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THE

APPLICATIONSBOOK

September 2015

www.chromatographyonline.com

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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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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

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Jan H. ChristensenDepartment of Plant and Environmental

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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

Consumer

Waste


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.


ENVIRONMENTAL


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

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ne

He

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Tolu

en

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Oct

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o-X

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No

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Similarity

(x 1,000,000)

TIC (1.00)

93 Phenol. 2.5-dimethyl-88

Compound name

De

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1-H

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(a)

(b)

a.u

.a

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(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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ENVIRONMENTAL


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.



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


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



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.



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


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


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.



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.


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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.



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



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.



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.



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.


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

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Pet

eBle

yer.c

om

unexpectedly attentive customer service. Check.

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we want to help you do great work with them.

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products.


PHARMACEUTICAL/DRUG DISCOVERY


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)




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


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.




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




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.




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.




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



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)



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)



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.


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Thermal Desorption

and Pyrolysis

Sample Preparation

and Introduction

Extraction techniques:

Twister®, SPE, SPME ... Liquid Addition

Derivatization


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