram of the control standard.
10.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0
Time (min)
a.u
.
Be
nze
ne
He
pta
ne
Tolu
en
e
Oct
an
e
p-X
yle
ne
o-X
yle
ne
No
na
ne
Un
de
can
e
Similarity
(x 1,000,000)
TIC (1.00)
93 Phenol. 2.5-dimethyl-88
Compound name
De
can
e
1-H
exa
no
l, 2
-eth
yl-
Un
de
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e
Do
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can
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Trid
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Tetr
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exa
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ine
, N
-cyc
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l-P
en
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eca
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ed
ioic
aci
d,
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(2-e
thyl
he
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est
er
(a)
(b)
a.u
.a
.u.
(x 1,000,000)
(x 1,000,000)Time (min)
Time (min)
TIC (1.00)
TIC (1.00)
3.5
3.0
2.5
2.0
1.5
1.0
0.5
2.00
1.75
1.50
1.25
1.00
0.75
0.50
0.25
2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 35.0 37.5 40.0 42.5 45.0 47.5
2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 35.0 37.5 40.0 42.5 45.0 47.5
50.0 52.5
Figure 4: Chromatograms of the upper layer material of fairing parts measured under (a) VOC and (b) FOG conditions
Table 1: VOC and FOG emission from the upper layer material
of fairing parts.
After unpackingAfter 7 days of
open storage
Emission VOC 1: 299 µg/g Emission VOC 1: 160 µg/g
Emission VOC 2: 290 µg/g Emission VOC 2: 156 µg/g
Emission FOG: 234 µg/g Emission FOG: 164 µg/g
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ES667260_LCESUPP0915_007_FP.pgs 09.01.2015 21:14 ADV blackyellowmagentacyan
ENVIRONMENTAL
8 THE APPLICATIONS BOOK – SEPTEMBER 2015
Used extensively from the 1940s through the 1960s,
organochlorine pesticides are chlorinated hydrocarbons used
in agriculture and mosquito control. Representative compounds
in this group include DDT, methoxychlor, dieldrin, chlordane,
toxaphene, mirex, kepone, lindane, and benzene hexachloride.
Many organochlorine pesticides have been banned in the
United States and Europe; however, they are still used in
developing countries. The chemicals can be ingested in fish,
dairy products, and other fatty foods that are contaminated.
The organochlorine pesticides accumulate in the environment,
presenting an ongoing issue with adsorption and accumulation
in the population as a result of ingestion.
Experimental Conditions
Instrument: TRACE — GC/POLARIS-Q
Carrier gas: He (1.5 mL/min)
Injector: Split/Splitless mode
Injector temp.: 275 °C
Split mode: Splitless (1 min split valve closed)
Split flow: 30 mL/min
Column: 30 m × 0.25 mm, 0.25-μm BP5MS
(P/N 054310)
Analysis of Organochlorine Pesticides (OCPs) on SGE BP5MS Trajan Scientif c and Medical
Trajan Scientif c and MedicalCrownhill Business Centre
14 Vincent Avenue
Crownhill, Milton Keynes
MK8 0AB, United Kingdom
Website: www.trajanscimed.com
Oven temp.: 60 °C (5 min) — 8 °C/min —
300 °C (10 min)
MS transfer line temp.: 300 °C
MS type: ITD
MS source temp.: 225 °C
MS acquisition mode: Segmented scan 45–450 amu
Figure 1: Sample chromatograms.
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THE APPLICATIONS BOOK – SEPTEMBER 2015 9
FOOD & BEVERAGE
The determination of aroma compounds in edible oils is important for
the manufacturers and vendors of these products. Off-f avours derived
from unsaturated fatty acid degradation such as, for example, hexanal,
2-(E)-nonenal, and 2,4-(E,E)-decadienal are of particular interest.
These compounds can compromise the taste and therefore the quality
of a product, even in the ng/g concentration range.
Sensitive and fast analysis methods are needed, ideally requiring
little or no sample preparation. A highly sensitive analysis method
based on direct thermal desorption of the oil in a standard microvial
with a purge slit placed just above the sample surface has been
developed. Oil samples were placed inside a thermal desorption unit
for thermal extraction in close proximity to the trap in which the aroma
compounds are subsequently trapped before being transferred for gas
chromatography–mass spectrometry (GC–MS) determination.
Instrumentation Thermal desorption of oil samples placed in microvials inside
thermal desorption tubes was performed using a GERSTEL Thermal
Desorption Unit (TDU). Analytes were refocused in a GERSTEL Cooled
Injection System (CIS 4), a PTV-type inlet, at low temperatures before
being transferred to the GC column. A 7890/5975 GC–MS system
from Agilent Technologies was used for separation and detection
of the analytes of interest. Thermal desorption tubes containing the
samples were delivered to the TDU automatically using a GERSTEL
MultiPurpose Sampler (MPS).
Analysis ConditionsTDU: Solvent venting; 30 °C; 200 °C/min; 90 °C (15 min).
PTV: Glass bead liner; 0.2 min solvent vent (30 mL/min);
split 3:1; -70 °C; 12 °C/s; 280 °C (30 min).
Column: 15 m ZB-FFAP (Phenomenex), di = 0.25 mm
df = 0.25 μm.
Pneumatics: He, constant f ow = 1.3 mL/min.
Oven: 35 °C (1 min); 4 °C/min; 120 °C (5 min);
50 °C/min; 250 °C (8 min).
MSD: SIM, cf. Table 1.
Determining Aroma Compoundsin Edible Oils by Direct ThermalDesorption GC–MS Using MicrovialsGerstel
Gerstel GmbH & Co.KG Eberhard-Gerstel-Platz 1, 45473 Mülheim
an der Ruhr, Germany
Tel: +49 (0)208 7 65 03 0 Fax. +49 (0)208 7 65 03 33
E-mail: [email protected]
Website: http://www.gerstel.com
Sample Preparation Edible oil was spiked with the analytes listed in Table 1 in the
concentration range between 10 and 1000 ng/g. A set of 30 mg
samples of the oil were weighed into individual microvials and placed
in thermal desorption tubes. Each tube was capped with a transport
adapter and placed in the autosampler tray for later desorption.
MeasurementsTen oil samples of 30 mg each were prepared in microvials with a
purge slit at 1 cm height measured from the bottom of the microvial
and individually desorbed in the TDU to determine the repeatability. A
resulting SIM GC–MS chromatogram is shown in Figure 1.
Results and DiscussionThermal extraction of aroma compounds from edible oils employing
microvials is highly feasible. The microvial prevents contamination
of the analysis system by high boiling matrix compounds while
allowing effective transfer of VOCs and SVOCs onto the analytical
column. After sample processing the microvial can be disposed of
and the desorption tube is ready to take up the next sample. Relative
standard deviations for 10 repeat measurements were between
7.2–16.8% with a median of 9.7%. This is highly acceptable
considering the complex matrix, the low concentrations, and the
straightforward sample preparation. A longer desorption time would
likely improve the relative standard deviations further.
Reference(1) GERSTEL AppNote-2014-03: Analysis of Aroma Compounds in Edible Oils
by Direct Thermal Desorption GC/MS using Slitted Microvials (http://www.
gerstel.com/pdf/p-gc-an-2014-03.pdf)
Dimethyltrisulfde
2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00
200000
400000
600000
800000
1000000
1200000
1400000
1600000
1800000
2000000
2200000
2400000
2600000
2800000
3000000
3200000
3400000
3600000
3800000
4000000
4200000
4400000
4600000
4800000
5000000
5200000
2,4-(E,E)-Nonadienal
3-Methylbutanal
gamma-decalactone
2-Ethylpyrazine
Methional
Hexanal
4-Heptanone
2-(E)-Nonenal
2,4-(E,E)-Decadienal
2-Methylpentanoic acid2,3-Dimethoxy-toluene2-Ethyl-3,5-
dimethylpyrazine
Figure 1: SIM chromatogram resulting from thermal desorption of a spiked edible oil inside a microvial with slit placed 1 cm from bottom.
Table 1: Analytes spiked into edible oil.
Compound RT (min) Quant. (m/z) Qual. (m/z)
3-Methylbutanal 1.194 58 57; 86
Hexanal 2.304 56 72; 82
4-Heptanone 2.787 71 114; 43
2-Ethylpyrazine 6.851 107 80; 53
Dimethyltrisulphide 7.540 126 79; 47
2-Ethyl-3,5-dimethylpyrazine 9.797 135 136; 108
Methional 9.992 104 76; 48
2-(E)-Nonenal 12.217 83 70; 96
2,3-Dimethoxytoluene 15.477 152 137; 109
2,4-(E,E)-Nonadienal 16.641 81 138; 95
2-Methylpentanoic acid 18.533 74 87; 43
2,4-(E,E)-Decadienal 19.431 81 152; 95
gamma-Decalactone 28.107 128 100; 85
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10 THE APPLICATIONS BOOK – SEPTEMBER 2015
FOOD & BEVERAGE
This application note describes the benef ts of using Markes’
Micro-Chamber/Thermal Extractor in conjunction with
thermal desorption (TD) and gas chromatography–mass
spectrometry (GC–MS) to analyze the aroma prof les of
cheese. Various cheeses are examined and compared, and
it is demonstrated how this ‘multi-hyphenated’ technique
allows rapid yet powerful assessment of the volatile organic
compounds (VOCs) released.
The Importance of Cheese Aroma
The annual value of cheese to the global economy runs into
many billions of dollars, and manufacturers expend a great
deal of effort in ensuring their product is of a consistently high
quality. Rigorous attention is paid to the ingredients, and the
appearance, texture, taste, and aroma of the final product.
The aroma profile is an important part of the consumer
experience of cheese, with a range of compounds responsible
for the wide variation of cheese odours. This presents analysts
with a substantial challenge when wishing to identify key aroma
components, many of which are present at trace levels and have
low odour thresholds relative to more abundant components such
as fatty acids.
Here we show how Markes’ technology can be used to
characterize the aroma profile from a range of cheese samples,
with analysis both of high- and low-concentration components.
Sampling Methodology
The sampling methodology used for the analysis of the cheese
samples described here combines three powerful techniques:
• Dynamic headspace sampling flushes the organic vapours
from the cheese onto a sorbent-packed tube, followed by
thermal desorption to concentrate these vapours into a
narrow band of carrier gas, suitable for introduction into a
GC–MS system. Here, we use Markes’ Micro-Chamber/
Thermal Extractor™, which is a stand-alone sampling
accessory for dynamic headspace sampling of organic
vapours from a wide variety of materials, including foodstuffs.
• Thermal desorption (TD) is a versatile “front-end” technology
for GC that is applicable to the analysis of volatiles in a
wide range of gaseous, liquid, and solid samples. Here, we
use Markes’ fully-automated 100-tube TD-100™ thermal
desorber, which allows full automation of both sample
desorption and re-collection.
• Time-of-flight mass spectrometry is a powerful alternative
to the quadrupole technologies often employed with GC.
In this work, Markes’ BenchTOF-HD™ instrument is used,
Rapid Aroma Prof ling of Cheese Using a Micro-Chamber/Thermal Extractor with Thermal Desorption–GC–MS AnalysisPaul Morris, Caroline Widdowson, and David Barden, Markes International
which provides the sensitivity otherwise only available
using a quadrupole instrument in SIM mode, but across
the full spectral range. Importantly, it also generates
spectra that match those present in reference (typically
quadrupole-acquired) libraries. Post-run data processing
used Markes’ TargetView™ software to remove baseline
interferences, deconvolve overlapping peaks, and match
spectra against those in the NIST database.
Experimental Procedure
A variety of cheeses (grated, 5 g per sample) were sampled using
the Micro-Chamber/Thermal Extractor (Figure 1) for 20 min
under a flow of dry nitrogen, with a chamber temperature of
40 °C used to generate a full VOC profile that reflects conditions
produced in the mouth. Samples were collected on to sorbent
tubes packed with quartz wool–Tenax® TA–Carbograph™ 5TD,
which is a sorbent combination that can handle the full range
of analytes expected to be present in the VOC profile of cheese.
The use of multiple sorbents in this way is only possible
because Markes’ TD systems are designed so that analytes
enter and leave the tube (or trap) at the end with the weakest
sorbents. This ensures that low-volatility “sticky” analytes are
retained on the weakest possible sorbent, so that when the gas
flow is reversed, they desorb easily. The tubes were analyzed
using an overall TD split of 51:1 (high split), 6:1 (low split).
Gas chromatography used a 30 m × 0.25 mm HP-Innowax
column to best handle the polar compounds expected, with a
temperature ramp from 40 °C to 260 °C, and an overall run
time of 36.0 min. Mass spectra were acquired over the range
m/z 33–350, with a data rate of 2 Hz (with 5000 spectra per
data point).
Figure 1: The Micro-Chamber/Thermal Extractor containing cheese samples ready for analysis.
ES665924_LCESUPP0915_010.pgs 08.31.2015 14:20 ADV blackyellowmagentacyan
THE APPLICATIONS BOOK – SEPTEMBER 2015 11
FOOD & BEVERAGE
Markes InternationalGwaun Elai Medi-Science Campus, Llantrisant, Wales, UK
Tel: +44 (0)1443 230935
E-mail: [email protected] Website: www.markes.com
Comparison of Different Cheeses
One of the benef ts of the Micro-Chamber/Thermal Extractor is that
several samples can be run under identical conditions quickly and
easily. Figure 2 shows the results of a comparison of four cheeses,
collected simultaneously from adjacent micro-chambers, and run
under identical TD–GC–MS conditions so that the peak sizes give
a good approximation of the relative abundances of the individual
components. Note the presence of the highly odorous component
dimethyl disulphide in the Emmental, and of two branched-chain
carboxylic acids in the Comté.
Analyzing High- and Trace-Level Components in a
Single Run
Markes’ TD splitting technologies offer a particular advantage for
aroma prof ling because of their ability to run a single sample twice,
using different split ratios to accurately measure trace-level and
high-concentration compounds in the same sample (Figure 3).
First, the sample is desorbed using a “high split”, with a small
volume being sent to the GC. This allows the high-concentration
components to be analyzed without overloading the analytical
system. The remainder is re-collected onto a fresh sorbent tube,
and then desorbed as before but using a “low split” — sending
a higher proportion of the sample to the GC. This allows the
trace-level components to be quantif ed more accurately.
Conclusions
In this application note we have shown how the Micro-Chamber/
Thermal Extractor allows rapid and straightforward sampling of
volatiles from a range of cheese samples. In conjunction with
TD–GC–MS, a wealth of information is provided that allows users
to identify key components and compare samples side-by-side.
The f exibility of this approach makes it suitable for a wide range
of sampling situations — from initial screening of “unknown”
samples to in-depth analysis of samples for quantitation of
trace-level components.
For more experimental results and details of the conditions
used, please refer to Markes Application Note 101, available at
www.markes.com.
(a)
High split
(b)
5
11
10
9
8
7
6
5
4
3
2
1
0
4
3
2
1
00 10 20
25 26 27 28 29
30
3-H
ydro
xybuta
n-2
-one
Ace
tic
acid
Buta
noic
aci
dD
imet
hyl
sulp
hone Ph
enol
Oct
anoic
aci
d
Bip
hen
yl
4-M
ethyl
phen
ol
Hex
anoic
aci
d
Car
bon d
isulp
hid
e
Buta
ne-
2, 3
-dio
ne
Hep
tan-2
-one
Lim
onen
e3-
Met
hyl
but-
3-en
-1-o
l
Nonan
-2-o
ne
3-H
ydro
xybuta
n-2
-one
Inte
nsi
ty (
x 1
07 c
ou
nts
)In
ten
sity
(x 1
07 c
ou
nts
)
Retention time (min)
0 10 20 30
Retention time (min)
Low split
Figure 3: Analysis of the VOC prof le of full-fat (extra-mature) Cheddar. (a) High-split (51:1) conditions provide an indication of the quantities of high-concentration components. (b) Low-split (6:1) conditions, to aid identif cation of trace-level components (see inset). Example peaks are highlighted; for a full peak listing, please see Markes Application Note 101.
3-M
ethyl
buta
n-1
-ol
Propan
-1-o
lD
imet
hyl
disulp
hid
e
3
2
1
00 10 20 30
Retention time (min)
ComtŽ
Low-fat Cheddar
Full-fat Cheddar
Emmental
Inte
nsi
ty (
x 1
08 c
ou
nts
)
3-H
ydro
xylb
uta
n-2
-one
Ace
tic
acid
Propan
oic
aci
d
2-M
ethyl
pro
pan
oic
aci
d
Buta
noic
aci
d
3-M
ethyl
buta
noic
aci
d
Figure 2: Parallel analysis of the VOC prof les of four cheeses under high-split conditions.
ES665929_LCESUPP0915_011.pgs 08.31.2015 14:20 ADV blackyellowmagentacyan
12 THE APPLICATIONS BOOK – SEPTEMBER 2015
FOOD & BEVERAGE
Separations of structurally similar compounds such as vitamin D2
and vitamin D3 are very challenging.
Vitamin D2
(ergocalciferol) and D3 (cholecalciferol) can be found in
different foods including fatty fishes, meat, egg, and some mushrooms.
Both compounds are (indirectly) involved in a number of biological
functions in the body, including bone metabolism and enhancement
of intestinal absorption of calcium, iron, magnesium, phosphate, and
zinc. A regular intake of vitamin D therefore is essential.
Standard C18 columns are not able to fully separate the two
vitamins. A very hydrophobic phase with a higher carbon coverage
and therefore a greater density of C18 chains is required. The highly
hydrophobic phase YMC-Triart C18 ExRS (carbon load 25%!) is able
to separate these two.
The isocratic high performance liquid chromatography (HPLC)
method for vitamin D2
and D3 separation, using a 5 μm YMC-Triart C18
ExRS column, can easily be transferred to a ultrahigh-performance
liquid chromatography (UHPLC) method using a 1.9 μm column,
thereby reducing the analysis time by 50%. Furthermore, the
resolution can be increased, resulting in a full baseline separation.
Vitamin D2 and D3 Separation by New Highly Hydrophobic UHPLC/HPLC PhaseYMC Europe GmbH
YMC Europe GmbHPhone: +49 2064 4270
E-mail: [email protected]: www.ymc.de
Columns: YMC-Triart C18 ExRS (5 µm, 8 nm),
150 × 3.0 mm
YMC-Triart C18 ExRS (1.9 µm, 8 nm),
75 × 2.1 mm
Part No.: TAR08S05-1503PTH/TAR08SP9-L5Q1PT
Eluent: THF/acetonitrile (10/90)
Flow rate: 0.425 mL/min
Detection: UV at 265 nm
Temperature: 30 °C
1. 2.
Vitamin D2
(Ergocalciferol)Vitamin D3
(Cholecalciferol)
H3C H3CCH3 CH3
CH2
CH3
CH3
CH2
H
H
H
H
H
HH
H
HO HO
Figure 1: Structures of vitamin D2 and vitamin D3.
-50%
HPLC method5 µm; 150 X 3 m ID
2
1
0.425 mL/min
0
0
10
20
30
40
mA
U
0
10
20
30
40
mA
U
2 4 6 8 10
Time (min)
HPLC method1.9 µm; 75 X 2.1 mm ID
Time (min)
12
0 1 2 3 4 5 6
0.21 mL/min
Figure 2: Easy method transfer HPLC UHPLC as a result of full scalability.
ES665920_LCESUPP0915_012.pgs 08.31.2015 14:20 ADV blackyellowmagentacyan
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ES667257_LCESUPP0915_013_FP.pgs 09.01.2015 21:14 ADV blackyellowmagentacyan
14 THE APPLICATIONS BOOK – SEPTEMBER 2015
GENERAL
There are currently over 300 nitrosamine compounds, of which
more than 90% are classified as carcinogenic or mutagenic. As
a bi-product of many manufacturing processes specific concern
has risen from the impact of cumulative exposure.
In rubber and latex manufacturing, accelerators are required
during the curing process to speed up the transformation from
the liquid state into the form-holding elastic material. This is
achieved by increasing the amount of cross-linking within the
original material. Typically sulphur cross-linking is utilized, and
common accelerators include dithiocarbamate, thiurams, and
benzothiazole.
When exposed to atmospheric nitrous oxide, during the mixing
or curing processes, these secondary amines undergo nitrosation
and produce nitrosamines.
These nitrosamines will remain within the rubber and latex
items unless extracted via chemical treatment. Concentration
levels found within consumer products are widely variable and
potentially continue to leach out.
The focus of this article is to determine a method suitable for
nitrosamine analysis with a brief study of nitrosamine levels found
in children’s toys and baby paraphernalia. Gas chromatography–
mass spectrometry (GC–MS) and liquid chromatography coupled
to tandem MS (LC–MS–MS) analysis can be costly to purchase,
complex to operate, and analytically complicated (often requiring
the use of single ion monitoring to achieve adequate sensitivity).
This analysis will focus on the suitability of utilizing a more
cost-effective and simpler analysis method employing the long-
established technique of gas chromatography–thermal energy
analyzer (GC–TEA). Sample preparation was achieved using UK
standard method — BS EN 71-12:2013 and co-referencing EN
12868:1999. N-nitrosodiethanolamine (NDELA) was derivatized
prior to analysis by the addition of BSTFA+TMCS (CAS no.
25561-30-2).
GC–TEA Detection of Nitrosamines within Toys and Rubber/Latex ProductsDonna Kinder, Ellutia
Ellutia Ltd12–16 Sedgeway Bus Pk, Witchford, Cambs, CB62HY, UK
Tel: +44 (0)1353 669916 Fax: +44 (0)1353 669917
E-mail: [email protected] Website: www.ellutia.com
Experimental ConditionsInstruments : Ellutia 200 Series GC and
810 Series TEA
Carrier gas : Helium 4.0 psi (constant f ow)
Injector : 150 °C Splitless (0.8 min split time)
Injection volume : 1 μL
Column : 20 m × 0.53 mm,1.0-μm DB-PSWAX
Oven temperature : 40 °C (1 min) 15 °C/min to 15 °C,
10 °C/min to 190 °C (1 min hold)
Results
Discussion/ConclusionThe results listed in Table 1 and illustrated in Figure 1 show how
nitrosamines can be successfully separated on an Ellutia 200 Series
GC and detected on the 810 Series TEA. The results also show
that whilst most products tested showed minimal nitrosamines,
N-nitrosodimethylamine (NDMA) and NDELA were present in
almost every sample at low levels. Balloons were the only sample
to have a positive response for any other nitrosamine, namely
N-nitrosodiethylamine (NDEA).
Current U.S. legislation (FDA and CPSC) for nitrosamines
recommends that baby products should contain less than 10 μg/kg
of any individual nitrosamine per item sold.
This study suggests that further monitoring of nitrosamines
within toys and other rubber/latex products is essential. Whilst
manufacturers continue to reduce the exposure risk, it is paramount
to reduce baby’s and children’s exposure to these carcinogenic
compounds through strict monitoring and control.
Time (min)N
DM
A
NM
EA
ND
EA
ND
PA
ND
BA
NP
IPN
PY
R
NM
OR
6 8 10 12 14 16
Vo
ltag
e (
mV
)
45
40
35
30
25
20
15
Figure 1: Chromatogram showing separation of common nitrosamines using an Ellutia 810 TEA.
Table 1: Nitrosamine results from various toys and common
rubber items.
Concentration measured in mg/kg
NDELA NDMA NDEATotal other
nitrosamines
Fingerpaints 3.3 2.2 n.d. n.d.
Toy 1 0.7 0.3 n.d. n.d.
Toy 2 0.8 0.1 n.d. n.d.
Looms 1.4 0.2 n.d. n.d.
Soothers 0.8 0.5 n.d. n.d.
Balloons n.d. 1.5 0.2 n.d.
Gloves 0.2 0.2 n.d. n.d.
Condoms n.d. 0.4 n.d. n.d.
ES665955_LCESUPP0915_014.pgs 08.31.2015 14:21 ADV blackyellowmagentacyan
THE APPLICATIONS BOOK – SEPTEMBER 2015 15
MEDICAL/BIOLOGICAL
Blood Alcohol Content Analysis Using Nitrogen Carrier GasEd Connor1 and Greg Dooley2, 1Peak Scientific, 2Colorado State University
Alcohol consumption can seriously affect the ability of a driver to
operate a vehicle and blood alcohol content (BAC) directly correlates
with this impairment. A number of nations have zero alcohol
tolerance for motorists, but the majority of countries worldwide
have a limit of between 50 and 80 mg alcohol per 100 mL blood,
or 0.05–0.08%. Results are used in court to provide quantitive
levels of BAC, which makes it one of the most commonly practised
analyses in forensic laboratories. The large number of samples and
requirement for speed of sample processing means that analysis
needs to be conducted quickly, whilst also giving reliable and
accurate results.
For analysis of BAC, headspace gas chromatography (GC) with
flame ionization detection (FID) is typically used. Headspace GC
allows the quantitative analysis of alcohol directly from blood samples.
Standard headspace systems use nitrogen for vial presurization, with
helium typically used for GC carrier gas. This application note looks
at the use of nitrogen for both vial pressurization and GC carrier gas.
Nitrogen offers a cost-effective, abundant alternative to helium for
carrier gas, whilst also providing a similar performance. Here we
compare analysis of real forensic blood samples, taken from motorists
suspected of driving under the influence of alcohol, analyzed using
nitrogen and helium carrier gas.
Sample Preparation
Using a Hamilton Microlab 600 Diluter, 200 μL of calibrators,
controls, or blood samples were aliquoted and dispensed with
2000 μL of internal standard solution into a 10 mL headspace
vial and capped. The internal solution consisted of 0.03% (v/v)
n-propanol/1M ammonium sulphate/0.1 M sodium hydrosulphite.
NIST traceable aqueous ethanol solutions from Cerilliant and
Lipomed were used as calibrators (10, 50, 80, 200, 300, 500 mg/
dL) and controls (20, 80, 400 mg/dL), respectively.
Experimental
Analyses were conducted using an Agilent 7890B GC with split/
splitless inlet and dual columns each connected to an FID detector.
Splitting of the samples onto the columns was via an Agilent
unpurged Capillary Flow Technology splitter. The GC was coupled
with an Agilent 7697A headspace sampler. Vial pressurization gas
for all tests was provided by a Peak Scientif c Precision Nitrogen
Generator. Carrier gas was provided by either the helium cylinder
or the Precision Nitrogen Standard Generator. The HS–GC–FID
system operating condtitions are displayed in Table 1.
The software used for analysis was Agilent MassHunter GC/
MS Acquisition and MSD ChemStation Enhanced Data Analysis
E.02.02.1431
Results
Calibration curves produced with helium and nitrogen carrier gas both
gave very good linearity with both curves having R2 values of 99.9999
(Figure 1). Blood alcohol levels of f ve blood samples were analyzed.
Ethanol-A - 6 Levels, 6Levels Used, 6 Points, 6 Points Used, 0 QCs
Ethanol-A - 6 Levels, 6Levels Used, 6 Points, 6 Points Used, 0 QCs
Rela
tive R
esp
on
ses
Relative Concentration
y = 0.002000* x -2.547391E-004R^2 = 0.99996690Type:Linear, Origin:Ignore, Weight: 1/x
y = 0.002037* x -4.208017E-004R^2 = 0.99996895Type:Linear, Origin:Ignore, Weight: 1/x
1.05
Nitrogen
Helium
1
0.95
0.9
0.85
0.8
0.75
0.7
0.65
0.6
0.55
0.5
0.45
0.4
0.35
0.3
0.25
0.2
0.15
0.1
0.05
0
-0.05
Rela
tive R
esp
on
ses
1.1
1.05
1
0.95
0.9
0.85
0.8
0.75
0.7
0.65
0.6
0.55
0.5
0.45
0.4
0.35
0.3
0.25
0.2
0.15
0.1
0.05
0
-0.05
-20 0 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540
Relative Concentration
-20 0 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540
Figure 1: Calibration curves for ethanol standards run using nitrogen and helium carrier gas.
Table 1: HS-GC-FID operating conditions.
Headspace sampler Agilent 7697A
Vial pressurization gas Nitrogen
Oven temperature 70
Loop temperature 70
Transfer line Deactivated fused silica, 0.53-mm i.d.
Transfer line temperature 90
Gas chromatograph Agilent 7890B
Carrier gas Helium Nitrogen
Detector FID
ColumnsDB-ALC1 (30 m × 320 μm, 1.8-μm)
DB-ALC2 (30 m × 320 μm, 1.8-μm)
Split ratio 10:1
GC oven start temperature 40 °C (3 min)
GC oven programme rate 40 °C min
GC oven f nal temperature 120 °C (1.2)
Method runtime 6.2 min
ES665933_LCESUPP0915_015.pgs 08.31.2015 14:21 ADV blackyellowmagentacyan
16 THE APPLICATIONS BOOK – SEPTEMBER 2015
MEDICAL/BIOLOGICAL
Peak Scientif c
Fountain Crescent, Inchinnan Business Park, Inchinnan, PA4 9RE, Scotland, UK
E-mail: [email protected]: www.peakscientific.com
Figures 2 and 3 show chromatograms from the DB-ALC1 and
DB-ALC2 columns, respectively for the separation and elution order
of analytes for the multi-component resolution mix when run using
nitrogen and helium carrier gas. Separation of potentially interfering
components such as methanol and 2-propanol was achieved within
3 min when using either carrier gas (Figure 2 and Figure 3).
Conclusions
Results of BAC analysis show that there is no difference in the
linearity of the calibration curve, or of the calculated ethanol content
of real blood samples regardless of whether nitrogen or helium carrier
gas is used.
As an abundant, inexpensive alternative to helium, which is
becoming increasingly more costly, there is no reason why nitrogen
cannot be used for BAC analysis in place of helium. Since nitrogen
is often used for vial pressurization in headspace samplers, the use
of a single gas source for vial pressurization, carrier gas and FID
make-up gas simplifies the lab’s gas sourcing and would allow total
gas supply from gas generators if the precision nitrogen was used in
conjunction with the Precision hydrogen and zero air generators for
GC–FID analysis.
Results of analyses of real blood samples (analyzed in duplicate)
run with both nitrogen and helium carrier gas gave equivalent results
with no differences found in the calculated ethanol concentrations
(Table 2). Of the five blood samples tested, one was over 0.2%, which
would result in a driving ban in almost every country worldwide. Two
samples were over 0.05%, which would result in a driving ban in a
number of countries. The other two samples were 0.014% and 0.023%,
which would be below the limit in the majority of countries worldwide.
FID1- A:Signal #1 ResolutionMix.D
FID1- A:Signal#1 ResolutionMix_test.D
x10 6
4.6
4.4
4.2
4
3.8
3.6
3.4
3.2
3
2.8
2.6
2.4
2.2
2
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
-0.2
x10 6
4.8
4.6
4.4
4.2
4
3.8
3.6
3.4
3.2
3
2.8
2.6
2.4
2.2
2
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
-0.2
-0.4
N-propanol
Ethyl Acetate
Ace
ton
e/A
CN
Iso
pro
pa
no
l
Eth
an
ol
Me
tha
no
l
Ace
tald
eh
yd
e
Paraldehyde
N-propanol
Ethyl AcetateAce
ton
e/A
CN
Iso
pro
pa
no
l
Eth
an
ol
Me
tha
no
l
Ace
tald
eh
yd
e Paraldehyde
Nitrogen DB-ALC1
Helium DB-ALC1
N-propanol
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9 3 3.1
Response Units vs. Acquisition Time (min)
3.2 3.3 3.4 3.5 3.6 3.7 3.8 3.9 4 4.1 4.2 4.3 4.4 4.5 4.6 4.7 4.8 4.9 5 5.1
5.000
3.385
2.038
1.788
1.487
1.205
0.943
0.633
0.852
0.942
1.085
1.335
1.605
1.825
3.097
4.8304.830
5.2 5.3 5.4 5.5 5.6 5.7 5.8 5.9 6
Figure 2: Results of resolution mixture run on a DB-ALC1 column using nitrogen and helium carrier gas.
Table 2: Blood alcohol analysis results from analysis conducted
with nitrogen and helium carrier gas.
Amount of ethanol detected (%)
Nitrogen Helium
Sample 1A 0.05749 0.05702
Sample 1B 0.05776 0.05689
Sample 2A 0.01438 0.01421
Sample 2B 0.01433 0.01417
Sample 3A 0.23587 0.23476
Sample 3B 0.23481 0.23323
Sample 4A 0.02295 0.02254
Sample 4B 0.02285 0.02255
Sample 5A 0.05890 0.05866
Sample 5B 0.05948 0.05867
FID2- B:Signal #2 ResolutionMix.D
FID2- B:Signal#2ResolutionMix_test.D
x10 6
4.8
4.6
4.4
4.2
4
3.8
3.6
3.4
3.2
3
2.8
2.6
2.4
2.2
2
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
-0.2
x10 6
5.25
5
4.75
4.5
4.25
4
3.75
3.5
3.25
3
2.75
2.5
2.25
2
1.75
1.5
1.25
1
0.75
0.5
0.25
0
-0.25
-0.5
N-propanol
Ethyl Acetate
Iso
pro
pa
no
l
Eth
an
ol
Me
tha
no
l
Ace
tald
eh
yd
e
Paraldehyde
Ethyl Acetate
Ace
ton
itri
leIs
op
rop
an
ol
Ace
ton
e
Eth
an
ol
Me
tha
no
l
Ace
tald
eh
yd
e
Paraldehyde
Nitrogen DB-ALC2
Helium DB-ALC2
N-propanol
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9 3 3.1Response Units vs. Acquisition Time (min)
3.2 3.3 3.4 3.5 3.6 3.7 3.8 3.9 4 4.1 4.2 4.3 4.4 4.5 4.6 4.7 4.8 4.9 5 5.1
4.653
2.362
2.262
1.733
1.508
1.273
1.005
0.630
0.907
0.835
1.147
1.355
1.560
2.025
2.108
4.487
5.2 5.3 5.4 5.5 5.6 5.7 5.8 5.9 6
Figure 3: Results of resolution mixture run on a DB-ALC2 column using nitrogen and helium carrier gas.
ES665932_LCESUPP0915_016.pgs 08.31.2015 14:20 ADV blackyellowmagentacyan
THE APPLICATIONS BOOK – SEPTEMBER 2015 17
MEDICAL/BIOLOGICAL
Low-molecular-weight heparins (LMWHs) are obtained by
fractionation or depolymerization of natural heparins. They are
def ned as having a mass-average molecular weight of less than
8000 and for which at least 60% of the total weight has a molecular
mass less than 8000.
Size-exclusion chromatography (SEC) has been the most
common way of measuring the molecular weight and molecular
weight distributions of LMWHs by using the two most common
detection technologies: ultraviolet (UV) coupled with refractive
index (RI) detection. However, these detectors embody a relative
method to determine molecular weights, requiring calibration
standards. A newer, absolute method involves the use of
multi-angle light scattering (MALS), which does not require any
standards. The European Pharmacopeia (EP) monograph for
LMWH specif es the use of the UV–RI detection method and
provides a known calibration standard. Many laboratories around
the world have adopted this method.
We previously developed an SEC–MALS method and found it
to be very suitable for the analysis of LMWHs. We have recently
adopted the UV–RI method described in the EP monograph and
compared the molecular weight results generated for LMWH using
each detection type. The adopted method uses an Agilent LC-1200
series HPLC, 0.2 M sodium sulphate pH 5.0 mobile phase, Tosoh
TSK-gel G2000 SWxl column with Tosoh TSK-gel Guard SWxl,
Waters 2487 dual wavelength UV detector, and Wyatt Optilab rEX
refractive index detector. For MALS analysis, the UV detector was
replaced with a Wyatt miniDAWN TREOS detector; all other method
aspects remained the same.
The results indicated that both detection types are suitable
and acceptable for the analysis of LMWHs. The molecular weight
and distribution results generated using each detection type are
comparable. This indicates that a SEC–MALS method could be
adopted in place of the SEC–UV–RI method currently required by
the EP monograph, and that it would result in less time because it
obviates the need for calibration standards.
This note was graciously submitted by Lin Rao and John Beirne
of Scientif c Protein Laboratories LLC.
Molecular Weight Determination of LMWH SEC–MALS vs. SEC–UV–RI Wyatt Technology Corporation
Wyatt Technology Corporation6300 Hollister Avenue, Santa Barbara, California 93117, USA
Tel:+1 (805) 681 9009 fax: +1 (805) 681 0123
Website: www.wyatt.com
Define Peaks: LMWH Sample
1.0
0.5
0.0
Rel
ativ
e sc
ale
5.0 10.0
Time (min)
15.0 20.0 25.0 30.0 35.0
LS dRI
Figure 2: Examples of LS and RI traces for an LMWH sample.
LS dRI UV
Define Peaks: LMWH Sample
0.8
0.6
0.4Rel
ativ
e sc
ale
0.2
0.0
5.0 10.0
Time (min)
15.0 20.0 25.0 30.0 35.0
Figure 1: Examples of UV and RI traces for an LMWH sample.
ES665953_LCESUPP0915_017.pgs 08.31.2015 14:21 ADV blackyellowmagentacyan
18 THE APPLICATIONS BOOK – SEPTEMBER 2015
MEDICAL/BIOLOGICAL
Membrane proteins — together with lipids — make up biological
membranes that are essential for life. In order to understand the
role of membrane proteins in assisting membranes to carry out
many different functions, it is of great importance to understand the
structure of those proteins.
Membrane protein is generally soluble only in the presence of
micelles; thus, it is very difficult to characterize the oligomerization
state of the membrane protein in a lipid-containing solvent. In this
application note we demonstrate the use of multi-angle light scattering
(MALS) detection in combination with UV absorption and differential
refractive index (DRI) detection to determine the molar masses (MM)
of both the core protein and the entire protein-lipid complex.
The chromatograms of one membrane protein were obtained from
size-exclusion chromatography (SEC). SEC can often refer to fast
protein liquid chromatography, or, FPLC (Amersham Biosciences,
Uppsala, Sweden). In this experimental set-up, a DAWN MALS
detector was coupled to a UV (280 nm) and DRI detector, and the
resulting traces are shown in Figure 1.
In order to keep the membrane protein in solution, it was
necessary to use a mobile phase that contained lipids at greater than
the critical micelle concentration. Since the membrane protein-lipid
complex has quite a different conformation and probably different
adsorption characteristics to the column packing than globular
standard proteins, the elution property of membrane proteins and
globular proteins are very different. As a result, the traditional column
calibration method fails to provide any estimation on molar mass
using elution time.
ASTRA software’s protein conjugate algorithm analyses the data
from the MALS, UV, and DRI detectors to determine molar masses of
the core membrane protein, lipid micelle, and protein-lipid complex,
as seen in Figure 2. These data suggest that the membrane protein
is in a monomeric state (62 kDa).
This example demonstrates clearly that the combination of MALS,
UV, and DRI detection is an unique and powerful tool in characterizing
membrane proteins in particular, and other modified proteins — such
as pegylated and glycosylated proteins — in general.
Membrane Proteins
Wyatt Technology Corporation
Wyatt Technology Corporation630 Hollister Avenue, Santa Barbara, California 93117, USA
Tel: (805) 681 9009 Fax: (805) 681 0123
E-mail: [email protected] Website: www.wyatt.com
BSA monomer= 67KDa
Protein complex=97kDa
Core protein= 62kDa
Figure 2: The analysis based on data from LS, UV, and DRI detectorsreveals molar masses for the core protein and protein-lipid complex are 62 and 97 kD, respectively. The results from BSA demonstrate that the SEC properties of these two protein samples are very different.
Light Scattering
signal
RI signal
UV signal
Figure 1: Chromatograms of a membrane protein obtained from a LS (top), DRI (middle), and UV at 280 nm (bottom) detectors.
ES665954_LCESUPP0915_018.pgs 08.31.2015 14:21 ADV blackyellowmagentacyan
The Most Interesting Manin Light Scattering.
We Call Him Dad.Dr. Philip Wyatt is the father of Multi-Angle Light
Scattering (MALS) detection. Together with his sons,
Geof and Cliff, he leads his company to produce the
industry’s most advanced instruments by upholding
two core premises: First, build top quality instruments
to serve scientists. Check. Then delight them with
For essential macromolecular and nanoparticle characterization—The Solution is Light™
© 2015 Wyatt Technology. All rights reserved. All trademarks and registered trademarks are properties of their respective holders.
phot
o: ©
Pet
eBle
yer.c
om
unexpectedly attentive customer service. Check.
After all, we don’t just want to sell our instruments,
we want to help you do great work with them.
Because at Wyatt Technology, our family extends
beyond our last name to everyone who uses our
products.
ES667262_LCESUPP0915_019_FP.pgs 09.01.2015 21:14 ADV blackyellowmagentacyan
PHARMACEUTICAL/DRUG DISCOVERY
20 THE APPLICATIONS BOOK – SEPTEMBER 2015
This application note shows the comprehensive 2D-LC analysis
of the traditional Chinese prescription Si-Wu-Tang and the
individual herbs contained in Si-Wu-Tang. The possibility
for authentication of traditional Chinese prescriptions is
investigated.
The authentication of traditional Chinese prescriptions is a
challenging task because of the highly complex nature and the
natural variability of the herbs used in Chinese herbal medicine
(CHM). Generally, chromatographic f ngerprinting is regarded as an
effective method for authentication (1–4). Si-Wu-Tang is composed
of the four herbs Radix Angelicae Sinensis, Rhizoma Chuanxiong,
Radix Paeoniae Alba, and Radix Rehmanniae Preparata. For each
herb, characteristic components are detected in Si-Wu-Tang
as a means of authentication. Additionally, changes following
the omission and replacement of one herb from Si-Wu-Tang are
examined.
Experimental ConditionsComprehensive 2D-LC analysis was achieved with the Agilent 1290
Inf nity 2D-LC Solution. In the f rst dimension an Agilent ZORBAX
RRHT SB-Aq column (2.1 × 100 mm, 1.8-µm) was used with a
gradient of water and methanol, each with 0.1% formic acid, at a
f ow rate of 0.05 mL/min. The second dimension separation used
an Agilent Poroshell 120 Bonus-RP column (3.0 × 50 mm, 2.7-µm)
with shifted gradients of water and acetonitrile, each with 0.1% formic
acid, at a f ow rate of 2.5 mL/min. Modulation was realized using the
Agilent 2-position/4-port-duo valve, equipped with two 40 µL loops.
A modulation time of 30 s was employed. Detection was performed
at 254 nm as well as using QTOF mass spectrometry in positive and
negative ionization mode. Samples were prepared as decoctions, in
the same manner as they are prepared for pharmaceutical use.
ResultsTo enable authentication of Si-Wu-Tang, a separate comprehensive
2D-LC analysis of each herb contained in Si-Wu-Tang was performed.
Detection of accurate masses by QTOF mass spectrometry in
connection with literature data enabled the tentative identif cation
of characteristic components of each herb. The peaks that were
tentatively identif ed and further high abundant peaks were
selected to construct a template for each herb. Each template was
then matched to the peaks detected in Si-Wu-Tang in terms of f rst
and second dimension retention times as well as agreement of
the base peak in the respective mass spectra. Figure 1 shows the
analysis of Si-Wu-Tang with the peaks matched from the templates
of the individual herbs. Several peaks detected in Si-Wu-Tang
can be attributed to more than one individual herb, for example,
senkyunolide A from Radix Angelicae Sinensis and Rhizoma
Chuanxiong.
Authentication of Traditional Chinese Prescriptions Using Comprehensive 2D-LCSonja Krieger, Agilent Technologies Inc.
Agilent Technologies Inc.5301 Stevens Creek Blvd., Santa Clara, California 95051, USA
Website: www.agilent.com
Generally, 75% or more of the template peaks could be matched
to peaks detected in Si-Wu-Tang. This shows the possibility to detect
characteristic components of an individual herb in a traditional
Chinese prescription. Additionally, adulteration through omission
and replacement of one herb from Si-Wu-Tang could be detected
by the matching of a considerably reduced number of template
peaks.
ConclusionsComprehensive 2D-LC is ideally suited for the analysis of complex
samples such as the traditional Chinese prescription Si-Wu-Tang.
The analysis of Si-Wu-Tang and its individual herbs provides a means
of authentication. Further, it is illustrated that one or a few marker
compounds for each herb are not suff cient for authentication when
those compounds are not uniquely contained in one herb.
References(1) P.S. Xie, and A.Y. Leung, Journal of Chromatography A 1216, 1933–1940 (2009).
(2) X.M. Liang et al., Journal of Chromatography A 1216, 2033–2044 (2009).
(3) D.Z. Yang et al., Journal of Chromatographic Science 51, 716–725 (2013).
(4) Y.Z. Liang et al., Journal of Chromatography B 812, 53–70 (2004).
Figure 1: Comprehensive 2D-LC analysis of a decoction from Si-Wu-Tang with MS detection in positive (a) and negative (b) ionization mode. Peaks matched from the templates of the individual herbs are marked: Radix Angelicae Sinensis (yellow), Rhizoma Chuanxiong (black), Radix Paeoniae Alba (white), Radix Rehmanniae Preparata (red).
8
Gallic acid
2–1
3
4
4 9
6–2–2
10–6
8
5 Albiflorin/Paeoniflorin
1
9
11
1Senkyunolide ASenkyunolide A
Z-LigustilideZ-Ligustilide
12
11 7
1023
Gallic acid
5
3
4 Echinacoside
1 14 7
23
15
8
16 7
2–114
12
Catechin
10 9
15
19
Ferulic acid
Ferulic acid
61
14 12 13 18
4 6
Albiflorin/
Paeoniflorin
(a)
(b)
ES665925_LCESUPP0915_020.pgs 08.31.2015 14:20 ADV blackyellowmagentacyan
PHARMACEUTICAL/DRUG DISCOVERY
THE APPLICATIONS BOOK – SEPTEMBER 2015 21
Gel permeation chromatography (GPC), also known as
size-exclusion chromatography (SEC), provides an easy
and effective way to measure the molar mass distribution
and the amount of free, unbound polysaccharide in iron
polysaccharide complexes.
Iron is an essential nutrient in the human body. In case of iron deficiency, complexes of a polysaccharide and iron are applied as drugs to enhance low iron levels. Suitable characterization of these complexes and their formulations are mandatory for regulatory reasons, quality control, and research. In the present investigation, iron polysaccharide complexes from different sources were analyzed on a GPC/SEC system with simultaneous ultraviolet/refractive index (UV/RI) detection.
Experimental Conditions:
GPC/SEC was performed using a PSS BioSECcurity SEC systemColumns: PSS SUPREMA, 5 µm, 30 Å + 2 ×1000 Å (8 × 300 mm, each) PSS SUPREMA precolumnEluent: 0.1 n NaNO3, in 0.01 m phosphate buffer at pH = 7Temperature: AmbientDetection: UV @ 254 nm, refractive index (RI)Calibration: PSS Pullulan ReadyCal standards Concentration: 2 g/L for dry material, approx. 50 g/L for formulationsInjection volume: 25 µLSoftware: PSS WinGPC UniChrom 8.2
Results and Discussion:
Figure 1 shows the overlay of the UV-chromatograms of the four different samples A, B, C, and D, while the inset of the figure shows the overlay of the simultaneously measured RI-traces for two of the samples (A and B), which show nearly identical UV-traces.
An advantage of this application is that the iron polysaccharide complex is selectively detected by the UV-detector operated at 254 nm (20–26 mL). All complexes reveal well shaped nearly Gaussian peak shapes, indicating that the PSS SUPREMA column combination is ideal for this molar mass separation range. By applying a calibration curve, established using PSS pullulan standards, the relative molar mass distributions as well as the molar mass averages and the polydispersities are derived.
While UV-detection is sufficient to differentiate between three of the four samples, samples A and B render identical elution profiles. However, when comparing the RI-traces of both samples, it becomes clear that sample A contains a significantly higher amount of the unbound polysaccharide.
We can therefore conclude that GPC/SEC with UV- and RI-detection does not only allow the molar mass distribution of iron polysaccharide complexes to be determined, but also provides information on the amount of free, unbound polysaccharide ensuring a more comprehensive characterization of the samples.
Investigation of Iron Polysaccharide Complexes by GPC/SEC Using RI- and UV-DetectionPSS Polymer Standards Service GmbH
PSS Polymer Standards Service GmbHIn der Dalheimer Wiese 5, D-55120 Mainz, Germany
Tel: +49 6131 962390 fax: +49 6131 9623911
E-mail: [email protected]
Website: www.pss-polymer.com
Figure 1: Comparison of the UV-traces of four different iron dextran samples used to determine the molar mass distribution of the iron complexes. While the UV-signals for samples A and B are nearly identical, the inset displaying the RI-traces shows that these samples differ in the amount of unbound polysaccharide.
ES665958_LCESUPP0915_021.pgs 08.31.2015 14:21 ADV blackyellowmagentacyan
22 THE APPLICATIONS BOOK – SEPTEMBER 2015
PHARMACEUTICAL/DRUG DISCOVERY
Pain management LC analyses can be diff cult to optimize
because of the limited selectivity of C18 and phenyl-hexyl
phases. In contrast, the selectivity of Raptor™ Biphenyl
superf cially porous particle (SPP) LC columns provides
complete resolution of isobaric pain medications with a
total cycle time of 5 min.
Accurate, reliable analysis of pain medications is a key component
in monitoring appropriate medical use and preventing drug
diversion and abuse. As the demand for fast, multicomponent
methods grows, LC–MS–MS methods are increasingly desired for
pain management and therapeutic drug monitoring because of the
low detection limits that can be achieved with this highly sensitive
and selective technique. However, despite the selectivity offered by
mass spectrometry, hydrophilic matrix components can still interfere
with early-eluting drug compounds resulting in ion suppression. In
addition, isobaric pairs must be chromatographically separated for
positive identif cation. The need for highly selective and accurate
methods makes LC column selection critical.
While C18 and phenyl-hexyl phases are frequently used for
bioanalytical LC–MS–MS applications, Restek’s Biphenyl phase
offers better aromatic retention and selectivity for pharmaceutical
and drug-like compounds, giving it a signif cant advantage over
other phases for the analysis of pain management medications or
other drugs of abuse. The Biphenyl phase, originally developed a
decade ago by Restek, has recently been combined with Raptor™
SPP (“core–shell”) silica particles to allow for faster separations
without the need for expensive UHPLC instrumentation. Here, we
demonstrate the fast, selective separation of commonly tested pain
drugs that can be achieved using the new Raptor™ SPP Biphenyl
LC column.
Experimental Conditions
A standard containing multiple pain management drugs was
prepared in blank human urine and diluted with mobile phase
as follows, urine:mobile phase A:mobile phase B (17:76:7).
The f nal concentration for all analytes was 10 ng/mL except
for lorazepam, which was 100 ng/mL. Samples were then
analyzed by LC–MS–MS using an AB SCIEX API 4000™ MS–MS
in ESI+ mode. Chromatographic conditions, retention times,
and mass transitions are presented here and in Tables 1 and 2:
Column: Raptor™ Biphenyl, 50 mm × 3.0 mm, 2.7-µm
Sample: Fortif ed urine
Inj. vol.: 10 μL
Inj. temp.: 30 °C
Mobile phase A: Water + 0.1% formic acid
Mobile phase B: Methanol + 0.1% formic acid
Results
As shown in Figure 1, 18 commonly tested pain management
drugs were analyzed, with the last compound eluting in less than
3.5 min, giving a total cycle time of 5 min on Restek’s Raptor™
SPP Biphenyl LC column. Analyte retention times are presented
in Table 2. Important isobaric pairs (morphine/hydromorphone
and codeine/hydrocodone) were completely resolved and
eluted as symmetrical peaks, allowing accurate identification
and integration. In addition, early-eluting compounds such as
morphine, oxymorphone, and hydromorphone are separated
from hydrophilic matrix interferences, resulting in decreased
ion-suppression and increased sensitivity. Similar analyses on
C18 and phenyl-hexyl columns often exhibit poor peak shape
and resolution (for example, peak tailing between closely
Accurate Pain Management Analysis in Under 5 Min on Raptor™ Biphenyl Superf cially Porous Particle LC ColumnsSharon Lupo, Ty Kahler, and Paul Connolly, Restek Corporation
Figure 1: Baseline resolution of isobaric pain management drugs in sub-5-min runs on the Raptor™ Biphenyl column.
Table 1: Mobile phase gradient.
Time (min) Flow (mL/min) %A %B
0.00 0.6 90 10
1.50 0.6 55 45
2.50 0.6 0 100
3.70 0.6 0 100
3.71 0.6 90 10
5.00 0.6 90 10
ES665928_LCESUPP0915_022.pgs 08.31.2015 14:20 ADV blackyellowmagentacyan
THE APPLICATIONS BOOK – SEPTEMBER 2015 23
PHARMACEUTICAL/DRUG DISCOVERY
Restek Corporation110 Benner Circle, Bellefonte, Pennsylvania 16823, USA
Tel: (800) 356 1688 fax: (814) 353 1309
Website: www.restek.com/raptor
eluting isobars), which makes identification and accurate
quantification more difficult.
Conclusions
Complete separation of critical pain management drug analytes
from hydrophilic matrix components and isobaric interferences
was achieved using the new Raptor™ SPP Biphenyl LC column in
less than 5 min. The fast, complete separations produced in this
method allow accurate quantif cation of pain management drugs
and support increased sample throughput and improved lab
productivity.
To learn more, visit www.restek.com/raptor
Table 2: Analyte retention times and transitions.
Peaks tR (min) Precursor Ion Product Ion 1 Product Ion 2
Morphine* 1.34 286.2 152.3 165.3
Oxymorphone 1.40 302.1 227.3 198.2
Hydromorphone* 1.52 286.1 185.3 128.2
Amphetamine 1.62 136.0 91.3 119.2
Methamphetamine 1.84 150.0 91.2 119.3
Codeine* 1.91 300.2 165.4 153.2
Oxycodone 2.02 316.1 241.3 256.4
Hydrocodone* 2.06 300.1 199.3 128.3
Norbuprenorphine 2.59 414.1 83.4 101.0
Meprobamate 2.61 219.0 158.4 97.2
Fentanyl 2.70 337.2 188.4 105.2
Buprenorphine 2.70 468.3 396.4 414.5
Flurazepam 2.73 388.2 315.2 288.3
Sufentanil 2.77 387.2 238.5 111.3
Methadone 2.86 310.2 265.3 105.3
Carisoprodol 2.87 261.2 176.3 158.1
Lorazepam 3.03 321.0 275.4 303.1
Diazepam 3.31 285.1 193.2 153.9
*An extracted ion chromatogram (XIC) of these isobars is presented in the inset of Figure 1.
ES665922_LCESUPP0915_023.pgs 08.31.2015 14:20 ADV blackyellowmagentacyan
PHARMACEUTICAL/DRUG DISCOVERY
24 THE APPLICATIONS BOOK – SEPTEMBER 2015
Antibody therapeutics are enjoying high growth rates,
the major areas of therapeutic application being cancer
and immune/inflammation-related disorders, including
arthritis and multiple sclerosis. In 2013, six of the top
ten best-selling global drug brands were monoclonal
antibodies (mAbs) and more than 400 monoclonals were
in clinical trials. The characterization of these complex
biomolecules is a major challenge in process monitoring
and quality control. The main product characteristics
that need to be monitored are aggregate and fragment
content, glycosylation pattern, and charged isoforms.
The standard method used in biopharmaceutical QC for mAb aggregate and fragment analysis is size-exclusion chromatography (SEC). A new series of 2 µm silica-based ultrahigh-pressure liquid chromatography (UHPLC) columns with 25 nm (250 Å) pore size can be applied to either increase speed or improve resolution of the separation of antibody fragments, monomers, and dimers.
Experimental
Columns: TSKgel UP-SW3000 (P/N 0023449), 2 µm Competitor Protein SEC Column, 1.7 µmColumn size: 4.6 mm × 15 cmEluent: 100 mmol/L phosphate buffer (pH 6.7) +100 mmol/L sodium sulphate
+ 0.05% NaN3
Flow rate: 0.35 mL/minTemperature: 25 °CDetection: UV @ 280 nm, micro f ow cell Sample (calibration): 1. thyroglobulin, 640,000 Da (1’ thyroglobulin dimer); 2. γ-globulin, 155,000 Da (2’ γ-globulin dimer); 3. ovalbumin, 47,000 Da; 4. ribonuclease A, 13,700 Da; 5. p-aminobenzoic acid, 137 Da Injection volume: 5 µLSample
(mAb analysis): therapeutic mAb (mouse-human chimeric) 1: trimer; 2: dimer; 3: monomer; 4: fragment Injection volume: 10 µL
Results
Figure 1 shows the calibration curves of the new TSKgel UP-SW3000 2 µm column and a commercially available 1.7 μm UHPLC column. The calibration of TSKgel UP-SW3000 shows a shallower slope in the region of the molecular weight of γ-globulin. These differences in the separation range and steepness of the curves are related to a slight difference in pore size (25 nm for TSKgel versus 20 nm for the 1.7 µm material).
The separation of an antibody sample on the new 2 µm packing compared to the competitor UHPLC column is depicted in Figure 2. The difference in pore sizes results in a better separation in the molecular weight range of antibodies, fragments, and aggregates. Based on the wider separation window the resolution between monomer and dimer as well as dimer and trimer is slightly higher with the TSKgel UP-SW3000 column,
UHPLC Analysis of Immunoglobulins with TSKgel® UP-SW3000 SEC Columns Tosoh Bioscience GmbH
1’
1
2’
2
3
5
7
6
5
4
3
2
12,0 3,0 4,0 5,0 6,0 7,0
4
Elution volume (mL)
Competitor SEC column
UP-SW3000
log
M
Figure 1: Calibration curves.
ES665976_LCESUPP0915_024.pgs 08.31.2015 14:22 ADV blackyellowmagentacyan
PHARMACEUTICAL/DRUG DISCOVERY
THE APPLICATIONS BOOK – SEPTEMBER 2015 25
Tosoh Bioscience GmbH
Im Leuschnerpark 4 64347 Griesheim, Darmstadt, Germany
Tel: +49 6155 7043700 fax: +49 6155 8357900 E-mail: [email protected]
Website: www.tosohbioscience.de
A)
(a)
(b)
UV
280 n
m
B)
1
1
2
2
3
3
(2 μm, 4.6 mm ID x 15 cm)
(1.7 μm, 4.6 mm ID x 15 cm)
Competitor Column
4
4
1
2 3
3
4
3
3.4 3.6 3.8 4.0 4.2 4.4
3.1 3.3 3.5 3.7 3.9 4.1
4
4
TSKgel UP-SW3000
Time (min)
5 6
Figure 2: Comparison of antibody analysis results: mouse-human chimeric mAb. 1: trimer; 2: dimer; 3: monomer ; 4: fragment.
although particle size is slightly larger than in the competitor
column. Moreover, the fragment peak is more clearly separated
from the monomer peak.
Conclusion
TSKgel UP-SW3000 is ideally suited for the analysis of the
aggregate and fragment contents of antibody preparations. It
features the same pore size as the renowned TSKgel G3000SWXL
and TSKgel Super mAb columns while improving resolution
through a smaller particle size. Based on the optimized pore size
and the high degree of porosity the resolution in the molecular
weight range of immunoglobulins is superior to a competitive
UHPLC column with slightly smaller particle and pore size.
Table 1: Comparison of resolution.
Column RS (peak 1/2) RS (peak 2/3)
TSKgel UP-SW3000 2 µm 1.52 3.56
Competitor UHPLC-SEC 1.7 µm 1.25 3.47
ES665977_LCESUPP0915_025.pgs 08.31.2015 14:22 ADV blackyellowmagentacyan
PHARMACEUTICAL/DRUG DISCOVERY
26 THE APPLICATIONS BOOK Ð SEPTEMBER 2015
Pindolol is a non-selective beta-blocker used for treatment of
hypertension and angina pectoris. It is applied as a racemate,
although only the (S)-form is the active stereoisomer.
YMC offers immobilized chiral phases that can be used either
in normal or reversed phase mode as well as in supercritical fluid
chromatography (SFC) mode. CHIRAL ART Cellulose-SC is a very
versatile phase to separate many chiral substances. YMC have
developed two isocratic applications to separate the pindolol
enantiomers in normal phase or reversed phase mode. Both methods
offer a high resolution. The normal phase application provides more
potential for a preparative scale up, while the reversed phase approach
separates the enantiomers in a shorter time.
Chiral Separation of Beta Blocker Pindolol EnantiomersYMC Europe GmbH
YMC Europe GmbHPhone: +49 2064 4270
E-mail: [email protected]: www.ymc.de
Normal Phase Method
Column: CHIRAL ART Cellulose-SC (250 × 4.6 mm,
5-μm)
Part No.: KSC99S05-2546WT
Eluent: n-hexane/ethanol/diethylamine (40/60/0.1)
Flow rate: 1.0 mL/min
Detection: UV at 265 nm
Injection: 10 μL (100 μg/mL)
Temperature: 25 °C
Reversed Phase Method
Column: CHIRAL ART Cellulose-SC (250 × 4.6 mm,
5-μm)
Part No.: KSC99S05-2546WT
Eluent: methanol/diethylamine (100/0.1)
Flow rate: 1.0 mL/min
Detection: UV at 265 nm
Injection: 10 μL (100 μg/mL)
Temperature: 25 °C
H3C
CH3
NH
NH
O
OH
Pindolol
Figure 1: Structure of pindolol.
mA
U
Time (min)
2.5
10.1Rs
α
200
100
0
0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0
Normal phase method
Figure 2: Normal phase method.
Reversed phase method
mA
U
Time (min)
150
100
50
0
0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0
1.5
4.2Rs
α
Figure 3: Reversed phase method.
ES665926_LCESUPP0915_026.pgs 08.31.2015 14:20 ADV blackyellowmagentacyan
www.gerstel.com
Heavy workload?
– of course!
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Laboratory Staff, the Workplace and the Environment.
What can we do for you?
Thermal Desorption
and Pyrolysis
Sample Preparation
and Introduction
Extraction techniques:
Twister®, SPE, SPME ... Liquid Addition
Derivatization
ES667259_LCESUPP0915_CV3_FP.pgs 09.01.2015 21:14 ADV blackyellowmagentacyan
The inventors of the Valco gas-tight valve for chromatography
45 years of experience in valves for chromatography
•Forinjection,streamselection,trapping, and column switching
•Boresfrom0.25mm(.010")to4mm(.156")
•:erodeadVolumelttingsfor1/�2",1/16", 1/8",or1/4"tubing
•Alloysandpolymercompositestomeet virtually any system requirement
•Manual,pneumatic,orelectrically-actuated
Valco Instruments Co. Inc.tel: 800 367-8424
fax: 713 688-8106
North America, South America, and Australia/Oceania contact:
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VICI AG Internationaltel: Int + 41 41 925-6200
fax: Int + 41 41 925-6201
Europe, Asia, and Africa contact:
Request a
catalog
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Multiposition stream selectors
Diaphragm valves
Sampling and switching valves
ES667261_LCESUPP0915_CV4_FP.pgs 09.01.2015 21:14 ADV blackyellowmagentacyan