3D-D

arstellung

, NASA

/JPL

-Caltech

World Premiere: AIM-9000

Multipurpose tool for tiny objects

lab4you: Anti-aging for batteries?

LC-MS method development for batteryelectrolytes

No carry-over: TOC determination

Wastewater treatment plant: high demandson equipment and sampling method

Pharmaceutical

Plastics and Rubber

CONTENT

Clinical

Chemical, Petrochemical,Biofuel and Energy

Manipulated steel – bi-axial defor -mation experienced by steel 2

No carry-over: TOC determination in process analysis – Wastewatertreatment plant: high demands onequipment and sampling method 8

Metallic contaminations in pharma-ceutical packaging – Content andimplementation of USP 661.1 withrespect to extractable elements 15

APPLICATION

Salty catalyst? – TOC analysis of salt-containing samples 10

Anti-aging for batteries? – LC-MS method development for battery electrolytes 12

Pyrolysis GC-MS user meeting – October 6th 2016, at Shimadzu’sLaboratory World, Duisburg,Germany 14

Healthy fat in chips and sausages? –A new method for extraction, digestion and analysis of fat in foodsamples 16

LATEST NEWS

MARKETS

Discover the future of LC-MS/MS –CLAM-2000 – fully automated samplepreparation module particularly forbiological applications 4

Multipurpose tool for tiny objects –AIM-9000 infrared microscope: automated microanalysis for conta-minations and microparticles

6

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SHIMADZU NEWS 2/2016

A s the low hanging light-weighting fruits are picked,automotive manufacturers now have to work that bit harderto shed the pounds. BMW is per-haps one of the more progressiveexamples of a manufacturer driv-ing composite R&D in a majorway. However, it is not simplyabandoning metal, and like manyothers, it is seeking out ways tolightweight vehicles using conven-tional steel.

This has led to BMW Mini initiat-ing a project between The Univer -sity of Oxford and DiamondLight Source – the UK’s nationalsynchrotron science facility locat-ed at the Harwell Science andInnovation Campus in Oxford -shire. The facility harnesses thepower of electrons and X-rays tohelp scientists and engineers gainnew insight and understandinginto the microscopic and internalstructures of materials.

BMW Mini wants answers to aphenomenon that has been wit-nessed, more broadly, since the1950s. It is seen when parts arestamp formed, a common automo-tive process, to make everythingfrom bonnets to doors.

The problem is, for any stampingprocess that is completed in morethan one stage, the deformationbecomes highly complex, particu-larly for a pressed part with strainapplied in two-axes. This resultsin a non-homogeneous arrange-ment of the crystalline and micro-scopic defect structures.

Recreating the bi-axial deformationexperienced by steel during stampforming is allowing researchers toengineer the microstructure and yieldlighter parts

Manipulatedsteel

Food, Beverages, Agriculture

Environment

Automotive

Universal testing machine AGS-X

APPLICATION

3SHIMADZU NEWS 2/2016

While it may sound arbitrary, theaffect can influence grain mor-phology, crystal orientation anddistribution – all of which havesignificant impact on mechanicalproperties, including most impor-tantly, how much the material willstretch before it fractures.

Leading the work is Dr DavidCollins, a researcher at the Uni -versity of Oxford, who explainsthe problem. He says: “They can’tstamp form stronger steels at themoment because of this affect.The metals they use on body pan-els are actually quite weak, say 10 % as strong as the strongeststeels on the market. Strongermetals are not ductile and can’t be stamped into the complicatedshapes needed.”

It means thicker sections have tobe used, so panels end up weigh-ing more. It’s something BMW iskeen to understand so it can ulti-mately reduce the weight of steelchassis.

The problem is complex, meaningtesting and analysis is far fromstraightforward. The deformationis bi-axial, meaning any obvioustraditional solution is not suitable.It led to Dr Collins applying touse the synchrotron to carry outtests and shed new light, or X-rays in this case, on the problem.How ever, he soon faced anotherproblem, the Shimadzu AGS-X 10 kN load frame that was avail-able, was a single axis machine.

Dr Collins decided to do what somany engineers have done beforehim – innovate. He designed amechanism to generate the bi-axialdeformation needed, which alsobolts straight on to the Shimadzumachine.

“I’ve built a mechanism that usesthe power of the load frame togenerate the stresses,” he says.“There are four diagonal rods atthe corners to change the angleand determine the ratio of howmuch it is deforming in eachdirection. I can make it morebiased towards the horizontal,vertical or even one side.

“We can tell BMW things like‘texture is important’, but wedon’t yet know how to optimizeit,” says Dr Collins. “Eventually,however, we hope to get enoughof an idea about the affect toapply it to different materials.We’re sticking with steel for themoment as we don’t want to endup blurring the problem withother complexities. But, there isno reason why this research couldnot be limited to any alloy.

For a lot of metals, no one hasproved if this phenomenon even

“I spent time in the workshopmachining all the componentsmyself. I started from scratch andit was a very steep learning curve.I broke a lot of tools and upset acouple of people, but I was verydetermined to make the mecha-nism and run the tests.”

The request to use the synchro-tron was successful and four daysof testing commenced. The resultsshow a series of concentric rings,with each ring corresponding to asignal coming from individual lat-tice planes. The shape of theserings, and their radius, gives animportant microscopic insight. “If you deform a bit of material,those rings change in diameter,”he says. “And you can then meas-ure how much strain is beingtaken up by individual planes. Soyou capture what is happening onthe atomic planar level.”

The cross shaped specimen is 1 mm thick, but in the centre thisis reduced to just 300 µm. If it wasa uniform thickness, the testwould simply pull the arms off. A cross is machined in the middle,which steps down to a thinner cir-cular cross section just 300 µmthick.

“One thing you can see is howstrain is being accommodated inthe individual grains based ontheir orientations,” says DrCollins. “You can monitor thecentre on a macroscopic level byputting a camera in front of therig. But with X-rays, they tell youwhat the strain is, on all of theindividual crystals.”

With the tests complete, the hardwork begins. After four intensedays of gathering data, it could bea year or more of analysis to findconclusive explanation of the bi-axial deformation phenomenon.What is known is that ‘materialtexture’, i.e. the orientation ofgrains in the material, has a bigeffect on the ductility of the mate-rials and how strain is accumulat-ed. As all sheet materials arerolled during production, it forcesmost of the crystals to align inone orientation.

exists or not, so there might bemassive benefits with lots of dif-ferent applications outside theautomotive world.”

Originally published in Engineering Materials

Magazine, Spring 2016

www.materialsforengineering.co.uk

AuthorJustin Cunningham

University of Oxford

The Diamond Light Source

Diamond Light Source is the UK’ssynchrotron. It works like a giantmicroscope, harnessing the powerof electrons to produce bright lightthat scientists can use to study any-thing from fossils to jet engines toviruses and vaccines.

The machine speeds up electrons tonear light speeds so that they giveoff a light 10 billion times brighterthan the sun.

These bright beams are then direct-ed off into laboratories known as‘beamlines.’

Here, scientists use the light tostudy a vast range of subject matter,from new medicines and treatmentsfor disease to innovative engineer-ing and cutting-edge technology.

http://www.materialsforengineering.co.uk

serum, blood plasma orurine in pharmaceuticaland medical depart-ments, or biologicalanalysis laboratories thatare dealing with issues ofvariability in analyticalresults or infection risk.It is a fully automatedsample preparation mo -dule that was developedbased on Shimadzu’sexperience to produceclinical analyzers. Theinstrument is able toperform the followingprocesses for samplepretreatment:

1. dispensing samples 2. dispensing reagents 3. stirring 4. suction filtration 5. incubation 6. automatic transfer of samplevials to an SIL-30AC auto-sampler after pretreatment.

The direct detection of diseasesrelated to biological compoundsin blood, urine, or other biologi-cal samples, the measurement oftrace concentration levels of drugsand other applications is possibleby mass spectrometry. By addi-tionally using the CLAM-2000unit in front of the LC-MS/MSsystem, data quality will not onlybe improved, but also throughputwill be increased. Simply, theblood collection tubes have to be

Figure 2: Workflow for simultaneous analysis of antiepileptic drugs in blood serum using the fully automated sample preparation

LC-MS/MS system

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4 SHIMADZU NEWS 2/2016

T he trend to use LC-MS/MStechnology instead of im -muno assays in clinical labo-ratories can be observed alreadyfor a few years. The shift to LC-MS/MS is constantly increasingfor several types of analysis, forexample immunosuppressants,vitamin D or steroids panels. Theprogress of LC-MS/MS in thatfield is mainly due to its abilityfor high sensitivity detection, highselectivity as well as the possibili-ty to multiplex compounds in oneanalysis without the risk of inspe-cific cross reactions inherent toimmunoassay tests.

Sample preparation is the bottleneck

LC-MS/MS has proven to be apowerful tool in clinical laborato-ries and the cost per analysis wasdecreased significantly - thanks toShimadzu’s UFMS (Ultra-FastMass Spectrometry) technologiesincluding ultra-fast polarityswitching (5 ms), short pause time(1 ms), multi MRM (555 per sec-ond), and fast scanning possibili-ties (30,000 Da/s), combined withthe well-known robustness ofShimadzu systems.

However, the bottleneck still re -mains sample preparation steps

like deproteinization and dilutionwhich is required to analyzeblood samples. These steps areoften tedious, introduce the risksof errors, and also increase thepotential health risk due to infec-tion for laboratory staff. To over-come this, Shimadzu has devel-oped the fully automated samplepreparation module CLAM-2000(Clinical Laboratory Automatedsample preparation Module).

Unique combination of sample preparation and LC-MS/MS analysis

The CLAM-2000 (figure 1) is anautomated pretreatment systemdesigned for customers that han-dle biological samples like blood

placed in the system and theCLAM-2000 will perform allother processes from sample pre-treatment to LC-MS analysisautomatically.

Unlike standard dispensing sys-tems or robots, which are basedon batch processing of 96-wellplates, the CLAM-2000 is com-pletely automated. No manualsteps are required after placing thesamples as even the transfer to theLC-MS/MS instrument is doneautomatically. It processes allindividual samples successively.Consequently, it results in uni-form pretreatment times for everysample, without slowing downprocessing speed, and improvesdata reproducibility and accuracy.

Fully automated analysis of blood serum

Figure 2 shows as an example the workflow for the simultane-ous analysis of antiepileptic drugs in blood serum using the CLAM-2000 coupled to a LCMS-8040 mass spectrometer.The blood collection tubes wereinserted into the CLAM-2000 andthe analysis was started on thetouch panel. Additional samplesmay be inserted while the instru-ment is already running.

Preparation of the next sample canalso be performed in parallel with

CLAM-2000* – fully automated sample preparation module particularly for biological applications

Discover the future of LC-MS/MS

Figure 1: CLAM-2000 fully automated sample preparation LC-MS/MS system

5SHIMADZU NEWS 2/2016

PRODUCTS

LC/MS/MS analysis, which cangreatly reduce the time requiredfor each sample analysis. In theinstant example, a per-samplecycle time including analysis ofnine minutes is achieved. Themass chromatogram (figure 3)shows the separation of sevenantiepileptic drugs and drug meta -bolites spiked into human bloodserum. Due to the selective detec-tion of the LC-MS based on massand structure, no interferencesfrom other components in theblood serum are apparent.

Calibration curves were preparedby continuous analysis with fullyautomated sample preparation andanalysis, and used to assess accu-racy and precision (repeatability).Good linearity was obtainedacross the set calibration curverange for each antiepileptic drug(figure 4), with accuracy within

100 % ± 15 % over the entiremeasurement range including theminimum limit of quantification.Similarly, precision was measuredat a % RSD of within 15 %,showing that good repeatabilitywas achieved (table 1).

Flexible and future-proof

The system is not limited to cer-tain methods and allows the use of homebrew methods as well ascommercially available kits as longas the manual preparation stepscan be adapted on the system. The required reagents just have to be inserted into the system, andthe respective method have to be assigned to the sample.

In addition, to keep a maximumdegree of flexibility and to be ableto adapt to future needs, theCLAM-2000 is compatible with

the full range of Shimadzu’s LC-MS/MS instruments:

• LCMS-8040• LCMS-8050• LCMS-8060

The CLAM-2000 can easily beadded to an already existing LC-MS/MS system. A simple GraphicUser Interface (GUI) via a touchpanel simplifies the monitoring ofthe unit (sample preparation plusLCMS system) as well as tables tofollow the work load of the sys-tem and tools to organize themaintenance. Samples can be easi-ly entered and tracked by the inte-grated barcode reader.

Consequently, the new CLAM-2000 is the first system inthe world able to perform all stepsfully automated from pretreat-ment of the sample to LC-MS/MSanalysis without any further man-ual interaction. It just requires thesimple task of placing the blood,or biological fluids collection tu -bes, reagents, internal standardsand specialized pretreatment vialsin the system and start.

It also features excellent manage-ment functions that can provide adramatically improved workflowwith better safety for clinical re -search and higher reproducibility.

*For Research Use Only. Not for use in diagnostic procedures. Not availablein the USA, Canada and China.

Figure 3: Mass chromatogram of seven antiepileptic drugs and drug metabolites in a

control serum sample

Figure 4: Calibration curves of seven antiepileptic drugs and drug metabolites

Further information

on this article

• Application News

High-Throughput

Optimization of

Therapeutic Drug Monitoring

Table 1: Results of validation test for simultaneous analysis of antiepileptic drugs

(x 100,000)

0.00

0.50

1.00

1.50

2.00

2.00 2.50 3.00 3.50 4.00 4.50

min

Area2,250,000

0250,000500,000750,000

1,000,0001,250,0001,500,0001,750,0002,000,000

0 250 500 750 Conc.

Area2,250,000

0250,000500,000750,000

1,000,0001,250,0001,500,0001,750,0002,000,000

0 250 500 750 Conc.

Area

0

100,000

200,000

300,000

400,000

500,000

600,000

700,000

0 250 500 750 Conc.

Area5,000,000

0

1,000,000

2,000,000

3,000,000

4,000,000

0 250 500 750 Conc.

Area

1,500,000

0

250,000

500,000

750,000

1,000,000

1,250,000

0 250 500 750 Conc.

Area

0

100,000

200,000

300,000

400,000

500,000

0 250 500 750 Conc.

Area

750,000

500,000

250,000

0

100,000

125,000

150,000

175,000

0 2,500 5,000 7,500 Conc.

Compounds

LevetiracetamFelbamateTopiramateCarbamazepine 10,11-EpoxideCarbamazepineTiagabineDiazepam

10 - 75025 - 1,000

500 - 10,0005 - 1,000

10 - 1,00050 - 1,0005 - 1,000

1025

5005

10505

100250

2,500100

100250250

7501,000

10,0001,000

1,0001,0001,000

Range(ng/mL)

QC samples concentration(ng/mL)

Accuracy (%) % RSD (n = 6)

LLOQ Medium ULOQ

94.698.6

102.392.9

90.698.598.1

106.1101.897.1

107.8

110.3101.9102.4

99.299.6

100.699.3

99.199.699.5

LLOQ Medium ULOQ

3.426.286.717.48

3.791.954.61

1.231.883.583.32

3.422.001.50

1.981.502.961.41

1.191.261.53

LLOQ Medium ULOQ

I nfrared microscopy is consid-ered to be the ultimate high-light in molecular spectrosco -py. With a few simple steps, com-plex questions about tiny sampleparticles can be easily answered.‘Tiny’ refers to ‘micrometer ran -ge.’ The measurement technologyor more specifically, the ap pliedradiation wavelength, limits thesample size. The mid-infraredrange of approx. 4,000 - 400 cm-1

can only be applied to measuringspots with a minimum size of atleast 5 µm, because 5 µm corre-sponds to the wavenumber 2,000 cm-1 in infrared spectrosco -py.

In the past, key words like con-taminations of products, forensicsor failure analysis have been used.

the definition of measurementspots, as well as for the measure-ment and identification of spectra.To analyze the tiniest spots, themicroscope must feature the ap -propriate measurement parame-ters.

The AIM-9000 achieves a signal-to-noise ratio of 30,000 : 1. It canperform and directly display high-speed measurements for 3D or 4Dmeasurements (mapping/screen-ing). An important advantage forthe user is simultaneous measure-ment and visual observation of thesample. Needless to say, the AIM-9000 microscope is availablewith numerous additional options,such as polarizers for observationand measurement, or wide-fieldcameras, ATR (attenuated total

Today, discussions focus on thetopic of microparticles and theiridentification. Particles that aresmaller than 250 µm are of partic-ular interest, for example particlesthat are found in the stomachs ofanimals (seals, dolphins, sea birds)or particles in foods (black parti-cles in honey, particles on pizzasor in filled bottles). The relevanceof this topic is covered by theapplication range of Shimadzu’snew AIM-9000 fully automaticFTIR infrared microscope.

Good measurement parameters for the analysis of the tiniest spots

The AIM-9000 is an automaticfailure analysis system and is opti-mized for automated observation,

PRODUCTS

6 SHIMADZU NEWS 2/2016

Multipurpose tool for tinyobjects AIM-9000 infrared microscope: automated microanalysis for contaminations and microparticles

Figure 1: AIM-9000 in combination with Shimadzu’s FTIR IRTracer-100

reflectance), grazing angle objec-tive and more.

AIMsolutions software and libraries

Automation control in the AIM-9000 is achieved via theAIMsolutions software, whichsupports the sequence of failuresearch, measurement and identifi-cation. For identification, theAIMsolutions software usesShimadzu’s standard library con-taining 12,000 spectra, the Tap-Water/Food ContaminantsLibrary, the Thermal-DamagedPlastics Library as well as theSadtler and the STJ Libraries. Thefailure analysis mentioned earlieris an automatic search function ofdistinctive regions in the sample.

Figure 2: Three steps to success for different tasks

Troubleshooting: Highest sensitivity, automatic zoom function and automatic detection of contaminants

7SHIMADZU NEWS 2/2016

PRODUCTS

IMPRINT

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PublisherShimadzu Europa GmbHAlbert-Hahn-Str. 6 -10 · D-47269 DuisburgPhone: +49 - 203 - 76 87-0 Fax: +49 - 203 - 76 66 25shimadzu@shimadzu.euwww.shimadzu.eu

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Depending on the size of thedefect, appropriate aperture sizescan be set.

Compatible with various detectors

The AIM-9000 works in conjunc-tion with Shimadzu’s FTIR

IRtracer-100 and IRAffinity-1Sspectrophotometers. To detect IRradiation (also called thermal radi-ation), the microscope can beequipped with various detectorsdepending on the heat sensitivityrequired. For high-sensitivitymeasurements, two MCT detec-tors with different measuring

ranges are available and for simpleand non-sensitive measurement, aTGS detector can be used.

The advantage of the TGS detec-tor is its wide measurement range.Its operation does not require anyliquid nitrogen. Its low signal-to-noise ratio, however, is a draw-back. While the TGS detector hasa signal-to-noise ratio of 100 : 1,the more sensitive MCT detector(detector cooling with liquidnitrogen is required) has a signal-to-noise ratio of 30,000 : 1. Im -portant for these key parametersis the observed area of 100 µm2.

Conclusion

The new Shimadzu AIM-9000infrared microscope can be fullyautomated to set apertures, to findsample spots and to measure andanalyze them. This considerablyreduces the microscopy workload.The result is a visible image of thesample area, the infrared spectraand the identification of materialsthat constitute the defects.

http://www.shimadzu.eu

APPLICATION

8 SHIMADZU NEWS 2/2016

important information on the re -maining organic contaminationand thus on the efficiency of theT he TOC (Total Organic Car -bon) parameter for organicpollution of wastewater can

serve as an indicator for accurateand efficient control of industrialprocesses. In addition to carryingout TOC analyses in the laborato-ry, TOC process analysis is in -creasingly in demand. UsingShimadzu’s TOC-4200 andswitch ing between measuringpoints, up to six sample streamscan be monitored – even in sample

streams of different concentrationlevels.

Practical example: wastewatertreatment plant

In industrial water purificationand wastewater treatment plants, a variety of processes are used topurify wastewaters in differentsub-processes (for instance, bio-logical or filtration processes).The TOC sum parameter provides

Wastewater treatment plant: high demands on equipment andsampling method

No carry-over: TOC determinationin process analysis

wastewater treatment plant. Atthe inlet of such plants, TOC con-centrations of well over 1,000 mg/L

MatrixTarget concentrationParameterAcid addedInjection volumeDilution

Ultrapure water1 mg/LNPOC100 µL50 µLNone

3 % NaCl solution1,000 mg/LNPOC100 µL50 µLFactor 10

Sample stream S # 1 S # 2

Table 1: Matrix and measurement conditions of the two sample streams

9SHIMADZU NEWS 2/2016

APPLICATION

––––– 1,000 mg/L TOC ––––– 1 mg/L TOC

TOC

[m

g/L]

Hig

h ra

nge

TOC

[m

g/L]

Low

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ge

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1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47 49 51 53 55 57 59 61 63 65 67 69 71 73 75 77 79 81 83 85 87

TOC-4200

Figure 1: ISP module of the

TOC-4200

as well as high salt loads can beexpected. Dissolved organic sub-stances are decomposed in biolog-ical intermediate stages. Floccu -lation or precipitation agents ad -ded at this stage modify the sam-ple composition (matrix).

At the outlet of such purificationor wastewater treatment plants,the TOC content of the effluentwater is usually less than 50 mg/L.

Figure 2: Measurement results of the two sample streams

The sample matrices and measur-ing ranges of these sample streamsthus vary widely. Monitoringthese sample streams using a sin-gle analyzer will place high de -

mands on the instrument and thesampling method used.

ISP module in the TOC-4200

The ISP module (Integrated Sam -ple Pretreatment) is the heart ofthe TOC-4200 series. It consistsof an 8-port valve and a syringepump (figure 1). The consistentuse of inert materials reduces therisk of cross-contamination.

In addition to automated samplepretreatment (acidification andsparging), the module enablesdilution of the sample. It is alsopossible to determine self-cleaningsequences for the module as wellas the sample inlets. The automat-ed dilution function serves notonly for measuring range exten-sion, but also for matrix reduc-tion. This reduces maintenanceand lowers operating costs.

Sampling method

Incorrect measurement valuesoften already occur due to carry-over effects in the sample inlet lineor in the sampler. Biologicalgrowth and deposits in the sampleinlet line constitute a further riskof contamination. To minimizethis, the sample should be directedto the sampler at a flow rate ofless then 1 m/s. Inspection open-ings and valves for manual or evenautomated flushing of the tubingshould be provided for. The sam-pler should be constructed of inertmaterials.

Automated rinsing steps betweenindividual samplings further re -duce time expenditure by theoperator. To reduce problems dur-ing operation, it is important topay particular attention to sam-pling during the project develop-ment phase.

Practical test

In a practical test, two samplestreams with different matrices

and TOC contents were analyzed(table 1). To demonstrate samplecarry-over between these samplestreams, they were alternatelysampled and analyzed 90 timesrespectively. The results of thesealternating measurements areshown in figure 2. From the meas-urement results for sample stream 1,a mean value of 1,02 ± 0,07 mg/LTOC and for sample stream 2, amean value of 992,7 ± 13,32 mg/LTOC was obtained.

Conclusions

Even with significant concentra-tion and matrix differences be -tween both sample streams, theTOC-4200 is operated free fromcarry-over thanks to its inert con-struction and automated rinsingfunction.

LATEST NEWS

10 SHIMADZU NEWS 2/2016

combustion tube, cause destruc-tive stresses upon cooling or heat-ing. Samples with high-salt con-tent thereby increase the need formaintenance and decrease the life-time of the analyzer.

Solution: temperature below the salt melting points

To prevent such problems, it isimportant to use combustion tem-peratures that are below the melt-ing points of common salts. Al -ready during the 1980’s, Shimadzuhas developed the 680 °C combus-tion method. The TOC analyzersoxidize the organic compounds incombination with a highly effec-tive platinum catalyst at a temper-ature of 680 °C. As shown in table 1, this temperature is belowthe melting points of commonsalts.

T he determination of theTOC (Total Organic Car -bon) parameter has becomefirmly established in many appli-cation areas. Whether in environ-mental analysis, pharmaceutical orchemical industries – TOC deter-mination is used wherever conta -mination by organic componentsplays a role.

In the most frequently used TOCdetermination method, the sampleis first treated with a mineral acidin order to convert the inorganiccarbon compounds, such as car-bonates or hydrogen carbonatesto carbon dioxide, which is thenremoved via the carrier gas stream.

An aliquot of the sample is subse-quently injected onto a hot cata-lyst where the organic compo-nents, in turn, are converted tocarbon dioxide and transported to

a NDIR detector via a carrier gas, which detects the resultingCO2.

Problem: effect of salt accumulation on the catalyst

While water evaporates duringcombustion oxidation and theorganic substances present inwater are completely converted tocarbon dioxide, salts such as sul-fates or chlorides accumulate onthe catalyst with each injection.

Salt buildup on the catalyst or inthe combustion tube is one of themost frequently occurring techni-cal problem in TOC analysis.Depending on the combustiontemperature, the salts can melt andclog the active surface of the cata-lyst. Residues of the molten saltson glass components, such as the

TOC analysis of salt-containing samples

Salty catalyst?

Figure 1: At 680 °C, the salts crystallize and trickle through the combustion tube into the salt trap (figure 1c). The figures show the catalyst before measurement and after measurement

of a 9 % salt solution (600 injections).

0

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Injection of 28 % NaCl in 5 mg/L KHP [Injection 50 µL; Acid 15 %]

Conc

entr

atio

n [m

g/L]

Are

a

50 100 150 200 250

Figure 2: The example shows the injection sequence of a 28 % sodium chloride solution with a TOC content of 5 mg/L. Even after 220

injections onto the salt kit, the analysis results remain stable. A standard was measured at the beginning, after 100 injections and at the

end of the injection sequence to verify the recovery. A blank value was determined at the beginning of the injection sequence.

11SHIMADZU NEWS 2/2016

LATEST NEWS

Analyzers of the TOC-L seriesalso feature an integrated samplepreparation module, which makesit possible to perform automaticdilution of the sample. The mod-ule cannot only be used to dilutethe TOC content of a sample, butalso to reduce the concentrationof the sample matrix.

NaCIKCINa2SO4MgCI2CaCI2K2SO4

801 °C773 °C888 °C708 °C782 °C

1,069 °C

Compound Melting point

Table 1: Melting points of common salts

Special kits for salt-containingsamples

Sometimes, the low TOC concen-tration in samples such as seawa-ter or pure salt solutions andbrines, do not allow for dilution.Special kits for salt-containingsamples are available for these sit-

uations. They consist of a com-bustion tube that has a specialgeometry. The tube is filled withdifferently sized catalyst beadsthat are fixed by a coarselymeshed ceramic lattice. Thisdesign allows the salts to crystal-lize and to trickle down the pack-ing of the catalyst tube as well asthe ceramic lattice without clog-ging the packed tube.

A so-called salt-trap is situatedunderneath the ceramic lattice(figure 1c). The salt tricklingdown the catalyst tube accumu-lates here. Experience shows thatthe use of this salt kit enablesmeasurement of sample amountsup to twelve times higher com-pared to the use of a conventionalcatalyst. Using a salt kit, it is pos-sible to carry out about 2,500injections of a seawater sample. It is even possible to carry outTOC analyses of highly concen-trated sodium chloride brines (28 %) using such a kit.

Also suitable for online process analysis

The use of a kit for salt-containingsamples is, however, not only use-ful for laboratory analysis. Also inprocess analysis, in which online

analyzers are working ‘around theclock’, these kits can be used inorder to increase the lifetime ofanalytical systems.

The kits have been proven partic-ularly suitable for use in waste-water control. To save water inindustrial processes, part of thewater is recovered from the waste-water. This is why the salt contentin wastewaters is often increased.

Conclusion

Salt-containing samples cause saltbuildup in catalysts during TOCdetermination and decrease thelifetime of the analyzer. A com-bustion temperature below themelting point in common saltsreduces the occurrence of saltmelts on the catalyst.

Additional kits for the determina-tion of salt-containing samplesenable failure-free analysis of sam-ples with high salt loads, such asseawater or brines, and increasethe lifetime of the analysis system– in the laboratory as well as inonline analysis.

TOC-L

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12 SHIMADZU NEWS 2/2016

Carola Schultz from the MEETBattery Research Center atMunster University, Germany, hasbeen the first of young scientistswho were awarded with lab timeat the Laboratory World in Duis -burg, Germany for her re searchproject. Here are some insights inher topic:

L ithium ion batteries are usedday-in-day-out in laptops,cell phones or digital cam-eras. They are also installed in e-bikes and electric cars, mainlydue to their low weight comparedto other types of batteries such asthe lead accumulator. They arecharacterized by their high specif-ic energy and high energy density.[1]

Figure 1 shows the functionalprinciple of a lithium ion battery.Often, the cathode consists of li -thium transition metal oxides. Theanode material is usually graphite.During the charge process, lithi-um ions move through the elec-trolyte from the cathode to theanode and are incorporated be -tween the graphite layers. Thecorresponding electrons travel to

the anode through an externalelectric circuit. During discharge,the process is reversed and usableelectrical power is supplied.

The anode and cathode are sepa-rated by an electronically insulat-ing separator. This way, the cell isprotected from short-circuiting,the same time it is permeable tolithium ions. The electrolyte en -ables ion transport between anodeand cathode.

The electrolyte contains 1M lithi-um hexafluorophosphate (LiPF6)as a conducting salt. The electro -lyte consists of cyclic and linearorganic carbonates such as ethyl-ene carbonate (EC) and dimethylcarbonate (DMC), ethylmethyl

carbonate (EMC) and/or diethylcarbonate (DEC). The use ofcyclic carbonates increases thesolubility of the conducting salt.Because EC is solid at room tem-perature, it is mixed with the lin-ear carbonates to decrease the vis-cosity. [1] The structures of theindividual components are shownin figure 2.

When the electrolyte is aging

The conducting salt, which ischemically and thermally unsta-ble, is subject to so-called agingduring the charge and dischargeprocess. This can lead to the for-mation of a variety of different(for instance phosphate-based [2]or carbonate-based) aging prod-ucts. An example is the formationof the aging product group ofpolycarbonates, shown in figure 3[3, 4]. This is further investigatedbelow.

The fact that batteries age maywell be confirmed by anyone whohas owned a battery for a while.The battery lifetime is limited be -cause repeated charging and dis-charging reduces the capacity ofthe battery. To understand theunderlying aging mechanisms andto be able to counteract them re -quires analytical methods for theidentification and quantificationof aging products.

Within the lab4you research pro-gram for young scientists, it wasthe aim to develop such a methodusing LC-MS. The focus of theinvestigation was on the electro -lyte. The following mass spectro -meters were used for methoddevelopment: the LCMS-IT-TOFand the LCMS-8040 (triplequadrupole LC-MS/MS).

Qualitative analysis using the LCMS-IT-TOF

The benefit of the LCMS-IT-TOFis the combination of 3D ion trapand a time-of-flight mass analyzer.This offers a high mass accuracyas well as a high mass resolutionin all MS and MSn stages.

Using the LCMS-IT-TOF, theaging products were qualitatively

LC-MS method development for battery electrolytes

Anti-aging for batteries?

Figure 1: Schematic representation of the functional principle of a lithium ion battery

Commercially available

lithium ion battery

Figure 2: Structures of the main components of an electrolyte

13SHIMADZU NEWS 2/2016

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analyzed, i.e. the structures wereelucidated with the help of theMSn function. An example for thestructure elucidation of manyaging products via MSn is thefragmentation pattern of diethylphosphate (DEP) in figure 4 withits corresponding spectra. Despiteits molecular mass of 154 amu, thestructure of DEP could be eluci-dated with the help of an MS4

analysis. The main molecule withm/z = 155.046 was detected viaMS1. The loss of a C2H4 groupwith m/z = 127.015 could bemeasured in the MS2 stage.Another C2H4 group was cleavedoff in the subsequent fragmenta-tion step. The resulting phosphor-ic acid in MS3 with m/z = 98.985is dehydrated in a final fragmenta-tion step, leading to a moleculewith m/z = 80.972 in the MS4

stage.

More aging products identified in the course of the lab4you program

As there are no commerciallyavailable standards for all agingproducts, the MSn function forstructure elucidation is cruciallyimportant and useful. Thus, dur-ing the lab4you program, manyaging products, which are notavailable as pure substances, couldbe identified. As an example, somepolycarbonates that were detectedin thermally aged (60 °C) electro -lytes as well as in electrolytes ex -tracted from cycled batteries arelisted in table 1.

It becomes clear that this sub-stance class of polymerized agingproducts alter the function of anaged battery by reducing the con-ductivity in the electrolyte andincreasing its viscosity. Further -more, organic electrolytes withLiPF6 are highly water-sensitive.Even brief contact between anelectrolyte and air causes the for-mation of many aging products,

such as DEP whose structure elu-cidation is shown in figure 4 (page14).

Charging and discharging of abattery as well as elevated temper-

atures (> 55 °C) lead to the forma-tion of aging products. This couldbe confirmed, among others, inthis study on the various agingprocesses in the battery and elec-trolyte.

Quantitative analyses using the LCMS-8040

The main components of the elec-trolyte as well as the commerciallyavailable aging products werequantified in the MRM modeusing Shimadzu’s LCMS-8040triple-quadrupole mass spectrom-eter. In addition, structural eluci-

dation via LCMS-IT-TOF wasconfirmed with LCMS-8040 andfurther supported by the extendedmass range – which in the triplequad is also available for masses < m/z = 50.

A total of nine substances, maincomponents as well as aging prod-ucts were quantified. These in clu -ded EC, DMC, EMC, DEC,DMDOHC, DEDOHC, triethylphosphate (TEP), dimethyl phos-phate (DMP) and DEP. A C18pentafluorophenyl phase (2.1 mmx 100 mm; particle size 2 µm) wasselected. �

Table 1: Polycarbonates that were detected via LCMS-IT-TOF in electrochemically and thermally aged battery electrolytes

Figure 3: Formation of carbonate-based aging products

Structure Ion m/z Formula

[M+H]+

[M+NH4]+

[M+Na]+

179.05196.08201.04

C6H10O6

C7H12O6

C8H14O6

C9H14O9

C10H16O9

C11H18O9

C12H18O12

C13H20O12

C14H22O12

193.07210.09215.05

207.09224.11

––

267.07284.09289.05

281.09298.11303.07

295.10312.13317.08

––372.11377.07

369.10386.13391.08

––400.14405.10

[M+H]+

[M+NH4]+

[M+Na]+

[M+H]+

[M+NH4]+

[M+Na]+

[M+H]+

[M+NH4]+

[M+Na]+

[M+H]+

[M+NH4]+

[M+Na]+

[M+H]+

[M+NH4]+

[M+Na]+

[M+H]+

[M+NH4]+

[M+Na]+

[M+H]+

[M+NH4]+

[M+Na]+

[M+H]+

[M+NH4]+

[M+Na]+

T ake this opportunity fordetailed discussions withexperts from research andindustry on the numerous pyroly-sis GC-MS applications.

The all-day event offers a variedprogram with lectures and practi-cal examples as well as cutting-edge information on instrumenttechnology and applications. In addition, the tour aroundShimadzu’s Laboratory World

Pyrolysis GC-MS user meetingOctober 6th 2016, at Shimadzu’s Laboratory World, Duisburg, Germany

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·· · Don’t miss this! Limited number of participants · · · Don’t miss this! Limited number of participants · · ·

time at Shimadzu’s cutting-edge‘Laboratory World’ in Duisburg,Germany. More information:www.shimadzu.eu/lab4you

AuthorCarola Schultz, MEET Battery Research

Center, Munster University, Germany

Literature[1] Winter, M. and J.O. Besenhard, Wieder -

aufladbare Batterien. Chemie in unserer

Zeit, 1999. 33(5): p. 320 - 332 (1999).

[2] V. Kraft, M. Grützke, W. Weber, J. Menzel,

S. Wiemers-Meyer, M. Winter, S. Nowak,

Journal of Chromatography A 1409: 201 -

209 (2015).

[3] T. Sasaki, T. Abe, Y. Iriyama, M. Inaba and

Z. Ogumi, Journal of Power Sources, 150,

208 (2005).

[4] C. Schultz, V. Kraft, M. Pyschik, S. Weber,

F. Schappacher, M. Winter, S.Nowak,

Journal of the Electrochemical Society

162, Nr. 4: A629 - A634 (2015).

14 SHIMADZU NEWS 2/2016

The mobile phase consisted ofwater mixed with 0.1 % formicacid and methanol. With this col-umn and mobile phase combina-tion, all nine substances could beseparated and quantification ofthese substances in various elec-trolytes could be carried out.

Conclusions

Method development for the iden-tification, separation and quantifi-

cation of electrolyte componentsas well as their aging productsusing LCMS-IT-TOF and LC-MS/MS could successfully becompleted. This method helps tounderstand battery aging and canthus contribute to ‘anti-aging’research for batteries.

For their research project, inter-ested students can apply untilOctober 31st 2016 to be selectedfor being awarded with free lab

Figure 4: Fragmentation with corresponding spectra of DEP

My experiences during the lab4youresearch programBecause of the interesting work, Ican highly recommend the lab4youprogram to everyone. This programprovides a unique possibility to carryout ones own research using theequipment available in Shimadzu’sLaboratory World, where participantscan specifically use those instru-ments that will deliver the best pos-sible results for their respective ana-lytes and their desired objectives.For all this as well as with regard tothe available measurement time,Shimadzu’s Laboratory World offersfar more opportunities than mostuniversities could ever offer.

I was able to work independently;there were no preconditions so thatI could make all my own decisions.

This had a positive effect on myresearch, but of course, it also re -quires very careful project planningin advance.

I have very much appreciated themany possibilities for my researchproject as well as the excellent tech-nical support regarding the equip-ment in the Laboratory World. Incase of problems or technical ques-tions, I was always helped immedi-ately by the friendly and helpfulproduct specialists or by the serviceteam. All in all, I had a great time atShimadzu who provided me withvaluable results, nice experiencesand, of course, interesting insightsinto the working world.

provides insights into this high-end testing facility for the entireanalytical instrumentation productrange on over 1,500 m2.

However, there will be sufficienttime for sharing experiences andfor discussions during the breaks.

Please register in time, as thenumber of participants is limited.The deadline for registration isAugust 31st, 2016.

We look forward to welcomingyou to this special event inDuisburg!

For registration and further infor-mation, please use the link below:

bit.ly/pyrolysis-day

www.shimadzu.eu

DEP

0

1x105

2x105

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4x105

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http://www.shimadzu.euhttp://bit.ly/pyrolysis-dayhttp://www.shimadzu.eu/lab4you

15SHIMADZU NEWS 2/2016

APPLICATION

The plastic materials may notleach more than 1 µg/g (1 ppmw)of the respective element, whichcorresponds to 0.4 mg/L in theextraction solution.

Furthermore, the PET and PETGare analyzed for antimony andgermanium. The limit values re -quired for these metals are thesame as for the previously men-tioned elements. The extractionprocess for antimony and germa-nium, however, is different. Alka -line extraction is used here: 20 gof the material to be tested isheated together with 0.01 N sodi-um hydroxide solution and is alsoheated at 50 °C for five hours andanalyzed within the same subse-quent stability period of the solu-tion of four hours.

Instrumental analysis implementation

In addition to the previouslymentioned elements and limit val-ues, lower limit values have beendefined for the other substanceclasses. In the extraction solu-tions, the limit value for chromi-um can be at 20 µg/L, while limitvalues for titanium, vanadium andzirconium can be at 40 µg/L.

To successfully implement theanalyses as described in the new USP chapters, the use ofShimadzu’s ICPE-9820 is advised,an optical emission spectrometerwith inductively coupled plasma.It features highly sensitive axialplasma observation and can, with-in the same method, also use radi-al view plasma observation formeasurement of higher con-centrations. Because in addition to trace elements, calcium for instance, which in the PVC substance class may be present in concentrations

Normativedirectives

While TOCanalyses serveas an indica-tor for ex -

tract able organicmaterials, the deter-mination of extract -able metals in mate-rials of construction(plastics) for theproduction of thepackaging or the

packaging systems is specificallyaddressed in chapter 661.1. Chap -ter 661.2, which addresses finishedpackaging systems, re quires thatonly materials of constructioncompliant with USP 661.1 be usedand metals are not regarded.

Application

In chapter 661.1, various extrac-tion solutions are used that varydepending on the element or typeof plastic to be investigated. Thesematerials of construction aredivided into three groups:

1. Polyethylene (PE), polyolefin(PO) and polypropylene (PP)

2. Polyethylene terephthalate(PET) and glycogen-modifiedPET (PETG)

3. Polyvinylchloride (PVC).

The most complex are the tests forGroup 2, PET and PETG, becausetwo different extraction solutionsare required: acid extraction andalkaline extraction.

In acid extraction, 20 g of thematerial to be tested is heatedtogether with 0.1 N hydrochloricacid (HCl) for five hours at 50 °C.After cooling, the solution is ana-lyzed within four hours for theelements aluminum, barium,cobalt, manganese, titanium andzinc.

Content and implementation of USP 661.1 with respect to extractable elements

Metallic contaminations in pharmaceutical packaging

of up to 35 mg/L in the extractionsolution, must also be determined.

The ICPE-9820 features, in addi-tion to full flexibility in the meas-uring range, a particularly low-maintenance Echelle spectrometer.In order to optimally use the en -tire measuring range (167 - 800 nm)at all times, conventional systemsare continuously purged withargon or nitrogen. Because of thesophisticated vacuum technologyof the ICPE-9820, this purgingprocess is no longer required, thuscompletely eliminating any addi-tional costs of consumables.

Conclusion

The ICPE-9820 is ideally suitedfor the implementation of USP661.1. Furthermore, Shimadzuoffers the ICPMS-2030, a massspectrometer with inductivelycoupled plasma, or as an alterna-tive, the AA-7000 atomic absorp-tion spectrophotometer.

All in struments can be operated ina regulated laboratory infrastruc-ture in accordance with FDACFR 21 Part 11. In addition toTOC, the package can be expand-ed with the IRAffinity-1S for theidentification of polymers or aUV-VIS spectrophotometer, likethe UV-1800 for implementationof the listed absorbance tests.

ICPE-9820

I n the pharmaceutical industry,plastic packaging is used invarious forms, such as infusionbags, bottles, cartridges or pre-filled syringes. For their particularpurposes, these packagings mustbe tested for suitability.

The United States Pharmacopeiahas published two new chapters(661.1 and 661.2) to this effect,which have been valid since May2016:

• Chapter 661.1 describes thecharacterization and testing ofthe individual plastic materialsof construction used in the pro-duction of plastic packaging.• Chapter 661.2 deals with therequired testing of the finalpackaging systems, as packagingoften consists of more than oneplastic material.

Characterization is carried out bythe identification and determina-tion of biocompatibility, physico-chemical properties and extract -able metals.

For the determination of physico-chemical properties, Shimadzuoffers two suitable TOC systems,the TOC-V and TOC-L, whichhave already been described inIssue 01/2016 of the ShimadzuNews. The present article containsan overview of the analysis ofextractable metals.

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16 SHIMADZU NEWS 2/2016

influence of heat. That’s why agentle microwave process hasbeen developed which is appliedin a closed system in two steps forvarious food compartments. Thisnew microwave procedure isfaster, cheaper and more environ-mentally friendly than the con-ventional ISO procedure.

It is assumed that the oxidationsensitive unsaturated fatty acidsare treated more gently in the mi -crowave process and oxidation isthereby minimized, resulting in ahigher analytical result of unsatu-rated fatty acids.

Methods

Conventional ISO procedureThe conventional standard proce-dure is based on the ISO extrac-tion method (ISO 8262) for totalfat determination in milk and milkproducts according to Weibull-Berntrop and the derivatization offatty acids in fatty acid methylesters (FAMEs) based on the ISOmethod (DIN EN ISO 12966-2).

The ISO 8262 method serves as abase and correlates with the ex -traction method of Weibull-Stoldtfor the determination of total fatcontent in food samples.

The ISO extraction method accor -ding to Weibull-Stoldt is based ona saponified digestion using hy -drochloric acid and water, whichreleases fat bound to proteins in

T he Regulation (EU) No1169/2011 of the EuropeanParliament requires thedetailed declaration of nutritionvalues for food as of 2016, espe-cially the differentiation of fat insaturated and unsaturated fattyacids [1].

This differentiation is importantin order to protect consumers.For example, the American Di -etet ic Association (ADA), theEuropean Food Safety Authority(EFSA) and the American “Aca -demy of Nutrition and Dietetics“recommend to cover less than 35 % of the body’s daily energyrequirements by fat; according toADA, less than 20 % should bemonounsaturated fatty acids(MUFA).

Determination of total fat contentas well as unsaturated and saturat-ed fatty acids in food is performedusing methods based on ISO stan-dards. Depending on the type of

(GC-FID). This analysis requiresthe derivatization of fatty acids infatty acid methyl esters (FAMEs)based on the ISO norm [4].

Unsaturated fatty acids are sensi-tive to oxidation processes gener-ated through contact with air and

food, methods like Röse-Gottliebor Weibull-Stoldt extraction areused for the determination of totalfat content [2, 3].

The following analysis of saturat-ed and unsaturated fatty acids isdone using gas chromatography

Healthy fat in chips and sausages?A new method for extraction, digestion and analysis of fat in food samples

Figure 1: Alcaline Derivatization of a Triglyceride with methanol to ester [5]

Figure 2: Acidic derivatization of a fatty acid to ester with methanol under formation

of water [6]

food samples. In the following hotfiltration, the fat released remainsin the filter. The filter includingthe fat is washed to neutral andthen dried. Finally, the fat is ex -tracted with a proper solvent in aSoxhlet extractor, taking severalhours. As soon as the extraction isfinished, the solvent is removedusing a rotation evaporator andthe remainder is dried. Determi -nation of total fat is performed byweighing the dried fat.

Based on the ISO procedure onsample preparation for GC analy-sis, the transformation of fattyacids in fatty acids methyl esters(FAMEs) requires a sequence of

alkaline and acidic derivatizationin water-free methanolic environ-ment.

The alkaline derivatization gener-ates the FAMEs out of the fattyacids bound as triglycerides andthe saponification of free fattyacids. The following acidic deriva-tization finally transfers the re -mai ning saponified fatty acids inFAMEs.

The reaction equations are dis-played in figure 1 and figure 2.

Microwave procedureThe new microwave procedure fordetermination of fat has been de -

veloped for the microwave systemDiscover SP-X® (CEM, KampLintfort). It consists of a micro -wave extraction method (MEM)and a microwave derivatizationmethod (MDM).

The MEM has been developedbased on the Weibull-Stoldtmethod and extracts the total fatin a food sample in a closed sys-tem under defined conditionsusing microwave radiation simul-taneously.

In a closed system (includingMDM), derivatization of the ex -tracted fatty acids is performedunder microwave radiation. Trans -formation of fatty acids in fattyacids methyl esters (FAMEs) alsorequires a sequence of alkaline andacidic derivatization in water-freemethanolic environment for sub-sequent GC analysis.

GC analysisAnalysis of FAMEs of the foodsamples has been done using a gaschromatograph GC-2010 Plus AFwith FID detector (ShimadzuEuropa GmbH, Duisburg, Ger -many).

Influence of the method of samplepreparation and derivatization onthe content of unsaturated fattyacids in the total fat content offood samples needs to be investi-gated. In order to compare thecontents of unsaturated and satu-rated fatty acids in both methods,

the total amounts of all fatty acidpeak areas are calculated (= 100 %).Peak areas of unsaturated and sat-urated fatty acids are then summa-rized, and the ratio of unsaturatedvs. saturated fatty acids is calculat-ed. This procedure has been ap -plied for both the ISO procedureand the microwave procedure.

Experimental part

ISO procedure

Extraction methodThe food sample is weighed in a600 mL glass beaker, in the expec-tation that a total fat weight of 2to 3 g will be present. The sampleis treated with 100 mL water and150 mL hydrochloric acid (25weight-%).

The glass beaker is covered with aglass lid, and the sample is thenboiled for 30 to 60 minutes. Afterthe digestion process, the hotsample is diluted with 100 mL ofwater and filtered in a 2 layerround filter which has been wet-ted before with hot water. After -wards, the filters and the residueare washed neutral with hot water.Finally, the filter paper and resi -due are dried in an oven for onehour at 105 °C. The dried filtersare then transferred in the extrac-tion sleeve and finally positionedin a 250 mL extractor (Soxhlet-Apparatus). �

17SHIMADZU NEWS 2/2016

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2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0

225,000

200,000

17,500

15,000

12,500

10,000

7,500

5,000

2,500

0

Time [min]

uV Max intensity: 62,263.072

Figure 3: Chromatogram of the 37 Standard-Mix identified FAMEs

2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5

200,000

175,000

150,000

125,000

100,000

75,000

50,000

25,000

0

Time [min]

uV Max intensity: 87,084.447

Figure 4: Chromatogram of fatty acids in a sausage sample, derivatization according to

ISO procedure and identified FAMEs

2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5

400,000

350,000

300,000

250,000

200,000

150,000

100,000

50,000

0

uV Max intensity: 82.382.253

Figure 5: Chromatogram of fatty acids in a sausage sample, derivatization according to

microwave procedure and identified FAMEs

Time [min]

C6:0C8:0C10:0C11:0C12:0C13:0C14:0C14:1C15:0C15:1C16:0C16:1C17:0C17:1C18:0

C18:1 cis+transC18:2 cis,cis

C18:2 trans,transC18:3

C18:3 cisC20:0C20:1C20:2

C20:3 cis11,14,17C20:3 cis 8,11,14

C20:4C20:5C21:0C22:0C22:1C22:2C23:0C24:0C22:6C24:1

FAMECaproicCaprylicCapricUndecanoicLauricTridecanoicMyristicMyrestoleicPentadecanoiccis-10-PentadecaniocPalmiticPalmitoleicHeptadecanoiccis-10-HeptadecanoicStearicOleic + ElaidicLinoleicLinolelaidicα-Linolenicγ-LinolenicArachidiccis-11-Eicosenoiccis-11,14-Eicosadienoiccis-11,14,17-Eicosatrienoiccis-8,11,14-EicosatrienoicArachidoniccis-5,8,11,14,17-EicosapentaenoicHenicosanoicBehenicEruciccis-13,16-DocosadienoicTricosanoicLignocericcis-4,7,10,13,16,19-DocosahexaenoicNervonic

Name of acidxxxxxxx

x

xxx

xx

x

xxx

x

xxxx

x

ISO

Identified findings of the methods

Microwave

x

x

xxx

xxxxxxx

xxxxxx

xxx

xx

xx

F [%] =E

m2 - m1 *100

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18 SHIMADZU NEWS 2/2016

The round bottom flask of theSoxhlet apparatus (250 mL) isfilled with solvent at a volumecorresponding to 1.5 times thevolume of the Soxhlet apparatus.The temperature of the solventpetrol ether is 40 - 60 °C.

The reflux condenser is then set,the solvent is boiled and the fat isextracted over three to six hours.After extraction, the remainingliquid is fully evaporated in therotation evaporator.

Afterwards, the open flask is fur-ther dried for one hour at 105 °Cin order to remove the extractionliquid completely. After cooling,the flask is weighed and the totalfat content determined.

ISO based fatty acid deriva-tization for GC analysisThe extracted fat is transferred ina 10 mL round flask and treatedwith 2 mL (0.2 mol/L) sodiummethanolate in methanol (8 g so -dium hydroxide are dissolved in1,000 mL methanol).

Boiling under reflux is performeduntil the solution is clear (5 to 20minutes). The boiling time de -pends on the chain length of thefatty acids, the longer the fattyacid, the longer the boiling takes.After wards the flask is removedfrom the heating source and twodrops of phenolphtaleine solutionare added.

Sulfuric acid (1 mol/L) is thenadded in methanol solution untilthe solution is clear; another 0.2 mL are then added.

The cooler is reinstalled, and thesolution is boiled under reflux forfive minutes. The solution is againre moved from the heating sourceand cooled down in water. 4 mLof a saturated sodium chloridesolution are now added and shookwell. Afterwards, 1 mL n-hexan isadded and the flask is shook againfor 15 seconds. The solution is leftto stand until both phases are sep-arated. Saturat ed sodium chloridesolution is again added until theaqueous phase reaches the lowerlevel of the flask neck, approx. 2 -3 mL. The upper phase now con-tains the FAMEs generated.

are treated in a glass vial with 10 mL water-free methanolicpotassium hydroxide solution (2.5 weight-%), and a stirrer chipis added.

The alkalic derivatization requiresthe following parameters on theDiscover SP-X

Final temperature: 90 °CRamp: 5 minutesHold time: 10 minutesStirrer: MediumPower: 200 Watt

After finishing the alkaline deriva-tization, the vial is cooled down ina water bath. 14.25 mL Methanoland 0.75 mL hydrochloric acid (37 weight-%) are added, and theacidic derivatization is generatedwith following parameters on theDiscover SP-X

stirrer for one minute. After -wards, this solvent will be trans-ferred in the aluminum bowl. Thiscold extraction is repeated three tofour times. The solvent with theextracted fat is dried in the heat-ing oven for twenty minutes at105 °C and the bowl finallyweighed. Calculation of the totalfat content is done by followingequation 1:

m1 = Mass in g empty bowlm2 = Mass in g bowl with dried

fatE = Mass in g weight sample

For the derivatization (MDM),one or two drops of the dried fat

Microwave method

Extraction method0,5 g of sample is weighed in an80 mL glass vial and treated with3,5 mL hydrochloric acid (37weight-%), 7,5 mL water and 5 mL n-Hexan. A stirrer chip isadded. The following parametersare set on the Discover SP-X.

Final temperature: 115 °CRamp: 4 minutesStirrer: StrongHold time: 15 minutesPower: 200 Watt

After the digestion and extraction,the solvent is transferred in a pre-viously weighed aluminum bowlin order to perform a cold extrac-tion. The digestion in the vial istreated again with 5 mL n-Hexanand then stirred on a magnetic

Table 1: Identified FAMEs of two procedures in direct comparison. Positive findings are marked with X.

19SHIMADZU NEWS 2/2016

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Final temperature: 120 °CRamp: 5 minutesHold time: 6 minutesStirrer: MediumPower: 200 Watt

After finishing the derivatization,the vial is cooled down in a waterbath. 10 mL n-Hexan are added,and the vial is turned twice frombottom to top. The generation ofoverpressure must be avoided.The glass vial is afterwards filledwith saturated sodium chloridesolution up to the top. The sol-vent needs to be transferred with apipette to a 10 mL GC vial, inclu -ding sodium sulfate or magnesiumsulfate as drying agent (approx.0.2 g) for possible water residue.This solution is now ready for theanalysis of FAMEs using GC-FID.

GC analysis:For identification and quantifica-tion of FAMEs, a 37 Standard-Mix (Supelco) has been used andanalyzed on GC-2010 Plus AFwith FID detector.

The following parameters havebeen set on the GC:

Injector: SPL (Split)Injection volume: 1 µLInjector temp.: 250 °CColumn type: FAME WAXInner diameter: 0.25 mmFilm thickness: 0.25 µmDetector temp.: 250 °CMobile Phase: HeliumCarrier gas speed: 35 cm / sec

The temperature program starts at130 °C with a hold time of oneminute. Afterwards, heating con-tinues with a heating rate of 5 °C/minute until reaching a tempera-ture of 240 °C which then staysconstant for ten minutes. Thetotal time is 33 minutes.

Results and Discussion

The gas chromatographic methodwith flame ionization detectionhas been optimized for separationof almost all fatty acid derivatedFAMEs.

From 37 fatty acids contained inthe standard mix, 36 fatty acidshave been identified. Two fatty

acids C18:1-cis and C18:1-transcould not be separated, as theyco-elute. But this has no influenceon the evaluation and differentia-tion between saturated and unsat-urated fatty acids since both co-eluting fatty acids are unsaturated.

Figure 4 (page 17) shows thechromatogram of a sausage samplewith the identified FAMEs whichhave been generated according tothe ISO procedure. Obviously,

fatty acids C16:0 und C18:0 aremainly present. In comparison tothe results of the ISO procedure,figure 5 (page 17) shows the chro-matogram of the same sampleusing the micro wave procedure.

A comparison of the chromato -grams (figure 4 and figure 5)shows a significant difference inthe FAMEs. The results on C18:1-cis+cis using the microwave pro-cedure are significantly higher incomparison to the ISO procedure.

Table 1 shows that the results ofthe ISO method have a significantdifference in comparison to themicrowave method. For a moreprecise comparison, the peak areasof saturated and unsaturated fattyacids are used and displayed indiagram 1, showing the differenceof the fatty acid ratios betweenthe ISO and the microwavemethod on sausage samples.

Comparison of both methodsshow that the result of unsaturat-ed fatty acids with the microwavetechnique is ten times higher thanthe ISO method. These elevated

results on unsaturated fatty acidscould be observed in the sameway during determination ofother food samples such as milkpowder, chocolate and potatochips, as displayed in diagram 2(page 20).

In all of the food samples deter-mined, use of the microwave pro-cedure has generated a higheramount of unsaturated fatty acids.This confirms the hypothesis that

the new microwave procedureprevents oxidation of unsaturatedfatty acids during the process, andthe more gentle treatment resultsin higher findings when using thenew microwave process.

Conclusion

The declaration of food samples isincreasingly more complex. Forconsumers, the declaration is veryimportant and therefore needs acertain transparency. The EU reg-ulation 1169/2011 demands a de -tailed declaration of food samples.Most important is the differentia-tion of total fat in saturated andunsaturated fatty acids, which isthe top criteria for the consumerto differentiate between healthyand unhealthy food, as has beenthe subject of two studies. Thedetermination of total fat contentas well as unsaturated and saturat-ed fatty acids is regulated in ISOnorms.

In this work, two methods havebeen developed. The first methodrepresents a procedure for diges-tion and extraction of the total fat

content in a closed microwavesystem, based on a method ofWeibull-Stoldt. Food samplesfrom different areas (dairy prod-ucts, luxury foods, meat and pas-tries) have been selected, and theapplication of the total fat deter-mination using the microwavemethod in a closed system hasbeen evaluated.

It has been found to be a universalmethod for the determination ofthe total fat content in differentfood areas.The second method correspondsto a microwave method, also in aclosed system for derivatization offatty acid in FAMEs which arethen analyzed in a GC-FID sys-tem.

The use of the developed micro -wave extraction method showsthat the method is best suited fordetermination of the total fat con-tent of food samples.

In the second method, food sam-ples have been analyzed for 37different fatty acid methyl esters.This enables a comparison be -tween the DIN method for diges-tion and extraction of the total fatcontent of a food sample with anISO method for derivatization ofextracted fatty acids in FAMEs,with the developed microwaveprocedure consisting of micro -wave extraction method (MEM)and microwave derivatizationmethod (MDM).

The focus of this comparison is onthe ratio between saturated andunsaturated fatty acids (milk pow-der, chocolate, sausage, chips).Results show that the ratio offatty acids differs between ISOand microwave procedure. Theproportion of unsaturated fattyacids is always higher when themicrowave procedure is applied.

The higher rate of findings ofunsaturated fatty acids confirmsthe sensitivity of the unsaturatedfatty acids related to the oxidationby air and heat influence. �

95

Saturated

ISO

Microwave

Unsaturated

5

59.0

40.9

0

20

40

60

80

100

Fatty acid

%

Comparison of the fatty acid ratios between ISO andmicrowave method: Sausage

Diagram 1: Comparison of the fatty acid ratios between ISO and microwave method on

sausage samples

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The DIN procedure is an opensystem and requires heat over along period of time which has aninfluence on the unsaturatedfatty acids (oxidation). The mi -cro wave system is a closed sys-tem with a significant shorterheat application and reduced oxi-dation of unsaturated fatty acids.The microwave procedure istherefore a more gentle and pre -ferable method as far as unsatu-rated fatty acids are concerned.

Read for youChromatography Today 5-6/16

AuthorsMarcel Pacheco-Moreno1, Jürgen Schram1,

Frank Scholten2, Ulf Sengutta2, Franz Kramp3,

Uwe Oppermann3

1 University of Applied Sciences,

Frankenring 20, D-47798 Krefeld, Germany

2 CEM GmbH, Carl-Friedrich-Gauß-Str. 9,

D-47475 Kamp-Lintfort, Germany

3 Shimadzu Europa GmbH

Literature[1]: Verordnung (EU) Nr. 1169/2011 des

Europäischen Parlaments und des Rates

vom 25. Oktober 2011 betreffend die

Information der Verbraucher über Lebens -

mittel und zur Änderung der Verord nun -

gen (EG) Nr. 1924/2006 und (EG)

Nr. 1925/ 2006 des Europäischen

Parlaments. at

[2]: LFGB. Bestimmung des Gesamtfettgehalts

in Fleisch und Fleischerzeugnissen –

Gravimetrisches Verfahren nach Weibull-

Stoldt. 3 (2014).

[3]: Matissek, R., Steiner, G. & Fischer, M.

Lebensmittelanalytik. (2010).

doi:10.1007/978-3-540-92205-6

[4]: Norm, D. Gas-Chromatographie von Fett -

säuremethylestern Teil 2: Herstellung von

Fettsäuremethylestern. 25 (Perinorm,

2011).

[5]: Reaktionsmechanismus_Veresterung.gif

(GIF-Grafik, 600 x 184 Pixel). at http://

www.mn-net.com/Portals/4/images/

Redakteure_Chroma/GC/Biodiesel-

Reaktion-DE600.gif

[6]: Veresterung_FAMES.jpg (JPEG-Grafik, 801

x 647 Pixel). at http://www.mobilohnefos-

sil.de/images/user/Biodiesel-Veresterung.

jpg

76

24

MP

ISO

MP

Micro

Choc

olate

ISO

Choc

olate

Micro

Saus

age

ISO

Saus

age

Micro

Chips

ISO

Chips

Micro

68 69 64

95

4159

90 95

32 31 36

5 510

Saturated fatty acids

0

20

40

60

80

100

%

Overview analyzed samples: ISO method vs.microwave method

Unsaturated fatty acids

Diagram 2: Overview of samples determined by comparing ISO procedure with

microwave procedure. Abbreviations: MP = Milk powder, ISO = ISO procedure,

Micro = Microwave procedure.

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