NEWS 01.2012 GB

NEWS

1/2012

NEWS

A good communicator:The new TOC-4200

Almost omnipresent:Determination of bisphenol A in plasticpackaging

Kaizen for maximumperformance: Nexera in continuousimprovement

Glass on the road in-creases driving safety:Particle measurementof glass beads

Low maintenance and inde-pendent operation as wellas fast determination of

TOC values – these are key fea-tures of today’s TOC processanalyzers. These requirementshave been implemented in thenew TOC-4200 system. It is usedfor continuous monitoring oforganic carbon pollution in awide range of applications.

Low-maintenance analyzer

The TOC-4200 is a powerfulanalyzer that applies catalyticcombustion at 680 °C. This tem-perature is below the melting

New design and softwaresimplify operation

The new design includes spatialseparation of electronic compo-nents, measuring system and flu-ids. For filling of standard solu-tions and hydrochloric acid, themain instrument can now remainclosed. The instrument is operat-ed via a color touch-screen. Theclear menu navigation simplifiesthe creation of calibration curvesand measurement methods.

Caviar and cigarettes – FTIR-ATR analysis of alginate on cigarette paper »4

Finding new antibiotic substances – LC-IT-TOF-MS of alkylated neomycin antibiotics »6

Kaizen for maximum performance – Nexera in continuous improvement »8

SQTs – excellent candidates preventing viral infections and cancer? – Biological activities of selected sesquiterpenes and terpenoids as well as natural products rich in these volatiles »10

Burning high-tech – Graphite tubes for electrothermal atomization »14

Replacement liver for simulation of keyhole surgery »16

Soft materials in hard drinks – Occur-rence of endocrine disruptors in foods »18

Glass on the road increases driving safety – Particle measurement of glass beads »20

Almost omnipresent – Determination of bisphenol A in plastic packaging »22

Determination of mineral oil hydro-carbons in vegetable oils »26

A good communicator – The new TOC-4200 »2

Argus eyes – New class of UV-VIS double-beam spectrophotometers »24

Light is difficult – Tips for the deter-mination of light elements »13

APPLICATION

The new process generation: TOC-4200

PRODUCTS Shimadzu News 1/2012

2

TIPS & TRICKS

PRODUCTS

A good communpoint of sodium chloride, pre-venting deactivation of the activecenters of the catalyst by a melt.

Use of a platinum catalyst en-sures complete conversion oforganic carbon compounds toCO2. The highly sensitive NDIRdetector allows small injectionvolumes (typically 20 – 50 μL),keeping the absolute sample saltload on the catalyst low. A fur-ther reduction can be attainedwith the integrated dilution func-tion, which considerably extendsthe lifetime of the catalyzer andthe associated maintenance inter-vals. The integrated dilutionfunction not only enables a meas-uring range up to 20,000 mg/L,but also helps in the creation ofmulti-point calibrations (max.five calibration points).

By default, TOC is determinedby the NPOC method (removinginorganic carbon prior to injec-tion). Depending on the applica-tion, users can also work with thedifference method (TOC = TC –IC) or the addition method(TOC = NPOC + POC).

Figure 1: TOC-4200 Shimadzu NEWS, Customer Magazine of Shimadzu Europa GmbH, Duisburg

Publisher:Shimadzu Europa GmbHAlbert-Hahn-Str. 6 -10 · D - 47269 DuisburgPhone: +49 - 203 - 76 87- 0 Telefax: +49 - 203 - 76 66 [email protected]

Editorial Team:Uta Steeger · Phone: +49 - 203 - 76 87- 410 Ralf Weber, Tobias Ohme

Design and Production:m/e brand communication GmbH GWADüsseldorf

Circulation:German: 7,670 · English: 21,850

©Copyright:Shimadzu Europa GmbH, Duisburg, GermanyApril 2012

Windows is a trademark of Microsoft Corporation. ©2012 Apple Inc. All rights reserved. Apple, the Apple logo, Mac, Mac OS andMacintosh are trademarks of Apple Inc.

IMPRINT

Figure 3: Sampling unit with homo-

genization

under the Modbus protocol.Additional protocols can howev-er be programmed optionally.Moreover, it is possible to querymeasurement values and theinstrument status via a web inter-face.

Sample preparation

An analysis system is only asgood as the sample preparation.The TOC-4200 series featuresseveral sample preparation sys-tems that can be optimally adapt-ed to the application range.

In the single stream option, thesample passes through a filterinto the sample chamber. A rotat-ing knife then homogenizes thesample before it is transferred tothe instrument for analysis. Inthis way, even samples containinglarge amounts of particulate mat-ter can be measured without dif-ficulty. After sampling, the cham-ber and the filter are cleanedusing rinse water. Depending onthe application, the rinse watercan be acidified to prevent thegrowth of algae.

When multiple sample streamsare being measured with one

A calendar function helps inplanning automated determina-tion of control samples or cali-bration curves. Measurement ofoffline samples is possible with-out having to stop the instru-ment. Online measurement isinterrupted only as long as theoffline sample is being measured.After completion, online meas-urement resumes automatically.

New communication possibilities

In a chemical industry park, thedistance to the process analyzerscan be long and it is thereforeimportant that the instrumentscan be controlled from a controlroom. Numerous alarm and sta-tus signals facilitate the detectionof exceeded limit values or main-tenance need. The remote func-tion enables users to start mea-surement or calibration and toselect a particular measurementstream. Until now, a separate cir-cuit was needed for each individ-ual control or measurement func-tion. Through the use of bidirec-tional serial communication, allof these functions can now beimplemented via a two-wire bus.By default, the instrument runs

3

PRODUCTSShimadzu News 1/2012

icator The new TOC-4200

Figure 2: Straightforward operation via touch-screen

instrument, the multi-samplestream unit (Figure 3) is used.Again, the sample is homoge-nized prior to analysis. The rins-ing function prevents carry-overeffects when changing samplestreams. The measuring programcan be selected individually foreach sample stream. The user canalso freely determine the meas-urement sequence of the samplestreams.

Various options extend theapplication range

The TN option allows simulta-neous determination of TOC andTNb (total nitrogen) within a fewminutes. The measurement sam-ple is injected onto the catalyst at720 °C. Carbon compounds arethen converted to CO2 and nitro-gen compounds to NO. Afterdrying, the carrier gas containingboth components first reaches theNDIR detector (where carbondioxide is detected) and subse-quently the chemiluminescencedetector. With the help of ozone,a chemiluminescence reactiontakes place which is then mea-sured. The peaks of both compo-nents are integrated independent-ly and the concentration is calcu-lated.

For continuous TOC analysis of samples with high salt loads (> 10 g/L), Shimadzu has devel-oped a salt kit. The combustiontube has a special shape and twodifferent catalyst spheres areused. This combination signifi-cantly extends maintenance inter-vals. Using this kit in a seawaterapplication, the lifetime of thecatalyst and the combustion tubecould be increased by a factor often.

When monitoring cooling water or condensate systems, the TOCcontents are generally much lower.Here, the high-sensitivity kit formeasuring TOC concentrationsbelow 1 mg/L will be helpful.

Prototype has passed operational reliability test

A prototype of the TOC-4200was tested in a German chemicalpark during a three-month trialunder real conditions. The samplewas a high salt concentrationwastewater. Thanks to the specialcombustion tube and catalyst fill-ing of the salt kit, the TOC-4200did not need any maintenance. In addition, its excellent perform-ance and ease of use was clearlydemonstrated.

APPLICATION Shimadzu News 1/2012

4

According to estimations(European Commissionstandardization mandate,

M/425), carelessly unattendedcigarettes cause some 14.000 firesevery year in the EU, with 7,000fatalities, 2,500 injuries andaround 50 million euro of materi-al damage. How can moleculargastronomy decrease the numberof these accidents?

Spherification is one of manyapplications of molecular gas-tronomy which combine uncon-ventional textures and flavors. It’sa process of turning liquid juiceinto juice-filled pearls. To pro-duce these pearls some alginate –a substance derived from algae –is simply dissolved in a juice.Droplets of the juice are thendropped into a calcium water

Caviar and cigaFTIR-ATR analysis of alginate on ci

bath. The calcium from the waterbath reacts immediately with thealginate in the juice to form afilm around the droplet. Spheresof juice are thus created whichlook just like caviar (Figure 1).How is fake caviar related to cig-arettes?

Fake caviar and its relation-ship to cigarettes

Cigarettes are a source of heatand therefore represent a firehazard. They can ignite materialssuch as furniture or textiles. Self-extinguishing cigarettes can re-duce the number of accidentscompared to unattended ciga-rettes. These cigarettes are pro-duced by adding two specialretardant bands to the cigarettepaper during manufacturing.

Figure 1: Alginate based Caviar prepared with fluoresceine excited with analytical

wavelength of 460 nm in a Shimadzu fluorescence spectrophotometer RF-5301PC:

it is fluorescent caviar filled into a 1 cm quartz cell

Figure 2: View of a cigarette and zoom onto one form of the cigarette paper. In this

case the rings which help to burn the cigarette continuously are apparent.

Figure 3: Structure of alginate acid base

of the sodium alginate salt

Figure 4: Structure of a common cellu-

lose molecule

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5

APPLICATIONShimadzu News 1/2012

These bands act as speed bumpsby decreasing the flow of oxygenthrough the paper to the burningtobacco. They slow down therate at which the cigarette burnsas the lit end crosses over. Theyare more likely to self-extinguish.These speed bumps are made of alayer of alginate on the cigarettepaper – the same alginate used forthe molecular caviar.

A simple application of the FTIRspectrophotometer in combina-tion with the single reflectionunit demonstrates the differencesof the materials. The single mate-rial spectra and the spectrum ofthe final paper layer are shownstep by step. All significant com-ponents such as cellulose in thepaper, sodium carbonate and algi-nate have broad signals in theinfrared spectrum. All have incommon the polysaccharide char-acteristics of the 6 ring structurein conjunction with -C-O-C- andthe -OH bonded groups.

The paper spectrum shows signif-icant signals for the whitener of

the sodium carbonate. These are a sharp signal at 700, at 900 and a broad signal at 1400 cm-1. This is reasonable based on the sub-structure of the carbonate groupwhich generates two -CO- andone -C = O bonding constellationand also distribution of electronsover this bonding system, re-sulting in the broad signal at 1400 cm-1. In the literature, thesymmetric valence vibration �sy(-COO-) is calculated as 1440 -1360 cm-1.

The spectrum of alginate onpaper shows a mixture of alginateand paper. It is possible to differ-entiate between both spectra.Even though both materials arebased on polysaccharide, theyhave differences in their molecu-lar structure which are visible inthe infrared spectrum. The signalat 1600 cm-1 is the -OH bondingin the huge molecules. Whencomparing the structure of thepolysaccharides the differentpositioning at the ring systemsgenerates the additional signal incomparison to the cellulose.

rettesgarette paper

Figure 5: Infrared spectrum of a white cigarette paper measured with single reflection

technique

Figure 6: Infrared spectrum of cigarette paper with a thin layer of Sodium-Alginate,

surface analysis with single reflection ATR technique

Figure 7: Infrared Spectrum of sodium alginate powder measured with single reflection

technique

-0.010

0.010.020.030.040.050.060.070.080.090.100.110.120.130.140.150.160.170.180.190.200.210.220.25

Cigarette paper, white DuraSampleIIR

4400 4000 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600 400

1/cm

Ab

s.

-0.0225-0.00750.00750.02250.03750.05250.06750.08250.09750.11250.12750.14250.15750.17250.18750.20250.21750.23250.24750.26250.27750.29250.30750.32250.33750.3525

Cigarette paper, white plus algenate Na DuraSampleIIR

4400 4000 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600 400

1/cm

Ab

s.

-0.01

0

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08

0.09

0.10

0.11

0.12

0.13

0.14

0.15

0.16

0.17

0.18

0.19

0.20

0.21

0.22

0.23

Sodium alginate, Sosa DuraSampleIIR

4400 4000 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600 400

1/cm

Ab

s.


6

Finding new antibiotic sLC-IT-TOF-MS of alkylated neomycin antibiotics

Dr. Martin Giera and

Prof. Dr. Wilfried M.A. Niessen

VU University Amsterdam,

Faculty of Sciences,

BioMolecular Analysis Group,

De Boelelaan 1083,

1081 HV Amsterdam,

The Netherlands

Structural elucidation ofsmall molecules is an inte-gral part of numerous life-

science areas. Although NMRspectroscopy undoubtedly playsthe most important role in theelucidation of analyte structures,mass spectrometry (MS) hasbecome widely applied in struc-tural elucidation wherever possi-ble. The success of MS basedstructural elucidation is based onseveral facts:

a) compared to NMR, only smallamounts of the analyte are nec-essary

b) the technique can convenientlybe coupled on-line to chro-matography based separations(LC or GC)

c) more and more knowledge hasbeen gained in the last decadeabout the fragmentation mech-anisms of particular analyteclasses, i.e. sugars, peptides orDNA/RNA molecules. Thepossibility to perform MS/MSor MSn experiments combinedwith high resolution MSinstruments has especially beencrucial for this development.

The following example shows the significance of the combina-tion of LC-MSn capabilities andhigh-resolution spectral data witha Shimadzu LC-IT-TOF-MSinstrument.

Case study

Finding new antibiotic substancesis a key challenge as microorgan-isms are becoming ever moreresistant to currently availabletreatments. Enhanced screeningand identification techniques forbioactive substances from diversesources therefore have to bedeveloped and applied. At theVU University Amsterdam, on-line screening strategies are de-veloped which can be combinedwith LC to assess bioactivity. In this case, the effect of singlealkylation of neomycin at its dif-ferent nitrogen atom sites on theantibacterial behavior of theformed derivative has been inves-tigated [1]. The reductive alkyla-

tion of the aminoglycoside anti-biotic neomycin with one equiva-lent n-octanal leads to the forma-tion of six regioisomers (Fig. 1).

The regioisomers were separatedby reversed-phase LC, and differ-ences in their antibacterial behav-ior were demonstrated [1]. Inorder to elucidate the compoundstructures an IT-TOF instrumentcoupled to LC was applied. Aschematic overview of the systemis shown in figure 2.

Hi-res MS data used for the molecular formula

In the first instance, all regioiso-mers displayed the same m/z val-ue of 727.4448. This proved thatthe mono-alkylation actuallyworked. The performance ofMS/MS still yielded identicalfragment ions with m/z 564.3628for all six regioisomers, renderingthe different species so far indis-tinguishable. In MS3, the first dif-ferences between the six regioiso-mers could be obtained. At thisstage, it could be deduced whichof the three rings was alkylated.While a ring-4 alkylation causedtwo fragments with m/z 273.217and 405.261, a ring-2 alkylationgave a fragment with m/z275.233, and a ring-1 alkylationresulted in the formation of twofragments with m/z 273.217 and435.317.

From left to right: Dr. Martin Giera, Prof. Dr. Wilfried M.A. Niessen,

Prof. Dr. Hubertus Irth

Figure 1: Reductive alkylation of neomycin. The alkylated derivatives have a single

n-octyl chain on one of the six nitrogen atoms, as exemplified for N-2. Ring numbers

(in the ring) and nitrogen numbers are specified.

7


ubstances

Figure 3: Fragmentation scheme of mono-N-alkyl neomycin derivatives in MSn

experiments, showing the different ring losses and the calculated m/z values of

the resulting fragmentions. R = n-octyl or H.

Figure 2: Schematic overview of the LC–IT-TOF-MS setup used in this study

In summary: high resolution MSdata allowed generation of themolecular formulae for the sub-stances formed. MS/MS fragmen-tation, as would also be available

on a Q-TOF instrument, resultedin the loss of one of the (unmodi-fied) terminal aminosugars, there-by providing no insights into themolecular structures. MS3 experi-

ments performed on the identicalMS/MS fragments provided thefirst structural insights: it enabledassigning of the ring number atwhich alkylation took place. A summary of the fragmentationpath up to MS3 is shown in fig-ure 3.

The final question to answer was:which nitrogen atom in the ringwas alkylated? MS4 experimentson the fragment ion with m/z275.233 (ring-2 alkylation) re-sulted in identical spectra, asexpected due to the symmetricalstructure of the N-1 and N-3derivatives. For the other fourregioisomers, MS4 experimentsdid lead to characteristic frag-ment ions and subsequentlyunequivocal assignment of thealkylated ring nitrogen atom.

Conclusion

High-resolution MSn experimentson an LC-IT-TOF-MS instru-ment can lead to a full structuralassignment of closely relatedcompounds. The assignment ofthe alkylated ring in the N-octyl

neomycin derivatives was accom-plished by investigating the sub-sequent cleavages of the glyco-sidic bonds. MS4 experimentswere needed to fully assign thestructures of four out of the sixregioisomers. To assign the othertwo, preparative LC and subse-quent NMR analysis would beneeded.

[1] Giera M et al. Rapid Commun

Mass Spectrom, 2010,

24:1439


8

Kaizen for maximum peNexera in continuous improvement

Kaizen is the Japanese philosophy of life andwork that strives for

continuous improvement. In thebusiness world, Kaizen standsfor continuous improvementprocesses. Every product andprocess has the opportunity forimprovement – even those whichare said to be the optimal solu-tion.

For Shimadzu’s Nexera system,Kaizen means that 18 monthsafter its first launch, a series ofmodifications and new solutionsincreases the applicability of theNexera family.

The quaternary gradient optionhas already been introduced andthe flow range has been increasedto a maximum of 10,000 mL/min.This has extended the range ofuse involving pumps for solventmixing and semi preparativeapplications.

To ensure that the pumps workreliably as the backbone of thesystem, modifications have beenmade to the pump head, pistonsuspension and valves. Whilemostly not visible to the user, the

reduced air bubble sensitivityand the improved lifetime areremarkable.

It’s all in the right mix

Gradient reproducibility andaccuracy are crucial when fastgradients are applied to shortcolumns. This requires as ‘per-fect’ as possible choice of themost appropriate components ofthe mobile phase. The micro-reactors used so far with 20 μLand 180 μL (TFA version) havemeanwhile been supplementedby a 40 μL and 100 μL versionand the design of the 180 μLmicro-reactor has been opti-mized to minimize baseline fluctuations caused by mixing.More choices, however, meanmore ‘difficult’ decisions for theuser as to which mixing volumerepresents the optimal solutionfor a specific application.

Autosampler as heart of the system

The heart of the Nexera system isand will be the autosampler. Fastand carryover-free injections haverepeatedly been emphasized as its

essential characteristics. In termsof changes, the major focus is ondurability and maintenance-freeoperation, without overridingexisting features or compromis-ing performance.

Many laboratories already use thepossibility to inject larger samplevolumes. When installing a largersample loop (max. 2.000 μL) inthe system, it is possible to injectthese larger volumes. However,flow rate, pressure range and col-umn dimensions must be takeninto account to prevent damageto the system and/or the column.

Depending on the application,the use of fixed sample loops isworth considering. System vol-ume is reduced, resulting inshorter gradient run times andshorten equilibration timebetween the analyses. Consider-ing about peak symmetry manyadvantages speak for this injec-tion principle. One importantpoint still remains, when talkingabout possible carryover fromsample to sample: the user shalldecide about the priorities forhis/her application and system, asa generic answer cannot be given.

Figure 1: Nexera pump with installed

quaternary gradient valve

Figure 2 a - c: performance test of the new 180 μL micro-reactor at (gradient A: 0.1 % TFA in water, B: 0.1 % TFA in acetonitrile,

C: 0 - 45 % B

Minutes Minutes Minutes

9


“Kaizen”, continuous improve-ment – this process will continue...

New detector cells

Detectors are the eyes of the sys-tem. Standard and semi-microflow cells are supplemented bycells with higher sensitivity.These cells can be considered asmidfield between the former two with respect to cell volume.A longer optical path comparedto the semi-micro cell and areduced diameter of the beampath compared to the standardcell in combination with strongerfocusing, allow greater sensitivi-ty. Two additional cells are avail-able for the UV detector and oneadditional cell is available for thephotodiode-array detector.

What is the optimal flow cell fora particular application? TheNexera system guide suppliedwith the system can provide aninitial answer. Even if the newflow cells are not yet specified,users are provided with a guide-line stating which cell underwhich conditions would be theoptimum choice.

rformance

Figure 3: Comparison of direct injection with loop injection

Figure 4: Comparison of the peaks obtained using the new flow cells versus the

semi-micro flow cell

Figure 5: Flow cell selection diagram from the Nexera system guide

-25,0000

25,00050,00075,000

100,000125,000150,000175,000200,000225,000250,000275,000300,000325,000

uV

0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1

Minutes

254 nm

Fixed loop inj.

Direct inj.

Column : Kinetex 2.1 mm, 50 mm, 2.6 μmMobile phase : A: Water, B: AcetonitrileGradient : 0 min (B: 15 %), 1 min (B: 80 %), 1.01 min (B: 15 %)

Methylparabene: 50 ppm, Ethylparabene: 50 ppmPropylparabene: 50 ppm, Butylparabene: 50 ppm

0

50,000

100,000

150,000

200,000

250,000

300,000

350,000uV

0.457 0.500 0.525 0.550 0.575 0.600 0.625 0.650 0.675 0.700

Minutes

0.1

1

10

100

0 0.5 1 1.5 2 2.5 3

Flow rate (mL/min)

Flow cell selection

Re

ten

tio

n t

ime

(m

in)

Cabreuva oil 0.4 0.4 0.4 0.4 0.8 0.8 0.8 0.8 0.8 0.8 0.8Cedarwood oil 0.2 0.2 0.2 0.2 0.4 0.4 0.8 0.8 0.8 0.4 0.4Juniper berry oil 0.2 0.2 0.2 0.2 0.4 0.4 0.8 0.8 0.8 0.4 0.4Myrrh oil 0.1 0.1 0.1 0.1 0.2 0.2 0.4 0.4 0.4 0.2 0.2

�-Bisabolol 0.1 0.1 0.1 0.1 0.2 0.2 0.4 0.4 0.4 0.4 0.4Cedrol 0.2 0.2 0.2 0.2 0.4 0.4 0.8 0.8 0.8 0.4 0.4(E,E)-Farnesol 0.4 0.4 0.4 0.4 0.8 0.8 0.8 0.8 0.8 0.8 0.8(E)-Nerolidol 0.1 0.1 0.1 0.1 0.2 0.2 0.4 0.4 0.4 0.2 0.2(Z)-Nerolidol 0.1 0.1 0.1 0.1 0.2 0.2 0.4 0.4 0.4 0.2 0.2Sabinene 0.4 0.4 0.4 0.4 0.8 0.8 0.8 0.8 0.8 0.4 0.4Thujopsene 0.2 0.2 0.2 0.2 0.4 0.4 0.8 0.8 0.8 0.4 0.4

BC ST STI SE CD EC PA PAI PF SA SAI

Cabreuva oil – – – – – – – – – – –Cedarwood oil 13.5 18.5 19 16.5 – 8 – – – 6 6Juniper berry oil 12.5 16 16.5 14.5 7 8 – – – 9 9Myrrh oil 13.5 22 24 21 6 10 – – – 9 10

�-Bisabolol 12.5 18 17.5 16.5 7 9 – – – 9 9Cedrol 13 15 16 14 – 6 – – – 7 7(E,E)-Farnesol 11 13 14 14 9 8 – – – – –(E)-Nerolidol 11 23 21 22 – 10 – – – – –(Z)-Nerolidol 11.5 24 21.5 22 – 11 – – – – –Sabinene 12 18 18 16.5 6 8 – – – – –Thujopsene 10.5 14 16.5 15 – 6 – – – 9 10



10

SQTs – excellent candidates preinfections and cancer?

Sabine Krist, Martina Hoeferl,

Leopold Jirovetz

Department of Clinical Pharmacy

and Diagnostics, University of

Vienna, Austria

Volatile organic compoundshave been the subject ofmany investigations. In

the last years, there has been astrong research focus on the biological activities of organicvolatiles. In this work, data con-cerning the antimicrobial activityas well as antiviral, anticancer andcytotoxic properties of selectedsesquiterpenes and terpenoids(SQTs) and natural products richin these volatiles is presented.

The test compounds were addedto broth containing 1.0 % (v/v)Tween 80 at the appropriate vol-umes to produce final concentra-tions of samples in the range of 1 to 1,000 ppm. The concentra-tion of pathogens in the mediumexceeded 106 -107 cfu/cm3. Theagar plates were inoculated bypipetting 0.1 mL of the cultureand 6 μL of the samples ontopaper discs (6 mm) and were thenincubated at 37 °C for 24 h.

Tested microorganisms

Gram-positive bacteria• Bacillus cereus (ATCC 11778)

BC• Staphylococcus aureus

(ATCC 6538) ST• Staphylococcus aureus

(food spoilage isolate) STI• Staphylococcus epidermidis

(clinical isolate) SE

Gram-negative bacteria• Citrobacter diversus (clinical

isolate) CD• Escherichia coli (ATCC 8739)

EC• Pseudomonas aeruginosa

(ATCC 9627) PA• Pseudomonas aeruginosa

(clinical isolate) PAI

Antimicrobial tests

For the antimicrobial tests, theagar diffusion disc method andthe agar dilution method wereapplied. These tests were carriedout against four strains of Gram-positive and seven strains ofGram-negative bacteria.

At first, the agar diffusion discmethod was carried out with 6 mm paper discs and 6 μL ofsample. After incubation of thebacteria at 37 °C for 24 h, thediameter of the inhibition zone(IZ) was measured. Afterwards,the agar dilution method wasapplied.

Biological activitieproducts rich in t

Table 1: Antimicrobial activity I – Inhibitory zones [mm] in agar diffusion assay

Table 2: Antimicrobial activity II – MIC [%] in agar serial dilution assay

Cabreuva oil 0.8 0.8 0.8 0.8 0.8 0.8 1 1 1 0.8 0.8Cedarwood oil 0.4 0.4 0.4 0.4 0.8 0.8 0.8 0.8 0.8 0.8 0.8Juniper berry oil 0.4 0.4 0.4 0.4 0.4 0.4 0.8 0.8 0.8 0.8 0.8Myrrh oil 0.2 0.2 0.2 0.2 0.4 0.4 0.8 0.8 0.8 0.4 0.4

�-Bisabolol 0.2 0.2 0.2 02 0.4 0.4 0.8 0.8 0.8 0.8 0.4Cedrol 0.4 0.4 0.4 0.4 0.8 0.8 0.8 0.8 0.8 0.8 0.8(E,E)-Farnesol 0.8 0.8 0.8 0.8 0.8 0.8 > 0.8 > 0.8 > 0.8 > 0.8 > 0.8(E)-Nerolidol 0.2 0.2 0.2 0.2 0.4 0.4 0.4 0.4 0.4 0.4 0.4(Z)-Nerolidol 0.2 0.2 0.2 0.2 0.4 0.4 0.4 0.4 0.4 0.4 0.4Sabinene 0.4 0.4 0.4 0.4 0.8 0.8 0.8 0.8 0.8 0.8 0.8Thujopsene 0.4 0.4 0.4 0.4 0.8 0.8 0.8 0.8 0.8 0.8 0.8


11


venting virals of selected sesquiterpenes and terpenoids as well as naturalhese volatiles

• Pseudomonas fluorescens (food spoilage isolate) PF

• Salmonella abony (ATCC 6017)SA

• Salmonella abony (clinical isolate) SAI

By applying disc diffusion test,the Gram-positive bacteria aswell as the Gram-negative bac-terium Eschericha coli wereinhibited by all essential oils andsingle compounds tested exceptcabreuva oil. For the Gram-nega-tive bacteria, Pseudomonasaeruginosa (ATCC 9627 and clin-ical isolate) and Pseudomonasfluorescens could not be inhibit-ed by any of the tested volatiles.The agar dilution assay, resultingin minimal inhibitory concentra-tion (MIC), revealed that sub-stance concentrations of 0.1 to0.8 % of the tested volatiles andessential oils were necessary inorder to achieve an overall reduc-tion of bacteria. The MinimalBacterial Concentration (MBC),defined as the lowest concentra-tion of an agent that shows noevidence of microbial growth inthe determination assay andwhich is also achieved by theagar dilution method, was be-tween 0.2 and 1 % for the testedSQTs and essential oils. Detailedresults are shown in tables 1-3.

Antitumor tests

Various organic volatiles weretested for their anti-tumor andantiviral potential, investigatedon HeLa (human cervix carcino-ma) cells. Jurkat E6.1 (humanleukaemic T cell lymphoblastderived from a patient with acutelymphoblastic leukaemia) cellswere included in the analyses to

account for the possibility ofcell-type specific activity of thesamples investigated. Cellulartoxicity of volatile compoundswas tested using the CytoTox-96® assay according to the stan-dard manufacturer’s protocol.The ED50 values were calculatedby regression analysis of the doseresponse curves generated fromthe data.

Tested cell lines:• HeLa (human cervix carcino-

ma)• Jurkat E6.1 (human leukaemic

T cell lymphoblast)• Cellular toxicity: Cytotox-96®

assay

All SQTs tested affected the via-bility of HeLa cells. A reductionof 50 % of HeLa cells, growingas a monolayer, was observedafter applying nerolidol (naturaland synthetic) and (E,E)-farnesolat concentrations (CC50) lessthan 5 μm. The CC50 of syntheticnerolidol in HeLa cells was al-most ten times less than the effectdose required to achieve 50 %cytotoxicity of the cells (ED50).A similarity in the activity againsttumor cells between nerolidol

and farnesol could be attributedto their similar chemical struc-ture. Tables 4 and 5 show detailedresults.

Antiviral tests

For virus infections, mouse poly-omavirus (MpyV) strain A2 wasused at the multiplicity of infec-tion (MOI)5 plaque-formingunits per cell. The titer of viruswas estimated by immunofluores-cence microscopy.

• Tested virusmouse polyoma virus (MPyV)

• Infected cells3T6 cells (Swiss albino mousefibroblasts)

• Assayplaque reduction assay

• Virus titrationimmunofluorescent stayingagainst early or late viral antigens

Together with a remarkable re-duction on the viability of tumorcells, antiviral activity of selectedvolatile compounds could bedemonstrated. The effective con-centration (CC50) of naturalnerolidol against mouse poly-

omavirus appeared to be morethan ten times lower comparedwith the cytotoxic dose (ED50),the synthetic nerolidol showed aCC50 three times lower comparedwith the ED50 (see table 6).

Cytotoxic tests

Today, little is known of thecytotoxic properties of volatilearoma compounds. The in-vitrocytotoxicity of promising vola-tiles was evaluated on cell viabili-ty of HeLa cells. Curcumin, anactive natural anticancer agentand Antimycin D, a well-estab-lished anticancer drug, were usedas references. �

Table 3: Antimicrobial activity III – MBC [%] in agar serial dilution assay

�-BisabololCedrol

39.0 ± 8.048.3 ± 5.8

33.6 ± 6.440.0 ± 8.1

Patchoulol (80 %) 48.3 ± 14.0 40.6 ± 11.7Santalol 29.6 ± 2.6 28.8 ± 13.3(Z)-Nerolidol 16.5 ± 6.7 20.4 ± 7.8Curcumin 15.0 ± 5.0 18.3 ± 5.7Actinomycin D 0.17 ± 0.05 0.056 ± 0.027

Cytotoxic activity

CCT50 (μM) CCP50 (μM)

�-Bisabolol < 10 < 10Cedrol < 10 < 5Patchoulol < 10 < 5Santalol < 20 < 20Nerolidol (synthetic) 3.2 ± 1.5 11 ± 1.8Nerolidol (natural) 1.2 ± 0.4 10.6 ± 3

Antiviral activity

CC50 (μM) ED50 (μM)

Nerolidol (synthetic) 1.5 ± 0.7 < 10 4.2 ± 1.4 < 10Nerolidol (natural) 4.2 ± 1.4 < 10 – –(Z)-Nerolidol < 5 < 10 – –(E)-Nerolidol < 10 < 10 – –

HeLa Jurkat

CC50 (μM) ED50 (μM) CC50 (μM) ED50 (μM)

�-Bisabolol < 5 < 10 < 20 < 20

�-Caryophyllene < 15 < 30 – –Caryophyllene oxide < 30 < 50 – –Cedrol < 20 < 20 < 50 –(E,E)-Farnesol < 5 < 10 – –Farnesol (isomer mix.) < 10 < 10 – –Longifolene < 30 < 50 – –Patchoulol < 10 < 10 < 10 < 10Santalol < 20 < 30 < 50 –Ylang fraction < 30 < 50 – –

HeLa Jurkat

CC50 (μM) ED50 (μM) CC50 (μM) ED50 (μM)


12

interaction with the cell mem-brane, leading to altered perme-ability and cell death, is consid-ered. Nerolidoles in particularmay act as prospective agents forcancer chemoprevention. Theother volatile compounds investi-gated (�-bisabolol, cedrol, san-talol and patchoulol) were suffi-cient to achieve 50 % of cytotox-icity on the HeLa cell line incomparable concentrations as(Z)-nerolidol. Detailed results areshown in table 7.

GC analysis

A Shimadzu GC-17A with FIDand a Shimadzu GCMS-QP5050were applied for the qualitativeand quantitative analysis of natu-ral products rich in sesquiter-penes and terpenoids investigatedin this study.

Vistas

The sesquiterpenes and ter-penoids (SQTs) caryophyllenes,�-bisabolol, cedrol, farnesol,

[2] J. Wanner, E. Schmidt, S. Bail,

L. Jirovetz, G. Buchbauer,

V. Gochev, T. Girova,

T. Atanasova and A. Stoyanova;

Chemical Composition and

Antimicrobial Activity of Selected

Essential Oils and Some of Their

Main Components. Natural

Product Communications 5,

1359 -1364 (2010).

[3] B. Ryabchenko, E. Tulupova,

E. Schmidt, K. Wlcek,

G. Buchbauer and L. Jirovetz;

Investigations of Anticancer and

Antiviral Properties of Selected

Aroma Samples. Natural Prod-

uct Communications 3, 1085 -

1088 (2008).

[4] B. Ryabchenko, E. Tulupova,

E. Schmidt, W. Jaeger,

G. Buchbauer and L. Jirovetz;

Cytotoxic Properties of Selected

Sesquiterpene Alcohols on

Human Cervix Carcinoma Cell

lines. Journal of Essential Oil

Bearing Plants 14, 316 - 319

(2011).

Remarks

In cooperation with Dr. Margit

Geissler, Shimadzu Europe GmbH

longifolene, nerolidol, patchoulol,sabinene, santalols, thujopsene aswell as natural products (essentialoils, oil fractions and oleoresins)rich in these volatiles possess var-ious biological activities. Many ofthese compounds show antimi-crobial properties against selectedGram-positive and Gram-nega-tive bacteria as was demonstratedby applying agar dilution andagar diffusion methods [1,2]. Fur-thermore, pronounced antiviral,anticancer and cytotoxic proper-ties against mouse polyomavirusinfected 3T6 cells and humancancer cell lines were shown[3,4]. SQTs may therefore beexcellent candidates in the pre-vention of viral infections andcancer.

References

[1] E. Schmidt, S. Bail, S.M. Friedl,

L. Jirovetz, G. Buchbauer,

J. Wanner, Z. Denkova,

A. Slavchev, A. Stoyanova and

M. Geissler; Antimicrobial

Activities of Single Aroma

Compounds. Natural Product

Communications 5, 1365 -1368

(2010).

The cytotoxic activity of volatilecompounds within 24 h after thetreatment was evaluated using theCytotoxic Detection Kit. Theinfluence of the tested volatileson viability of HeLa cells wastested using cell ProliferationReagent WST-1 after 24 h ofincubation.

• Tested cell lines:HeLa (human cervix carcino-ma)

• Cellular toxicity and cell lysis:Cytotoxicity Detection Kit(LDH), Roche Diagnostics

• Cell proliferation, growth, viability and chemosensitivity:Cell Proliferation ReagentWST-1, Roche Diagnostics

Among the volatile aroma com-pounds tested, the sesquiterpenalcohol (Z)-Nerolidol demon-strated the highest cytotoxicactivity, comparable to the refer-ence compound curcumin at aconcentration of 15 μm. As a pos-sible mechanism of the cytotoxic-ity of sesquiterpenes a direct

Table 4: Antitumor activity I – CC50 = concentration required to reduce cell number

to 50 %; ED50 = effective dose required to achieve 50 % cytotoxicity

Table 5: Antitumor activity II – CC50 = concentration required to reduce cell number


Table 6: Antitumor activity III – CC50 = concentration required to reduce infected cells


Table 7: Cytotoxic activity – CCT50 = concenrtation required to achieve cytotoxicity

of 50 %; CCP50 = concentration necessary to achieve a decrease in cytoproliferation

by 50 %

13

TIPS & TRICKSShimadzu News 1/2012

Figure 1: Fluorescence yield as a function of the element number. Lighter elements

have a lower yield, heavier elements a higher yield.

Figure 2: Transmission capacity of various polymer films as a function of the wave-

length of the incident X-ray radiation. Lighter elements exhibit a higher wavelength and

a lower energy. They are found at the right-hand side of the graphic.

Fluorescence yield

An additional difficulty whenmeasuring lighter elements is thefluorescence yield of these ele-ments. In an X-ray fluorescencespectrometer, the sample is excit-ed with X-rays. The sample, inturn, emits fluorescence radiation– as well X-rays. As this emittedradiation is characteristic for therespective element, its identity aswell as its concentration can bedetermined unequivocally. Thefluorescence yield of lighter ele-ments is, however, much lowerthan that of heavier elements.Sodium will always have a lowerfluorescence yield than lead.

Sample vessel

If the sample is transferred to asample vessel, its base usuallyconsists of a thin polymer filmwhich is highly transparent to

equal or heavier than sodium(Na).

For instance, Shimadzu’s EDX-800 is capable of carrying outmeasurements starting from car-bon (C) due to the use of a spe-cial window sensitive enough totransmit enough radiation fromlighter elements.

Air

Air also reduces the amount offluorescence radiation that hitsthe detector. In this case, it ishelpful to remove air out of thesample compartment. Modernspectrometers, such as the EDXinstrument series, offer twooptions: either the sample com-partment is evacuated or it isflooded with helium. Evacuationis practical, simple and can beimplemented almost without run-ning costs but is, however, onlysuitable for solid samples. Liquidsamples are measured in a heliumatmosphere. In practice, it hasproven useful to measure sam-ples, for which the content ofaluminum/silicon or lighter ele-ments needs to be determined,under vacuum or helium condi-tions.

X-ray fluorescence analysisis a powerful method inelemental analysis for

reliable determination of concen-trations in the low ppm-rangewithout sample preparation. Inaddition, virtually all elements ofthe periodic table can be meas-ured simultaneously. Results canalready be available in less thantwo minutes.

The measuring range of X-rayfluorescence spectrometers oftenstarts with sodium (Na), some-times magnesium (Mg) and some-times only with carbon (C). It isinteresting to note that in mostcases the heaviest element meas-urable is always uranium (U).

Why is it so difficult to measurelithium (Li) using an X-ray fluo-rescence spectrometer? Because it is, in general, more difficult tomeasure the lighter elements –several challenges need to bemastered.

Detector window

The first challenge is the detectorwindow which often consists ofberyllium (Be) and only transmitssufficient radiation of elements

Light is difficult Tips for the determination of light elements

0

Wavelenght [Å]

Energy [keV]

Tra

ns

mis

sio

n [

%]

20

40

60

80

100

5 10 15

20 10 5 3 2 1.5 1.2 1 0.8

0

Element number [Z]

Flu

ore

sc

en

ce

yie

ld [

%]

100

0

10 20 30 40 50 60 70 80 90 100

Na [11] Fe [26] Pb [82]X-rays. But caution is required aswell. There are various films thatdiffer in their resistance to chemi-cals, but also in their degree oftransparency. Mylar is often usedas standard film due to its goodchemical resistance. When Mylaris used for the measurement ofaqueous sodium solutions, how-ever, sodium may not be includedin the analytical results. The trans-parency of Mylar with respect toX-rays is indeed good. For thecase described here it is appro-priate to use Ultralene instead ofMylar, as its transparency ismuch higher.

When an X-ray system, such asShimadzu’s EDX instrumentseries, offers sophisticated fea-tures such as special windowsand helium or vacuum options,the tips mentioned above willenable simple measurement oflight elements.


14

Burning high-tech

In graphite furnace technolo-gy, it is important to selectthe graphite tube according

to the target element and samplecomposition. In fact, a wide vari-ety of graphite tubes are used inelectrothermal atomization. TheAA-7000 atomic absorption spec-trometer in combination with thehigh-sensitivity digitally con-trolled GFA-7000 graphite fur-nace atomizer can be operatedwith the following tube types:high-density graphite tube,pyrolytic-coated graphite tube,“fork”-platform graphite tubeand omega platform tube®.

Made of ordinary graphite, thehigh-density tube is widely used.Since the hexagonal graphitecrystal structure has porous char-acteristics, the injected samplesolution permeates into thegraphite tube wall during theheating process. On the otherhand, the pyrolytic-coating tubehas a metallic shining surface,made by formation of a pyrolyticlayer through chemical vapor

element in case of high concen-tration samples.

For example, in case of alu-minum, iron and copper,although a level of 1 ppb can bemeasured using the pyrolytic-coated tube, the sensitivity can bereduced by a factor of 100 to alevel of 100 ppb using the high-density tube.

Figure 3 shows an example ofcalibration curves for Cu meas-urements using the high-densitytube and the pyrolytic-coatedtube. The measurement points are20, 40 and 60 ppb in the case ofhigh-density tube and 2, 4 and 6 ppb in the case of pyrolytic-coated tube, although similarabsorbance values are shown.

Pyrolytic-coated tube shows sharp peaks for carbide-forming elements

In general, the pyrolytic-coatedgraphite tube is the most effectivesolution for elements which easi-ly form carbides as a reactionwith the graphite in an uncoatedtube. Nickel (Ni), calcium (Ca),titanium (Ti), silicon (Si), vanadi-um (V) and molybdenum (Mo)are elements typically showingthis effect. In the high-densitytube, a sample easily permeatesinto the graphite, resulting in alarger contact area between theelement and carbon. In thepyrolytic-coated tube, however, asmaller contact area can suppresscarbide formation, and a highersensitivity is obtained as a result.When the peak profiles are com-pared, a sharp peak is observed inthe case of pyrolytic-coated tube,while a broad peak is observed inthe case of high-density tube.Figure 2 shows the example forcopper.

deposition. Since the density ofthe surface is higher than that ofa high density tube, sample per-meation into the wall is lowerand the generation of the atomiccloud during the atomizationstage is improved. The platformtube refers to a graphite tube inwhich a plate with a basin for thesample (platform) is mounted.The Shimadzu platform tube hasa platform made of 100 % pyro-lytic carbon. Figure 2 shows acomparison of peak profiles ofpyrolytic-coated tube and thehigh-density tube.

High-density tube specializes in elements with low atomization temperatures

The high-density tube is used formeasurements of various ele-ments, especially for those show-ing low atomization tempera-tures, such as cadmium (Cd), lead(Pb), sodium (Na), potassium(K), zinc (Zn) and magnesium(Mg). It is also useful to reducethe measurement sensitivity of an

Graphite tubes for electrothermal atomization

Figure 1: AA-7000 atomic absorption spectrometer with GFA-7000 graphite

furnace atomizer

Figure 2: Comparison of peak profiles of

pyrolytic-coated (6 ppb) and high density

tube (60 ppb)

0.0 25

Pyro tube (Cu: 6 ppb) : High density tube

(Cu: 60 ppb)

Cu 60 ppb

Cu 6 ppb

0.000

0.100

0.200

0.300

0.400

0.500

15


Acid concentration affects sensitivity and reproducibility

Furthermore, variation of theacid concentration has a signifi-cant effect on the sensitivity andreproducibility of the analyticalresult. The effect of nitric acidconcentration on the sensitivityof lead is shown in figure 4.When comparing differentgraphite tubes, the sensitivityvaries more drastically as the acidconcentration changes. In thecase of a pyrolytic-coated tube,however, the analytical result is

Figure 3: Sensitivity comparison of graphite tube types Figure 4: Effect of nitric acid concentration on Pb sensitivity among graphite tube types

(Pb: 5 ppb, 10 μL injection)

easily affected by the acid con-centration in the sample. Com-pared to platform or high-densitytubes, the sensitivity varies moredrastically as the acid concentra-tion changes.

As a feature of the platform tube(Figure 5), the sample solution isinjected onto the platform andhas no direct contact with thewall of the graphite tube. It isthen heated according to the ele-ment-specific heating program.

During electrothermal atomiza-tion, the graphite tube is heated

0 10 20 30 40 50 60 70

Cu concentration [ppb]

Ab

so

rpti

on

0

0.1

0.2

0.3

0.4

0.5

0 2 4 6 8 10HNO3 concentration [%]

Ab

so

rpti

on

0

0.05

0.1

0.15

firstly by the wall. Therefore, in a standard graphite tube such asthe high-density or the pyrolytic-coated tube, the sample is heatedand atomized as the wall is heat-ed. In a platform tube, however,the sample is atomized after thetemperature of the entire tubereaches the atomization tempera-ture, so the sample is atomizedunder optimum temperature dis-

tribution. When measuring withplatform tube, the atomizationpeak becomes broader althoughthe platform material is pyrolyticgraphite (Figure 6).

Platform tube is the effec-tive solution for complexmatrix samples

The platform tube requires thetemperature for ashing and atom-ization to be set at a value of 100°C to 200 °C higher than normalpyrolytic-coated tubes. The dif-ference in the heating characteris-tics of the platform tube in com-parison to a standard tube mini-mizes the influence of matrixeffects of complex samples, so thebackground signal is separatedclearly from the element signal,especially in combination withmatrix modifiers such as palladi-um, iridium, rhodium and others.Use of the platform tube andmatrix modification is thereforethe most effective solution for thedetermination of elements insamples with complex matrix,such as biological sample, wastewater and seawater.Figure 5: Omega platform tube® Figure 6: Comparison of peak profiles

of pyrolytic-coated (6 ppb) and platform

graphite tube (60 ppb)

0.0 25

Platform gra-

phite tube

Pyrolytic coated graphite

tube

Pyro tube (Cu: 6 ppb) : Platform tube

(Cu: 6 ppb)

0.000

0.100

0.200

0.300

0.400

0.500

ble at all or only with consider-able restrictions.

The liver is in the way

During a gall bladder removal,the liver plays an important role,especially due to the anatomicalproximity of both organs. Duringsurgery or simulation, the livermust be held to the side for a rel-atively long time to obtain accessto the gall bladder, which is whya uniform replacement material isrequired that is equipped withsimilar gripping properties. Forthis purpose, tensile strength testswere carried out on pig liver sam-ples in order to determine themechanical material parametersof an average pig liver.


16

Replacement liver for sim

Sylvia Donner, Marc Kraft

TU Berlin, Germany

ZMMS, GRK prometei/

FG Medizintechnik

In medicine and medical tech-nology, animal tissues – inmost cases pig tissue – are

often used for testing products ordevices that are intended for useon humans. Like all natural mate-rials, animal tissue is also subjectto strong fluctuations, and (me-chanical) material parametersvary strongly. Reproducible ex-perimental conditions can there-fore only be guaranteed to a lim-ited extent. Storage conditions,age of the sample and the particu-larity of the animal are some ofthe most important factors influ-encing the properties of animalmaterials.

In order to simulate a so-calledkeyhole surgical procedure – alaparoscopic gall bladder removal– a replacement tissue for the liv-er had to be found. In this inter-vention, the gall bladder posi-tioned under the liver is removedvia three to four small skin cut-tings in the abdominal wall. In

contrast to conventional proce-dures, the abdominal wallremains virtually closed.

An advantage for patients, a challenge for surgeons

Patients benefit greatly from thisprocedure – compared to opensurgical procedures the pain ex-perienced is significantly less, andleads to a better cosmetic resultdue to reduced scarring.

On the other hand, the freedomof movement and vision for thesurgeon is severely limited. Sur-gery is only possible by usingspecialized instruments (Fig. 1).Performing surgery under theseconditions presents physical chal-lenges for the surgeon, some ofwhich can be attributed directlyto the instruments used [2]. Thisis why guidelines for the designof laparoscopic (gripping) instru-ments are currently being devel-oped to take into account notonly the safety of the patient butalso the ergonomic aspects forthe surgeon. However, for ethicalreasons measurements during realsurgical procedures are not feasi-

So far, relatively little researchhas been carried out on materialparameters of animal tissues. Thetechniques described in a stan-dard work by Yamada [5] are nolonger state-of-the-art. Forinstance, Yamada describes tensiletests using suspended weights ona relatively small number of sam-ples. The tensile properties werebased on tensile tests carried outin 1953 on several rabbit livers.Due to the methodology theywere presented in g/mm2. Inmore recent research, these classi-cal tensile tests were abandonedin favor of other methods. Maz-za, for instance, used compres-sion tests on the liver of livingpatients to determine the materialparameters in 2008 [3].

Figure 1: Surgical instruments used in keyhole surgery (‘minimal invasive surgery’)

Figure 2: Shimadzu’s AGS-X universal testing machine specially prepared for the

experiments and used in the tensile tests

17


How to find ‘replacement livers’

In order to determine the materi-al parameters, experiments had tobe carried out. Due to the lack oftest specifications, own test rou-tines had to be developed. Thesewere built upon existing stan-dards, such as DIN EN ISO 527for the determination of tensileproperties of plastics [1]. Thetensile tests were carried out atroom temperature (approximately20 °C), on a 20 N ShimadzuAGS-X universal testing machine(Figure 2).

Clamping a total of 52 standard-ized pig liver samples with por-tions of exterior tissue presenteda challenge. Due to the particularconsistency of the animal tissue,existing clamping devices couldnot be used. The spring gripsused next destroyed the samplesbecause the local pressure loadwas too high. This is why ownclamping devices have beendesigned to clamp all samples(Figure 3). The samples werestretched with the AGS-X uni-versal testing machine at a rate ofone Newton per minute untilrupture. Own MATLAB® algo-

rithms (The MathWorks) havebeen used to analyze the rawdata.

As expected, the stress-straincurves obtained exhibited strongscattering. For the evaluation, itwas therefore necessary to firstlydefine rogue results for which themaximum stress lies outside therange between the lower andupper quarter of the samples.

Of the remaining data sets, meanvalues were established and abest-fit curve, a polynomial ofthe 9th order, was approximated.The curve had a linear range andthe elastic modulus could there-fore be determined as 6.28*10-2

N.mm2 (62.8 kPa) at an elonga-tion of 54 % (Figure 4). Usingthese values, it was possible tofind a replacement material – aPUR-ether foam – possessingsimilar mechanical tensile proper-ties.

The ‘replacement liver’ found thisway is currently used in surgicalsimulations (Figure 5) as well asfor the continued study oflaparoscopic gripping devices.The replacement material there-

fore also serves in additionalstudies to formulate a definedload for the medical instrumentindustry. In this way, it is possi-ble to investigate locking mecha-nisms that enable tissues to beclamped over longer periods oftime without excessive strain. Thetarget is to develop a grippingmechanism that will, on the onehand, meet ergonomic re-quirements for the protection ofthe doctor as well as being easyto apply and to release. On theother hand, a longer clampingduration shall not lead to anydamage of the patient’s tissues.

In contrast to the use of animalmaterial, all studies on the use ofsynthetic materials are repro-ducible. In addition, the resultscan be compared with each other.Realistic replacement materialstherefore offer an importantfoundation for all relevant studies.

Literature

[1] DIN EN ISO 527-1: 2010 Kunst-

stoffe – Bestimmung der

Zugeigenschaften/Plastics –

Determination of tensile proper-

ties

[2] Donner, S.; Kraft, M. (2010):

Potentiale zur verbesserten

Gestaltung von minimalinvasiven

Chirurgieinstrumenten – Auswer-

tung einer Befragung unter

laparoskopisch operierenden

Chirurgen. In: Grundlagen –

Methoden – Technologien/5.

VDI Fachtagung Useware 2010.

Baden-Baden, 13. und 14. Okto-

ber 2010. Düsseldorf: VDI-Ver-

lag, S. 179 -188

[3] Mazza, E.; Grau, P.; Hollenstein,

M.; Bajka, M. (2008): Constitu-

tive Modeling of Human Liver

Based on in Vivo. MICCAI

2008, Part II, LNCS 5242,

2008: 726 -733

[4] Ohara, T (1953): On the com-

parison of strengths of the vari-

ous organ-tissues. J. Kyoto Pref.

Med. Univ., 53: 577-597

[5] Yamada, H (1970). Strength

of Biological Materials.

The Williams & Wilkins Compa-

ny, Baltimore, 1970

Figure 3: Clamped pig liver sample Figure 5: Simulation of a laparoscopic gall

bladder removal using artificial materials

Figure 4: Stress-strain diagram for pig liver: representation of the mean values of the

measurements without outliers (blue), the best-fit polynomial (red) as well as the linear

range (green)

-0.01

0

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0 0.5 1 1.5 2 2.5 3

Strain

Stress-strain diagram for pig liver

Str

es

s i

n N

/mm

2

ulation of keyhole surgery


18

Soft materials in hard drinks Occurrence of endocrine disruptors in foods

Plastics such as polyethy-lene, polypropylene andpolyvinyl chloride are

long-chain polymers. In additionto the chemical composition ofthe polymer, its desired proper-ties also depend on the admixtureof additives. The most widelyused additives are plasticizerssuch as phthalates, bisphenol Aand nonylphenol.

This group of plasticizers con-tains substances with hormone-like properties. These endocrinedisruptors are considered as criti-cal due to their ability to migrateand their toxic potential. It istherefore important to keep anycontamination with endocrinedisruptors as low as possible forrisk groups such as infants, chil-

dren, adolescents and pregnantwomen (ALARA principle*). An overview of the tolerableintake levels of the main phtha-late DEHP – diethylhexyl phtha-late – is presented in table 2.

Plasticizers are everywhere

The phthalic acid esters (phtha-lates) mainly used as plasticizersare found in paints, varnishes,adhesives, plastics, gaskets, cos-metics, toys, packaging and inmedicinal products – to mentionjust a few. Due to the large-scaleuse of phthalates in all types ofplastics, many of these com-pounds are ubiquitous. In housedust they have been detectedrepeatedly [1, 2].

Figure 1: Calibration of DEPH (diethylhexyl phthalate) within the range of 5 pg/μL - 2500 pg/μL

To detect an increased phthalatecontamination, its concentrationis determined using analyticalinstrumentation. For many appli-cation areas, appropriate testingprocedures have been describedand are required by law (e.g. EPA506 (5), DIN EN ISO 18856:2005for water analysis).

For instance, PVC (foils, floor-ing) often contains 20 % to 30 %plasticizers. They can be consid-ered as main components. Analy-sis using FTIR-ATR and GC/MSis easy. The ubiquitous occur-rence (such as DEHP) hardlyaffects the result, as a contamina-tion is only expected in trace-amounts and this is, in the double-digit percentage range,negligible.

Phthalates detectable even in PET beverage bottles

Other plastics, such as PET(polyethylene terephthalate)should not contain any phtha-lates. Due to improper recycling,however, thermal stress or migra-tion from the imprint, phthalatecontamination cannot be exclud-ed. These are mainly trace-amounts that may be releasedinto beverages such as mineralwater or juices. After consump-tion they can be incorporatedinto the body. Table 1 shows theanalysis results of a study on acommercial cola flavor soft drink.Several phthalates could bedetected in trace amounts. Theanalytical determination was car-

0.0 1.0 2.0 [*10 ]̂0.0

2.0

4.0

ID#: 9 Mass: 149.00 Name: Bis(2-ethylhexyl phthalate) DEHP f(x) = 1.699990* + 0.000000 – rr1 = 0.999609 rr2 = 0.999218 Zero Through: Through[*10 ]̂

19


ried out using GC/MS after sam-ple preparation. The small amountof analyte requires a high sensi-tivity of the instrumental system.The combination of Shimadzu’sGC-2010 Plus with the QP-2010Ultra MSD easily meets this re-quirement.

Figure 1 shows the ubiquitouspresence of the phthalate DEHPusing a calibration curve. The lin-ear range of the detection methodis limited by the carry-over oftrace components in the lowerconcentration range, despite theuse of high-purity grade labora-tory chemicals. Special precau-tions during sample preparationas well as in the selection ofmaterials and laboratory chemi-cals are therefore needed to pre-vent erroneous measurements asmuch as possible.

In the usual representation of alinear calibration curve – particu-

larly over a wide range – system-atic deviations are hardly recog-nizable. In contrast, the represen-tation of the ratio area ratio/con-centration against log concentra-tion in figure 1 shows that a linear relationship is no longerpresent from approximately

Figure 2: Example of a SIM chromatogram of a phthalate mixture, approx 2.5 ng/μL;

Conditions: split ratio 1: 25, septum Supelco Thermogreen LB2, column Macharey &

Nagel Optima delta 3; 10 m x 0.1 mm ID x 0.1 μm film thickness

0.075 mg/L. Here, the overallDEHP contamination becomesevident. The detectable unilateraldeviations from the horizontalthat can be determined in thelower concentration range arecaused by the “blank content.”

Strategies for the preven-tion of phthalate contamina-tion

In principle, the determination ofphthalates in trace quantitiesrequires the use of phthalate-freeinstruments and materials – usu-ally glass and aluminum [3].These materials can be cleanedeasily using thermal decontami-nation [4].

Suitable methods have beendescribed in the DIN EN ISO18856 :2005 standard. Solventsmust be tested prior to use todetermine if they are free fromphthalates. Septa and liners mustmeet these requirements as well.Additional measures such assilanization (hexamethyldisila-zane) also help in reducing themeasurement uncertainty. Byusing fast GC, multiple determi-nations can be realized withinshort analysis times (see Fig. 3).

3.0 4.0 5.0 5.8

(nm)

Intensiy 3,445,588 TIC

ID# Analysis Result Unit Method

12456789

10

Dimethyl phthalate (DMP)Diethyl phthalate (DEP)Nonylphenol (NP)Di-n-butyl phthalate DBPBisphenol ABis(2-ethylhexyl) adipate DEHABenzyl butyl phthalate BBPBis(2-ethylhexyl) phthalate DEHPDi-n-octyl phthalate DNOP

0.651.070.971.55N.D.1.280.251.870.46

μg/Lμg/Lμg/Lμg/Lμg/Lμg/Lμg/Lμg/Lμg/L

GC-MSGC-MSGC-MSGC-MSGC-MSGC-MSGC-MSGC-MSGC-MS

Institution TDI’s Value (μg/kg KG/d) Year

NL-RIVMUS-EPAWHOEU-CSTEEECB/EU

D-UBABfR andEFSA

TDIaRfDcTDIaTDIaTDIa

TRDdTDIa

a: Tolerable daily intakeb: Maximum permissible risk level c: Reference dose (for chronic exposure)d: Tolerable resorbed dose e: Minimal risk level (for chronic exposure duration)

420253720 (Infants 0 - 3 months and women of childbearing age)25 (Infants > 3 - 12 months)48 (Rest of the general population)5050

20021991200319982004

20032005

Endocrine disruptors

Endocrine disruptors occupy specific receptors, replace hormones and upsetthe hormone balance in the human body. This can lead to impairment ofgrowth, development and reproductive processes. Examples are develop-mental disorders, birth defects, premature puberty in girls, reproductive prob-lems as well as a decrease in sperm count.

Chromatographic conditions:

Shimadzu GC QP-2010 Plus withMSD QP-2010 Ultra, columnMacherey & Nagel Optima delta-3 -0.10 μm 10 m x 0.1 mm ID, split 1 :25,purge 3 mL/min, linear velocity 30 cm/s, temperature program 130 °C - 0.2 min, 40 °C/min to 270°C, 60 °C/min to 320 °C - 3.0 min

Table 1: Content of phthalates, bisphenol A and nonylphenol in a cola-flavored soft drink

in a polyethylene (LD-PE) bottle (excerpt from analysis report)

Table 2: Overview of tolerable intake levels of DEHP(6)

References

[1] Fromme, H., et al., et al.

Occurence of phthalates and

musk fragrances in indoor air

and dust from appartments and

kindergartens in Berlin (Ger-

many). Indoor Air, 2004, 14,

188-195

[2] Scharf, S., Uhl, M. and Hohen-

blum, P. Hausstaub – Ein Indika-

tor für Innenraumbelastung.

Umweltbundesamt Österreich,

Wien, 2004, 258

[3] Brüll, U., Alberti, J. and Furt-

mann, K. Phthalatanalytik in

Wasser und Sediment. Lan-

desumweltamt NRW, 45023

Essen

[4] Saido, Katsuhiko, et al., et al.

Thermal Stability of Phthalic

Esters. Journal of the American

Oil Chemists’ Society, 1984,

(61[5]), 945-949


20

Glass on the road increases driv

“To see and be seen” in roadtraffic means more safetyfor all. At night, reflectors

improve the visibility of non-selfilluminating objects and roadusers. Roughly three types of re-flection occur: diffuse reflection,mirror reflection and retroreflec-tion.

In diffuse reflection, the mainbackscatter takes place perpendi-cular to the material, independentof the incident direction of theradiation. Examples are milk,wall paints or paper. In mirrorreflection, the reflected lightretains its parallelism. The angleof incidence is the same as theangle of reflection.

In the case of retroreflection, asmuch as possible of the lightemitted from the source is re-flected back to its origin. Lightfrom a car headlight, for example,is reflected toward the driver’seye. The principle of retroreflec-tion is applied successfully intraffic signs and pavement mark-ings.

the thinly applied and still wetpaint to attain the desired visibili-ty, also at night.

There are, however, problemswith these types of pavementmarkings under wet road condi-

under wet road conditions in thedark. Indeed, there are two differ-ent types of markings.

Marking techniques in the pastused glass beads and antiskidmaterials that were scattered onto

Glass beads in retro-reflective materials

One possibility for the manufac-ture of retroreflective materials isthe embedding of small glassbeads within the surface of thematerial. In addition, the backsurface of these beads can becoated with a reflecting layer. Assoon as light hits the beads, theincident light is reflected back inits original direction due to thedual refraction within the bead.

Pavement markings often seem tobe visible to varying degrees

Figure 1: Diffuse, mirror and retroreflection

Figure 2: Performance of different marking types on a dry or wet road

Particle measurement of glass beads

Figure 3: Size distribution of the glass beads

tions, when a thin film of watercovers the road as well as themarkings. The water film reducesretroreflection and decreases visi-bility of the markings at nightconsiderably in wet conditions.

Literally outstanding – a new marking method

For these reasons a new markingmethod has been developed thatensures good visibility, also atnight and under wet conditions.This method has, therefore, large-ly replaced all conventionalmethods.

Virtually all markings of the old-er type have meanwhile beenreplaced on German highways.

Markings of the newer type havebeen applied on approximately 70 % of all German roads. Theyprotrude from the water film onthe road by several millimeters,ensuring that retroreflection con-tinues to occur within the glassbeads.

Particle measurement of the glass beads

The beads typically exhibit adiameter of approx. 50 μm. Inorder to verify the size distri-bution of the beads, particlemeasurement was carried outusing Shimadzu’s SALD-7101.

The glass beads were suspendedin water using several drops of atenside. In order to prevent thebeads from sinking to the bottomof the measuring cell (BC-71),they were kept in motion using astirrer.

As can be seen from the spectrumand as expected, a size distribu-tion of the beads around 50 μmcould be measured. Using the

1 5 10 50 100 500

Particel diameter (μm)

No

rma

lize

d p

art

ice

l a

mo

un

t (C

um

/Dif

f)

0

10

20

30

40

50

60

70

80

90

100

0

10

20

30

40

50

60

70

80

90

100Q3 (%) q 3 (%)

12

47.14647.957

44.66844.668

47.24548.718

0.1340.123

1.80 - 0.00i2.00 - 0.00i

#

blackred

ColorMedian D

[μm]

Modal D

[μm]

Mean D

[μm]Std. dev. Ref. Index

21


ing safety

SALD-7101 system including theBC-71, it was possible to reliablymeasure the size distribution ofglass beads.

0.00.10.20.30.40.50.60.70.80.91.01.11.21.31.41.5

2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5

cle lenses, household equipmentand in protective helmets – wher-ever durable or long-lasting mate-rials are needed. On the otherhand, BPA-containing epoxyresins are used as internal coat-ings in many steel cans for foods


22

Almost omnipresent

Troisi Jacopo, Di Fiore Raffaele

Laboratorio Chimico Merceolo-

gico, Az. Spec.

CCIAA Napoli, Italy

Palumbo Giancarlo

Università Federico II, Dottoran-

do di Ricerca in "Scienza dell'Ali-

mentazione e della Nutrizione”,

XXV ciclo, coordinatore:

Prof. Franco Contaldo, Tutor:

Prof. Maria Valletrisco

Bisphenol A (BPA) is mainlyused in the production ofplastics; its by-products

have been commercially availablefor over 50 years. BPA is alsoapplied in the synthesis of poly-esters, polysulfonates, as antioxi-dant in some plasticizers and asinhibitor against PVC polymer-ization.

Above all, however, BPA is thekey monomer in the productionof epoxy resins and the majority

Determination of bisphenol A in plastic packaging

and beverages. BPA is also a pre-cursor of flame-retardants andhas been used in the past as a fun-gicide.

Suspected since the 1930’s to beharmful to humans, concernsregarding the use of BPA-con-taining plastic materials have in-creased considerably in 2008, asmany governments commissionedstudies on the safety of BPA.Some distributors have with-drawn products containing BPAfrom the market. BPA seems tobe mainly responsible for numer-ous impairments in male andfemale sexual development duringthe fetal stage, as well as a declinein fertility during adulthood.

BPA and hormonal activity

Worldwide production of BPAwas estimated at two million tons

of polycarbonates. Polycarbon-ates are transparent and virtuallyunbreakable and are thereforefound in numerous childrensproducts as well as in bottles,sports goods, health care, dentaland optical equipment, in specta-

Figure 1: Gas chromatogram (TIC) of a BPA and BPA-d16 standard

0.05.0

10.015.020.025.030.035.040.045.050.055.060.065.070.075.080.085.090.095.0

100.0105.0

% Base Peak 224/1, 643,137

50.0 75.0 100.0 125.0 150.0 175.0 200.0 225.0 250.0 275.0 300.0 325.0

0.02.55.07.5

10.012.515.017.520.022.525.027.530.032.535.037.540.042.545.047.550.052.555.0

% Base Peak 213/1, 251,33

50.0 75.0 100.0 125.0 150.0 175.0 200.0 225.0 250.0 275.0 300.0 325.0 350.0

0.00

0.25

0.50

0.75

1.00

1.25

1.50

1.75

2.00

(x 10)

0 100 200 300 400 500 600 700 800 900 1,000

Conc. ratio

Are

a r

ati

o

23


in 2010. BPA alters the activity ofthe endocrine system by activat-ing hormone receptors. In case ofelevated doses, this can have neg-ative consequences for health.Experimental studies have shownthat BPA mimics the activity ofestrogen (essential for brain de-velopment) to such an extent thateven the smallest doses can com-pletely inhibit the activity ofestrogen and its effects on neu-ronal growth. Although BPA-based polymeric packaging mate-rials are considered to be stableunder normal circumstances, theycan still release small but toxico-logically significant amounts ofBPA so that an increased riskexists that beverages or foods arecontaminated with this substance.

BPA has also been associated withthe development of numerousother clinical conditions of thereproductive organs, the prostateand the female breast. The studiesconducted, show clear evidence of

endocrine effects and make it pos-sible to define a daily maximumrecommended dose of 0.05 mg/kgbody weight [1].

Long-term risks cannot be ruled out

In September 2008, the US Na-tional Institute of EnvironmentalHealth Sciences issued a brief forthe assessment of human healthrisks that is based on the result ofthe complex points of contactbetween food, consumer productsand living conditions [2]. Thedocument compared the effects ofBPA in experimental studies andthe associated ‘dosage-response’data with existing information onthe level of risks of human expo-sure to the compound, includingthe limited epidemiological stud-ies and biological monitoring car-ried out to date.

The conclusions rule out any riskto reproductive health and preg-

Figure 3: GCMS calibration curve of BPA

Figure 2: Mass spectra of BPA (a) and BPA-d16 (b)

nancy in adults. Nevertheless, theconcerns on long-term risks tothe endocrine, neurological andreproductive development as aresult of exposure in utero and/orduring childhood remain.

As of 28 January 2011, Europehas banned BPA-containing babybottles according to the EuropeanUnion directive 2011/8/UE. Since1 March 2011, the manufacture ofBPA-containing baby bottles hasbeen prohibited and from 1 June2011, this ban also applies to saleand import.

Research group poly-carbonate/BPA

The American Chemistry Coun-cil, Plastics Europe and the JapanChemical Industry Associationhave joined forces to form a poly-carbonate/BPA research group(www.bisphenol-A.org). Thisgroup has also defined the deter-mination of BPA under differentconditions (environment, biologi-cal and plastic materials) [3].According to these criteria, gaschromatography coupled to massspectrometry (GC-MS) is themethod of choice for the determi-nation of BPA, whereby confir-mation is based on the ratio ofthe signals at 228 m/z and 213

a

b

m/z. Nevertheless, a large part ofscientific work published to datehas reported the use of HPLC-MS-MS methods for the determi-nation of BPA. This discrepancymay be due to the need to detecteven the smallest amounts, whichhas been difficult to achieve withthe GC-MS systems.

Materials and methods

Two methods for the determina-tion of BPA are presented: a GC-MS method using a GC-MS-QP2010 system and an LC-MSmethod using the single QuadLCMS-2010 system. In both casesthe detection limit is approxi-mately 0.1 μg/L. �

Conventional diffraction grating

Low stray light diffraction grating

LO-RAY-LIGHTM

Al mirror(reference data) Groove density : 1200 grooves/mm

Blazing wavelenght : 250 nmGroove density : 1800 grooves/mm

Blazing wavelenght : 250 nm

Conventional diffraction grating

Low stray light diffraction grating

LO-RAY-LIGHTM

Al mirror(reference data)

10-7 10-510-6 10-7 10-510-6

Stray light values Stray light values

2,000

2,500

3,000

3,500

4,000

4,500

5,000

5,500

6,000

6,500

7,000

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0

Minutes

BPA determination usingGC-MS

The GC-MS method is based ona Supelco® SLB®-5ms columnwith a length of 15 m and aninternal diameter of 0.1 mm witha film thickness of 0.1 μm. TheGC oven program includes aninitial phase of 1.5 minutes to 160 °C and two heating rates:first 20 °C/min up to 260 °C,then 40 °C/min to 320 °C. Theinjection temperature is 260 °Cand the linear carrier gas velocity(helium) is 50 cm/sec. This set-ting requires a pressure of morethan 700 kPa and a linear flow of0.86 mL/min. The total cycletime is 8 min; the maximumretention time is 5.9 min.


PRODUCTS Shimadzu News 1/2012

24

Figure 1: Representation of stray light and comparison between a conventional grating,

a LO-RAY-LIGH grating and an aluminum reference

Argus eyesNew class of UV-VIS double-beam spectrophotometers

Shimadzu’s role as world-wide market leader in UV-VIS double-beam spec-

trophotometers means continu-ous challenges for R&D to meetthe high expectations of usersfrom all over the world and to setstandards in instrument develop-ment. Shimadzu designs not onlycomplete analytical systems butalso the optical components for a

spectrophotometer. This articlefocuses on a patented develop-ment in diffraction grating tech-nology.

New holographic exposuremethod

Shimadzu has optimized thequality of the edges for the angleof reflection in the sawtooth pat-

tern using a new holographicexposure method. This LO-RAY-LIGH® principle is a patenteddevelopment that produces anoutstanding grating quality. Untilnow, the generally accepted rulewas: the more lines to a grating,the sharper the spectral image.With the LO-RAY-LIGH® grat-ing technology, this has changed.The production process of thegrating results in a high gratingimage precision which also yieldsa spectral resolution sharpnessrendering better characteristicswhen compared to a conventionalgrating.

The objective in the developmentof these gratings was a significantreduction in stray light.

Sharper image

Figure 1 shows the stray lightvalues for a conventional gratingand the LO-RAY-LIGH grating.In the logarithmic scale it isapparent that the LO-RAY-LIGH grating enables an excep-tionally high reduction of straylight.

For the qualification of a grating,the intensity of the first-orderlight is plotted against delta (nm).

From figure 2 it becomes evidentthat the LO-RAY-LIGH gratingproduces first-order light muchmore sharply and with less straylight over the entire range. In thediagram, a grating of groove den-sity 1,200 grooves/mm was com-pared with a blaze wavelength of250 nm.

New optics improve quality

The quality of the optical compo-nents becomes noticeable duringthe measurements. Shimadzu’sUV-2700 spectrophotometer isequipped with double monochro-mator optics containing two ofthese new gratings. The effect issignificant. An example is the lin-earity measurement of a KMnO4dilution series where the differ-ence to Shimadzu’s previous UV-2550 model from the sameclass becomes evident. While theUV-2550 already exhibits astrong noise at six absorptionunits, the new UV-2700 UV-VISspectrophotometer featuresexceptional linear characteristicsup to eight absorption units.

With respect to the number ofgrid lines, the UV-2550 exhibits1,600 lines/mm and the new

These fast analysis times requirehigh data acquisition rates. Twodata acquisition channels wereused, one in the SIM mode andthe other in the SCAN modebetween m/z = 50 and m/z = 500.The so-called ‘event time’ for theSCAN mode was set to 0.1 sec(corresponding to a scan rate of5000 amu/sec, half of the maxi-mum rate attainable using the QP-2010). In the SIM mode, theions m/z = 213 and m/z = 228 forBPA and m/z = 224 for the inter-nal standard were measured.

Calibration was carried out usingeight standard solutions at a con-centration of 0.1 μg/L up to 1,000μg/L. This method has beenproven to be extremely reliable.Figure 4: LCMS chromatogram of a BPA and BPA-d16 standard

400.00 500.00 600.00 700.00(nm)

Ab

s.

0.000

2.000

4.000

6.000

8.000

9.000

400.00 500.00 600.00 700.00

(nm)

Ab

s.

0.0

2.0

4.0

6.0

8.0

9.0

Groove density : 1200 grooves/mmBlazing wavelenght : 250 nm

-60 -40 -20 0 20 40 60

� � (nm)

Inte

ns

ity

(n

orm

ali

zed

at

inte

ns

ity

of

firs

t o

rde

r li

gh

t)

10-7

10-6

10-5

10-4

10-3

10-2

10-1

1

requirement for the analysis ofsamples from different origins.Since the first investigations havebeen carried out, BPA appears tobe widely present in many every-day objects – even though in lowconcentrations. Only throughefficient quantification of BPA

0.0

0.5

1.0

1.5

2.0

2.5

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4

Area ratio [*10^1]

Conc. (ratio) [*10^3]

We will gladly send you additional infor-

mation. Please enter the corresponding

number on the reply card or order via

Shimadzu’s News App or

News WebApp. Info 402

25


PRODUCTSShimadzu News 1/2012

Figure 5: LCMS calibration curve of BPA

Figure 4: UV-VIS spectra of a potassium permanganate dilution series of 6 to 1 absorp-

tion units measured using the UV-2550 equipped with conventional gratings

Figure 3: Measurement of a KMnO4 dilu-

tion series of 8 to 1 absorption units

using the UV-2700 spectrophotometer

equipped with a double monochromator

and LO-RAY-LIGH grating

Figure 2: Representation of the intensity of the first-order light as a function of the

distance to the origin of the wavelengths in nm

UV-2700 instrument exhibits1,300 lines/mm. This reducedgrid line number results in bettermeasurement characteristics forthe new grating compared withthe conventional grating featuringa higher grid line number.

With this objective in mind whendeveloping the instrument, theUV-2700 is recommended forapplications with high absorp-tions, high linearity and low straylight:

• polarizing films• transmission of functional films• thickness determination of thin

films• protein and nucleic acid deter-

mination• environmental analysis –

quantification of inorganiccompounds in water

• food analysis – quantificationof vitamins, food additives andminerals.

Measurement was performedusing an internal deuterated stan-dard (BPA-d16).

Determination of BPA usingHPLC-MS

The HPLC-MS method, on theother hand, is based on a 50 mmx 2 mm Shim-Pack XR-ODS col-umn. Chromatographic separa-tion is carried out using a water/methanol gradient. The gradientprogram includes an initial phaseof one minute using 20 % metha-nol with an increase up to 50 %within 5 minutes, then an addi-tional ramp up to 100 % in a fur-ther five minutes, followed by areturn to the initial conditions(20 % methanol) in a further two

minutes, and finally an equilibra-tion of three minutes. The totalanalysis time is 15 minutes. Theflow rate is 0.2 mL/min.

Also in this case, data acquisitionwas carried out in the SCANmode (measuring negative ions)between m/z = 180 and m/z =480 as well as in the SIM mode(negative ions) m/z = 227.20 andm/z = 242.30. The ion source wasadjusted to - 3.5 kV, the desolva-tion line to 250 °C. Flow rate ofthe nebulizer gas was 1.5 L/min.

Conclusions

The possibility to quantify BPAwith increased sensitivity in bothGCMS and LCMS is an essential

present in various sources will itbe possible to assess the actuallevel of risk to humans and theeffect of BPA as an endocrinedisruptor.

References

[1] EFSA panel on food additives,

flavourings, processing aids and

materials in contact with foods –

Efsa Journal Jan 2007doi:

10.2903/jefsa.2007.428

[2] NTP-CERHR monograph on the

potentional human reproductive

and developmental effect of

Bisphenol A – NIH Publication

No.08-5994 Set 2008

[3] www.bisphenol-a.org/pdf/

criteria_102002.pdf

-0.50.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.0

uV (x 100,000)

0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0 55.0

Minutes


26

Determination of minervegetable oils

Manos Barmpounis,

Dimitris Georgantas

Applications Department of

N.Asteriadis S.A., 31 Dervenion

Str. & Poseidonos Str.,

144 51 Metamorfossi – Athens,

Greece,

e-mail: [email protected]

Vassiliki Panagiotopoulou,

Vassilis Tzamtzis

GeneralChemicalState Labora-

tory, 16 An. Tsocha Str.,

11521 Athens

Today food contaminationthrough paraffins of min-eral oil origin happens

rather often. Although it is diffi-cult to identify the exact sourcesof contamination, it is wellknown that many occur due tothe wide range of applications ofthis petroleum derivative in manyfields. Packaging additives, lubri-cants, cosmetics additives and

pesticides can release paraffinwaxes and oils. As a result, manycases of mineral oil contamina-tion in food relating to edibleoils, bakery products, packagedfoods and recycled cartons arereported in literature.

The presence of mineral oils infood can produce undesired toxi-cological effects. Acceptable dailyintakes have been established bythe Joint FAO/WHO ExpertCommittee on Food Additives(JECFA) as 0 - 20 mg/kg bodyweight for high viscosity mineraloils, 0 - 10 mg/kg body weight forlow and medium density class Imineral oils and 0 - 0.01 mg/kgbody weight for low and mediumdensity class II and class III min-eral oils [1].

In spring 2008, nearly 100,000 tof sunflower oil were found to becontaminated in Ukraine withmineral oil at concentrations

often above 1,000 mg/kg. Fol-lowing this discovery, the Euro-pean Commission with DGSANCO requested the nationalfood authorities to withdraw thecontaminated Ukrainian oil fromthe market. In June 2008, anadministrative limit of 50 mg/kgwas specified for mineral paraf-fins in crude as well as in refinedUkrainian sunflower oil.

The contaminated sunflower oilcase highlights the importance ofhaving a reliable, sensitive andsimple method to determine min-eral oil hydrocarbons in vegetableoils.

Principle

The saturated hydrocarbons ofthe sample are isolated by silicagel column chromatography anddetermined by capillary gas chro-matography using an InternalStandard Method.

Figure 1: Chromatogram of a standard sample

-2.00

0.00

2.00

4.00

6.00

8.00

10.00

12.00

14.00

0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00

0.00

0.25

0.50

0.75

1.00

1.25

1.50

1.75

uV (x 100,000)

10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0

Minutes

al oil hydrocarbons in

27


Apparatus• Erlenmeyer flasks, 25 mL• Test tubes with glass stop• Glass column for liquid chro-

matography, internal diameter(15 - 20 mm), length 30 - 40 cm,fitted with suitable frit andstop-cock

• Micro-syringe for on-columninjection, 10 μL, with a hard-ened needle

• Rotary evaporator• Analytical balance for weighing

to an accuracy of within 0.1 mg• Usual laboratory glassware

(variable volume automaticpipettes etc.)

Instrumentation• Gas chromatograph Shimadzu

GC-2010 Plus, OCI/PTV-2010injector and FID-2010 Detector

• Capillary column (MEGA1, 10 m, 0.25 mm ID, 0.1 μm filmthickness)

• Pre-column 0.53 ID 5 m deacti-vated (MEGA retention gap,0.53 ID)

Temperature program• PTV: 65 oC (5 min), 30 °C/min

to 180 °C, 6 °C/min to 350 andhold 40 min

• Column Oven: 65 °C (5 min), 5 °C/min to 350 and hold 10 min

Figure 2: Chromatograms of standard samples (data comparison) Figure 3: Calibration curve in mg/kg

Figure 4: Chromatogram of a blank sunflower oil

• FID temperature: 360 °C• Total flow: 5.5 mL/min• Carrier gas: Helium

Reagents• Silica gel: Silica gel 60, Merck

extra pure for column chro-matography (code 107754).Activated at 300 °C for 24 hand cooled to room tempera-ture in a desiccator

• n-hexane for chromatography(Unisolv by Merck is pro-posed).

• Internal standard, n-eicosane 20 μg/mL, by dilution from thestandard 2000 μg/mL in isooc-tane (Neochema Cat. No14700-0230).

• Paraffin oil, Merck, ProductNumber 1.07160 (for calibra-tion curve).

• 0.2 g of paraffin oil is diluted in100 mL of hexane, creating a2,000 mg/L standard �

-0.1

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1

1.2

1.3

1.4

1.5

1.6

5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0 55.0

uV (x 1,000,000)

Minutes

-0.50.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.0

0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0

uV (x 100,000)

Minutes


28

1,000mIS

m �c =

Aparaf

AISb

a �

Figure 6: Integration of peaks of the spiked sample

Table 1: Validation data of the method

50150

mg/Kg

43

RSD %

86

Uncertainty % (k = 2)

99 %99 %

Recovery

Figure 5: Chromatogram of a spiked sample integrated as single peak

Procedure – Sample pretreatment

Suspend 15 g of the above pre-pared silica gel in excess n-hex-ane, stir thoroughly with glassrod and introduce into the col-umn. Allow to settle while gentlytapping the silica by passing smallquantities of n-hexane in order tomake the chromatographic bandmore homogeneous. Weight 1 gof the sample into a 4 mL vialwith screw cap, add 1 mL of theinternal standard solution andmix thoroughly [2].

The sample solution should betransferred to the chromato-graphic column with the aid oftwo 1-mL portions of n-hexane.After introduction of the sampleinto the silica, the mineral oil hydrocarbons are eluted using 50 mL of n-hexane. Eluent flowshould be set to 10 drops/15 sec.The resultant fraction is collectedin a 100 mL Erlenmeyer flask andevaporated in a rotary evaporatoruntil the solvent volume isapproximately 1 mL. The rest ofthe solvent is removed by nitro-gen stream until dryness. Theisolated mineral hydrocarbonsare transferred to a 2 mL auto-sampler vial using 500 μL of hep-tane.

Finally, inject 1 to 4 μL of thesolution in the gas chromato-graph. As can be seen in Figure 1,

mineral oil hydrocarbons pro-duce a broad unresolved peakusually referred to as “hump”[3,4,5,6] instead of forming sharppeaks in GC. The hump and theISTD peak are integrated to create the calibration curve (Fig-ure 3). In the case of an unknownsample, the hump and peaks areintegrated as a single peak(resolved and unresolved peaks)using the manual integration barof the GCsolution program (Fig-ure 5).

All resolved peaks and the ISTDpeak (above the hump) are thenintegrated using the proper driftin the GC solution program (Figure 6). Using a calculator, thearea of the hump (unresolvedpeaks) is determined by subtract-ing the area of resolved peaksfrom the area of resolved andunresolved peaks.

The mineral concentration is calculated using the followingequation:

where: c = mineral concentration

(mg/kg)m = weight of the sample (g)mIS = weight of the internal

standard (mg)Aparaf = area of the mineral oil

AIS = area of the internal stan-dard

A = slope of the calibrationcurve

b = y-intercept of the line

Validation data

Two spiked samples of concen-tration of 50 and 150 mg/kgrespectively were used. Six repe-titions of each sample were per-formed and the results are shownin table 1. The limit of detection(LOD) was found to be 10 mg/kgand the limit of quantitation(LOQ) was found to be 20 mg/kg.

Conclusions

Mineral oil hydrocarbons weresuccessfully determined by theabove method, using a ShimadzuGC-2010 Plus gas chromato-graph. The method showed goodrepeatability and linearity.

References

[1] JECFA (Joint FAO/WHO Expert

Committee on Food Additives)

(1995) Summary of Evaluations

Performed by the Joint FAO/

WHO Expert Committee on

Food Additives

www.inchem.org/documents/

jecfa/jeceval/jec_1655.htm

[2] Official Method for stigmatadi-

enes and waxes, Regulation

EEC 2568/91

[3] L. Karasek, T. Wenzl, F.

Ulberth, EUR 23811 EN – Joint

Research Centre – Institute for

Reference Materials and Mea-

surements, Proficiency test on

the determination of mineral oil

in sunflower oil

[4] M. Biedermann, K. Grob, Eur.

J. Lipid Sci. Technol. 111

(2009) 313-319, How “white”

was the mineral oil in the con-

taminated Ukrainiansunflower

oils?

[5] D. Fiorini, A. Paciaroni, F. Gigli,

R. Ballini, Food Control 21

(2010) 1155-1160, A versatile

splitless injection GC-FID

method for the determination

of mineral oil paraffins in veg-

etable oils and dried fruit

[6] K. Grob,M.Vass, M. Bieder-

mann, H-P Neukom, Food Addi-

tives and Contaminants 18

(2001) 1-10, Contamination

of animal feed and food from

animal origin with mineral oil

hydrocarbons

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