New testing methodology Polarizing sunglasses block the …€¦ · polarized light is very low. The black sunglasses show transmis-sion values of around 20 % for vertical polarized

Weight reduction

New testing methodologyfor composite material

Clear view in wet conditions

Polarizing sunglasses block theglare from wet surfaces

Less expensive and easy

Analysis of dioxins in foods and feeds using GC-MS/MS

Pharmaceutical

Plastics and Rubber

CONTENT

Clinical

Chemical, Petrochemical,Biofuel and Energy

Clear view in wet conditions –UV-Vis spectroscopy: Polarizing sunglasses block the glare from wet surfaces 2

What’s in the water? –Water analysis of humic acid withfluorescence spectroscopy 6

Quality control of zeolites for washing powder with EDX-8000P 9

Purification made easy – Prepara-tive purification of Ibuprofen andits related substances 12

New anti-doping method for equestrian sports 14

Less expensive with easier handling – Analysis of dioxins in foods and feeds using GC-MS/MS 16

Organic plastic in beverage bottles 22

Automotive industry – New testing methodology for weightreduction 24

Ethanol as a blending componentfor petrol – Determination of higher alcohols and volatile impurities by gas chromatogra-phic method 26

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Mission for a good cause –Social Day 2018: Shimadzu Europeplants 1,500 new trees 5

Pyrolysis GC-MS user meeting –March 28, 2019 at Shimadzu’s Laboratory WorldDuisburg, Germany 28

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MARKETS

Speeding up with Velox Core Shell –The new “Velox Core Shell“ LC columns offer more applicationpossibilities 20

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Food, Beverages, Agriculture

Environment

Automotive

Clear view in wet conditionsUV-Vis spectroscopy: Polarizing sunglasses block theglare from wet surfaces

A s the days become longer,sunglasses are a commonsight again. Today, there

are no limits to design and colorof frame and glasses.

But not all design choices arepurely aesthetic: Yellow glassesfor example enhance contrastwhile the advantage of greenglass es is lower color distortion.

Figure 1: Investigated sunglasses. Upper half: Models without polarizing filters,

lower half: Models with polarizing filters.

APPLICATION

3SHIMADZU NEWS 1/2019

Figure 3: Polarization by reflection on a surface. Light with polarization vertical

to the surface enters the surface, meaning that the horizontally polarized light is

reflected. The degree of polarization depends on the angle of incidence.

Figure 2: Vector representation of an electromagnetic wave with propagation direction

C, wavelength λ, electric field vector E and magnetic field vector B.

Figure 4: Polarizer (left) and depolarizer (right). The line on the polarizer mount indicates

the polarization direction of the transmitted light.

A depolarizer (figure 4, right side)converts polarized light into com-pletely unpolarized light [2].

Setup and samples

For the measurements describedhere, a Shimadzu UV-3600 Plus

spectrophotometer was used. Thebaseplate of the rotating film hold-er accessory was used for mount-ing the polarizer and depolarizer.This setup is shown in figure 5(page 4). To compare the effects ofpolarized and unpolarized light, afoil polarizer (spectral range 400 -700 nm) and a Hanle depolarizer(spectral range 180 - 2,500 nm)were used.

Two different sunglasses with po -lar izing filters and two differentsunglasses without polarizing fil-ters were investigated. The sampleswere carefully positioned so thatthe frame didn’t block the samplebeam. Of each sample, the trans-mission spectrum was measured in the wavelength range of 400 -700 nm in four different configu-rations:

• without any additional optics inthe light path

• with unpolarized light (depolar-izer in position A) �

Important for the necessary pro-tective functions are the shading(attenuation of the transmittedlight) and the wavelength rangecovered. Both are optimized bythe choice of base material andcoating. Quality glasses block alllight of wavelength 400 nm andbelow for complete UV protec-tion.

A special case are polarizing sun-glasses, promoted for watersportsand car driving purposes, just toname two examples. In this case,polarizing filters are built into theglasses which block horizontallypolarized light. Sunlight is gener-ally unpolarized, but the reflec-tion on a smooth surface causes apolarization horizontal to thatsurface. Polarizing filters are sup-posed to protect the user fromthis glare.

Polarization on a surface

Light can be described as an elec-tromagnetic wave, as shown infigure 2, with propagation direc-tion C, wavelength �, electric fieldvector E and magnetic field vectorB perpendicular to E. The direc-tion of the electric field vector isalso described as polarization. A beam of light is composed ofmany such waves with the samedirection of propagation. Depend -ing on the light source, the prop-erties of these photons can be verydifferent (e.g. sunlight) or uniform(e.g. laser).

The beam of light is polarizedwhen some polarization directionsare filtered out and one polariza-tion direction becomes dominant.One example is reflection on asmooth surface, as shown in figure3. The light with polarization hor-izontal to that surface is reflectedto a higher degree [1], e.g. bysnow or water, but also by theoptical element of a UV-Vis spec-trophotometer, such as a mirror orgrating. The degree of polarizationdepends on the angle of incidencein both cases.

This polarization of the samplebeam must be compensated forwhen a sample with polarizingproperties is examined. A polariz-er (figure 4, left side) is transpar-ent only to light with one polar-ization direction and is used toenforce a complete polarization ofthe sample beam. By this, polar-ization effects (e.g. by reflectionfrom the sample) are eliminated.

Figure 5: Experimental setup. A: Mounting position for polarizer or depolarizer,

B: Sunglasses, C: Sample beam, D: Reference beam.

• with vertical polarized light(polarizer set to 0° in positionA)

• with horizontal polarized light(polarizer set to 90° in positionA).

Polarizing filters for selective absorption

Figure 6 shows the transmissionspectra of the sunglasses describedabove. The samples are labelled bytheir glass color as orange (upperleft), green (upper right), black(lower left) and multicolored (low -er right). The spectra are arrangedanalog to the pho tos of figure 1(page 2).

Only the spectra of polarizingglasses without additional opticsshow peaks at 470, 580 and 665 nm.Transmission in these measure-ments is also below the valuesmeasured for the same glasseswith a depolarizer in the lightpath. This shows that the samplebeam is horizontally polarized tosome degree. As the degree ofpolarization depends on the angleof incidence to the polarizing ele-ment, this absorption is only indi-

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4 SHIMADZU NEWS 1/2019

Further information

on this article:

• Application:

shimnet.shimadzu.

local/product/Spectro/

Spectro_UV/Application_Notes/SCA-

100-022_Sunglasses.pdf

rectly dependent on the wave-length. The spectra measuredwith out depolarizer are influencedby such polarization effects.

For the orange and green sun-glasses, all four spectra are con-gruent. The polarization of thesample beam does not have any

influence on the measurements.This is different to the spectra ofthe black and multicolored sun-glasses. As expected from sun-glasses promoted for watersports,the transmission for horizontallypolarized light is very low. Theblack sunglasses show transmis-sion values of around 20 % forvertical polarized light in thismeasurement range, but only 10 %for unpolarized light and less than5 % for horizontal polarized light.

This effect is weaker with themulticolored sunglasses. The spec-tra measured with unpolarizedand vertically polarized light arenearly congruent. The attenuationof horizontally polarized light isstill remarkable though. The bestattenuation is observed in thespectral range of 500 - 620 nm,while the black sunglasses showan even attenuation over the wholespectral range up to 650 nm.

Conclusion

Many projects in the optic indus-try are concerned with polarizedlight. Using a depolarizer willensure that the sample is irradiat-ed with completely unpolarizedlight. Measurement artifacts aretherefore avoided and the trueproperties of polarizing samplesare elucidated.

Literature[1] https://en.wikipedia.org/wiki/

Polarization_(waves)

[2] https://en.wikipedia.org/wiki/Depolarizer_

(optics)0

20

40

60

80

100

0

20

40

0

20

40

100

0

20

40

Sunglasses Orange

Wavelength [nm] Wavelength [nm]

Wavelength [nm] Wavelength [nm]

Sunglasses Green

Sunglasses Black Sunglasses Multicolored

Tran

smis

sion

[%

T]Tr

ansm

issi

on [

%]

Tran

smis

sion

[%

T]Tr

ansm

issi

on [

%]

400 500 600 700 400 500 600 700

400 500

––– Normal ––– Unpolarized ––– Vertical polarized ––– Horizontal polarized

600 700 400 500 600 700

Figure 6: Transmission spectra of the four sunglasses from figure 1 (page 2), each measured in four configurations and in the spectral

range of 400 - 700 nm. Upper half: Samples without polarizing filters, lower half: Samples with polarizing filters.

LATEST NEWS


T he social day, at whichShimadzu employees sup-port social projects, has

become a tradition within thecompany since 2013. Once again,volunteers have been activelyinvolved in their community andin December 2018 helped to refor-est an area in the city forest ofDuisburg with 1,500 new trees.This will contribute to the long-lasting woodlands development.So far, the heavily thinned outarea was covered only with birchand bracken.

The 1,200 young European oakand 300 sweet chestnut, littleleaflinden and hornbeam have beenselected to fit the natural treespecies spectrum, growing well inthe sandy soil. Forests with theseindigenous trees serve as a habitatfor many rare and endangered ani-mals, plants and fungus species.Additionally, oak and linden treesare particularly long-lived, sofuture generations will also enjoythis biotope. The planted wood-

land is part of the DuisburgSechs-Seen-Platte (Six Lakes Re-gion), a recreational area with anetwork of hiking trails of 18 kilo-meters.

The two-three-year-old trees witha size of about 1.50 m were plant-ed relatively densely, so they candecide among themselves which of

Social Day 2018: Shimadzu Europe plants 1,500 new trees

Mission for a good cause

them grows best in the first 20 to30 years. This replicates naturethat produces an unimaginablylarge number of seedling offspringby natural seeding from the moth-er trees, which evolve over theyears so that only a few treesremain and their number contin-ues to reduce further as theygrow. Usually the trees prevail

which secure the best spot in thesun over the longest period,receiving the most sunlight in thelong term.

Social Responsibility

‘Realizing wishes for the well-being of mankind and the Earth’is one of Shimadzu’s establishedcorporate principles. Engaging incommunity, society and the envi-ronment expresses this claim atthe local level.

In the year of its 50th anniversaryin Europe, Shimadzu gladly sup-ported this long-term project andthanks all 30 participating employ -ees of Shimadzu Germany andShimadzu Europa for their indi-vidual social commitment.

APPLICATION


water. Humic acid is not a stablemolecule. It oxidizes throughoxygen and decomposes chemical-ly into tryptophan and L-tyro-sine. It could be deduced fromthis reaction that if no morehumic acid is present, the neces-sary amount of oxygen in thewater is also consumed [1], [2], [3].

In the following application, itwas interesting to determine thechange in humic acid content byfluorescence spectroscopy. Humicacid, tryptophan and tyrosineshow a fluorescence spectrumunder excitation in the ultraviolet

W ater is the basis of all life,all of nature and its habi-tats. When processed

into food as drinking water, it’s asubstance that is strictly tested.However, water also has an im-

into the ground andthen into waterbodies or deeperwells. It absorbs soilcomponents andcarries them in solu-tion or as suspendedparticles. There areboth visible andinvisible portions.

Visible particlesusually give thewater an earthycolor, an appearancethat is normal foroutdoor ponds orlakes (figure 1).However, if a slighttint in a bottle ofdrinking water isfound, it is per-ceived purely sub-jectively as deviat-ing from norm andexperience. Drin-king water is ex-pected to be color-less and clear.

The warm summerof 2018 brought asupply of still min-eral water with aslight tint (figure 2).This had to beinvestigated withfluorescence spec-troscopy.

It is known from the literaturethat water can contain humic acid,a natural degradation product oforganic sources such as leaves andgrasses. This occurs naturally withopen water, and the acid serves asfood for living organisms in the

portant task in chemistry since itis a good solvent and absorbsmany substances.

The most common way of waterto enter the natural cycle is as rain

What’s in the water?Water analysis of humic acid with fluorescence spectroscopy

Figure 2: Still mineral water with

a slight coloration from a delivery in

summer 2018

Table 1: Intensities of the fluorophores in the samples and references

A

B

C1

C2

FluorophoresHumic acid

20 mg/L

0

0

100

51

Tyrosine

1 mg/L

0

583

0

0

Tryptophan

1 mg/L

976

0

0

0

Bi-distilled

0

0

0

0

Mineral

water

0

0

27

17

Well water,

standing,

garden hose

34

44.5

34.9

30

Well water,

freshly pumped,

garden hose

0

0

36.6

26.5

Pond water

standing

86.1

45.7

24.9

17.3

Pond water with

fresh water exchange

38.6

32

82.6

40

Samples and references

Figure 1: Autumn view of a city park with a pond supplied with fresh water

range. Due to their chemical struc-ture, the complex molecules havemany ring systems, and individualelectrons can be excited to ener-getically higher states. The energyabsorbed in this way is releasedagain via radiation of photonswhen the electron returns to itsenergetic ground state.

For this fluorescence analysis arapid screening was used, endingin an EEM view (ExcitationEmission Matrix). Fluorescencespectroscopy is a very sensitiveand selective measurement tech-nique. It can make traces of fluo-rescent-active substances visible ina mixture.

Samples

Various samples were collected forthis study:

• A chemically pure water – double distilled,

• Well water, standing, from a garden hose,

• Well water, freshly pumped,from a garden hose,

• Pond water standing,• Pond water with fresh water

exchange, and • Mineral water without carbon

dioxide (still).

Reference materials were solutionsof tyrosine (1 mg/L), tryptophan(1 mg/L) and humic acid (20 mg/L,pH 8 to 9).

All liquids used were transferredinto a fluorescence cell. In thestandard version, the cuvettes havefour polished windows and a layerthickness of 10 x 10 mm. Thequartz in this cuvette is fluores-cence-free. The windows are need-ed because fluorescence is a scat-tered light which is detected at anangle of 90 degrees from the irra-diation direction of the excitationsource. The reference material wasbalanced and diluted with doubledistilled water. This water was alsotested to exclude possible contami-nation (see figure 3).

Analysis of DOM in natural water

“Dissolved Organic Matter” wasinvestigated with the ShimadzuRF-6000 fluorescence spectropho-tometer. �

bidest_water_180814_114003.fs3f - CorrectionDataEM Wavelenght (nm ) / EX Wavelenght (nm)

EM: 600.0/EX:250/-11.5

400 400

300

200

100

0

350

300

250250 300 350 400 450 500 550 600

humic_acid_20mg/L_180814_134403.fs3f - CorrectionDataEM Wavelenght (nm ) / EX Wavelenght (nm)

EM: 600.0/EX:250/43.9

400 400

300

200

100

0

350

300

250250 300 350 400 450 500 550 600

Tryptophan_1mg/L_180814_130607.fs3f - CorrectionDataEM Wavelenght (nm ) / EX Wavelenght (nm)

EM: 600.0/EX:250/-1.9

400 400

300

200

100

0

350

300

250250 300 350 400 450 500 550 600

Tyrosine_1mg/L_180814_115700.fs3f - CorrectionDataEM Wavelenght (nm ) / EX Wavelenght (nm)

EM: 600.0/EX:250/6.5

400 400

300

200

100

0

350

300

250250 300 350 400 450 500 550 600

fresh_water_source_1_180817_101228.fs3f - CorrectionDataEM Wavelenght (nm ) / EX Wavelenght (nm)

EM: 600.0/EX:250/-5.1

400 100

80

60

40

20

0

350

300

250250 300 350 400 450 500 550 600

fresh_water_source_2_180817_103733.fs3f - CorrectionDataEM Wavelenght (nm ) / EX Wavelenght (nm)

EM: 600.0/EX:250/-1.2

400 100

80

60

40

20

0

350

300

250250 300 350 400 450 500 550 600

waste_water_source_1_180817_105907.fs3f - CorrectionDataEM Wavelenght (nm ) / EX Wavelenght (nm)

EM: 600.0/EX:250/-1.2

400 100

80

60

40

20

0

350

300

250250 300 350 400 450 500 550 600

waste_water_source_2_180817_111956.fs3f - CorrectionDataEM Wavelenght (nm ) / EX Wavelenght (nm)

EM: 600.0/EX:250/5

400 100

80

60

40

20

0

350

300

250250 300 350 400 450 500 550 600

Figure 3: Representation of the EEM matrix of double distilled water (top left), humic acid (top right), tryptophan (bottom left)

and L-tyrosine (bottom right), the scaling of the representation was set to 0 - 400 intensities (red corresponding to very intense,

black up to no intensity).

Figure 4: Representation of the EEM matrices of standing well water in a hose (top left), flowing well water from a hose (top right),

standing pond water (bottom left) and flowing pond water (bottom right).


APPLICATION

APPLICATION


In a quick screening, EEM matri-ces of the samples and referenceswere created. The EEM matrix isformed when the excitation wave-lengths are increased slowly insmall steps and the respective flu-orescence spectrum is applied.Depending on the recordingspeed, it can take approx. 2 min-utes (60,000 nm/s) or approx. 13minutes (2,000 nm/s) to create thematrix.

From the reference measurements,it was possible to obtain the ana-lytical wavelengths for the identi-fication of the proteins involved.Distilled water shows no presenceof a fluorophore, as was to beexpected.

The excitation wavelength (EX) at275 nm and the emission wave-length range (EM) at 340 - 381 nmwere found for tryptophan. ForL-tyrosine the EX are at 275 nmand EM at 310 - 320 nm. Thehumic acid has two active surfacesin the EEM at EX 300 - 370 nmand EM 400 - 500 nm, as well asEX 240 - 260 nm and an EM of450 - 500 nm.

In the same way, the five samplesfrom different sources were exam-ined. All samples had lower con-centrations than the referencematerial. It became apparent (figures 4 [page 7] and 5), that themineral water contained a lowconcentration of a molecule simi-lar to humic acid. In the standingwaters, the signal group of humicacid has decreased strongly.Tryptophan and L-tyrosine-simi-lar substances predominate. Bothfresh waters, on the other hand,contain mostly proteins similar

ment processes and also for con-tamination of natural water.

Literature[1] Hudson, N., Baker, A. and Reynolds, D.

(2007). Fluorescence analysis of dis-

solved organic matter in natural, waste

and polluted waters – a review. River

Research and Applications 23: 631-649.

[2] Yan, Y., Li, H. and Myrick, M. L. (2000).

Fluorescence fingerprint of waters:

excitation-emission matrix spectroscopy

as a tracking tool. Applied Spectroscopy

54(10): 1539-1542.

[3] AD-0133, Shimadzu Asia Pacific, 2016

to humic acid. Table 1 (page 6)shows the detected intensities ofall samples and references in-volved to make the quantitativeaspect visible.

Conclusion

The Excitation Emission Matrix(EEM) of fluorescence spectros-copy is a very fast technique forobtaining an overview of dis-solved organic components (DOM)in water. With this option, thewater quality can be monitored,for example, in wastewater treat-

Further information

on this article:

• Application:

AD-0133: Excitation-

Emission Matrix (EEM)

Fluorescence Spectroscopy for Analysis

of Dissolved Organic Matter (DOM) in

Natural Water and Wastewaters

• Application:

SCA_105_010: Dissolved organic matter

analysis (DOM) and its appearance

under different environmental condi-

tions – f luorescence EEM matrices of

different sources

EM Wavelenght (nm ) / EX Wavelenght (nm)Mineral_water_180814_140456.fs3f - CorrectionData

EM: 600.0/EX:250/-0.1

250 300 350 400 450 500 550 600

400

350

300

250

100

80

60

40

20

0

EM Wavelenght (nm ) / EX Wavelenght (nm)bidest_water_180817_095419.fs3f - CorrectionData

EM: 600.0/EX:250/-8.3

250 300 350 400 450 500 550 600

400

350

300

250

100

80

60

40

20

0

Figure 5: Comparison of the EEM matrices of still mineral water from the bottle (summer 2018) and double-distilled water from the

laboratory. The mineral water exhibits fluorescence activity in the area of humic acid-like proteins.

A fast alternative for routine qual-ity checks is energy-dispersive x-ray fluorescence spectroscopy(EDX), e.g. with the EDX-8000P,a system penetrating samples withso-called hard x-rays of up to 50 keV. Here, the zeolite powderis filled in a special sample cupwithout pretreatment. Digestionof the sample is not required, andmeasurement time is on the scaleof a few minutes including prepa-ration.

The EDX-8000P detector is opti-mized for light elements such assodium, aluminum and silicon.For this measurement under vacu-um condition, the cups are closedby a seal permeable to air to pre-vent bursting of the cup by thesudden pressure change and spill-ing of the sample.

Quick measurements

The EDX-8000P has another ad-vantage for this application: thesample composition can be deter-mined by fundamental parameters(FP) methods without the need forstandards. �

Hard x-ray for softwaterQuality control of zeolites for washing powder withEDX-8000P

M any washing powderscontain zeolites for watersoftening. This role was

fulfilled in the past by phosphates,leading to a notorious growth ofalgae and severe damage of aquaticecosystems. Even though naturalzeolites exist, they are specificallysynthesized with defined stoi-chiometry and structure [1]. Onewidely used example is Zeolite Awith an equal content of aluminaand silicone. The advantage fortechnical applications is a well-defined anionic cage structuremade from ([AlO2]12[SiO2]12)12-

blocks as shown in figure 1.

Molecules such as water that aresmall enough to enter the poresare bound there until the zeolite isheated. The effective pore diame-ter for use as molecular sieve is on

the scale of 0.3 - 0.5 nm and de-pends on the cations bound to theanionic cage, since they block thepores to some degree depending on their size, charge and bindingforce.

For the application as water soften-er, only sodium and potassium arevalid cations. During the laundry,they are exchanged easily by calci-um or magnesium from the hardwater, which have a higher bindingforce to the zeolite cage. Withoutfree Ca2+ or Mg2+, the formationof lime is prevented. To ensure thebest product quality for each targetapplication, the elemental composi-tion (aluminum (Al), silicon (Si),sodium (Na) and possibly otherelements) of these zeolites must becontrolled.

Easy sample preparation

A common method for precise ele-mental analysis is inductively cou-pled plasma optical emission spec-troscopy (ICP-OES or short ICPE).For mineral samples, such as zeo-lites, a thorough digestion is need-ed. Sample preparation takes sometime and requires the use of chemi-cals such as hydrofluoric acid.

Figure 1: Structure of Zeolite A. Al and Si atoms (nodes) are connected by shared O

atoms (lines), leading to a cage of alternating AlO4 and SiO4 tetrahedrons. Only some

atoms are labelled to indicate the binding motif.

Figure 2: Zeolite powder prepared for EDX measurement. Left: open cup, right: sealed

cup for vacuum measurements.


APPLICATION

For the mean deviation shown intable 3, relative deviation of eachEDX value from the ICPE valueof the same sample was calculatedfor each method and sample (seeformula 1 page 11).

The average of these deviationsover all samples was then calculat-ed for each method (see formula 2page 11).

When the interfering element ishard to identify, testing differentcorrections as in this example andfinding the method with the small-est deviation is a good startingpoint. The correcting elementshould have a small coefficient(±0.001) to avoid overcompensa-tion.

Conclusion

The EDX-8000P is the ideal in-strument for quick quality checks.The fundamental parametersmethod can be enhanced by addi-tional data, e.g. from loss of igni-tion measurements to measureunknown samples without theneed for calibration. Using matrixcorrections, analysis of more com-plicated minerals is possible. Thisway, the EDX method applyinghard x-rays supports applicationsfor water softening.

AcknowledgementsWe gratefully acknowledge the assistance

and supply of samples from our colleagues

in Shimadzu d.o.o. Sarajevo, Bosnia and

Herzegovina.

Literature[1] Zeolithe – Eigenschaften und technische

Anwendungen, Lothar Puppe, Chemie in

unserer Zeit 1986, https://doi.org/

10.1002/ciuz.19860200404

[2] X‐ray fluorescence analysis applying the-

oretical matrix corrections. Stainless

steel, Willy K. De Jongh, X-Ray Spectro-

metry 1973, https://doi.org/10.1002/xrs.

1300020404

APPLICATION


Two such fundamental parametersare the order number and concen-tration of the elements included,which are given as results from theintensities of characteristic lines.Other fundamental parameters, likethe sample shape, density or fixedconcentrations, are either addedmanually or calculated from scatterpeaks to support the algorithm.

FP methods are used for quickscreening and depend strongly onhow well the sample is describedwithin the FP model. A higherquality quantitation of the elementsis possible using calibration curves.Here, four samples of Zeolite 3Awith different ratios of sodium topotassium were analyzed with an

FP method and a quantitativemethod calibrated with ICPEresults.

Figure 3 shows a typical EDX-spectrum of a synthetic zeoliteafter 100 seconds measurementtime. All peaks detected over thedefined measurement range are fitted and analyzed by an FPmethod. The rhodium lines arebackground from the x-ray tubeand not used for the quantitativeanalysis.

Table 1 compares ICPE resultsgiven by the manufacturer of thesamples to semi-quantitative andquantitative EDX measurementsfor all Zeolite 3A samples.

The FP values for aluminum andsilicon show a good match withthe ICPE values, while the valuesfor sodium and especially potassi-um show a bigger deviation. Withthe calibration curve method, onlya small deviation from the ICPEvalues was found for all oxides.

To calibrate the EDX method,ICPE results of the same sampleswere used as standards. Calibra-tion curves are shown in figure 4.While quantitation of aluminumand silicon by this calibrationcurve method seems to be inferiorto the FP method at first glance,all standards had nearly identicalcontents of these elements, sothere was not enough difference inintensity for a linear fit.

Matrix corrections

One important raw material forzeolite is the aluminum or baux-ite, where the content of alumi-num and undesired oxides must bemonitored to optimize the processparameters. The analytical meth-ods used for artificial zeolites areeasily adopted for the qualitycheck of natural bauxite. The pos-sible influence of other oxides onthe aluminum intensity musthowever be considered.

To show the effect of differentmatrix corrections, table 2 lists theresults for five different samplesof bauxite measured with differentmethods. The same samples wereused as standards. Iron was foundto be the interfering element inthe case of aluminum in bauxite.This was derived from the obser-vation that the change of the alu-minum line intensities in sampleswith constant concentration ofaluminum correlated to a changeof the iron concentration.

To test the matrix correction, theDe Jongh (dj) method [2] wasused with one element for calcula-tion of the correction factor. Eachelement was applied once for thecorrection and the aluminum con-centration in each sample was re-calculated once with each method.Table 3 shows the coefficient andaverage deviation from the ICPEvalue for each correcting element.As expected, the best results arefound with iron as element for thecorrection.

0

1

2

3

4

5

6

Energy [keV]

Inte

nsit

y [c

ps/μ

A]

0.5 1 1.5 2 2.5 3 3.5 4

Figure 4: Calibration curves for the EDX method from table 1

Figure 3: EDX spectrum of Zeolite A3 sample 1 at 15 kV tube voltage (C - Sc channel),

100 s live time and vacuum. The rhodium lines are scattered radiation from the x-ray

tube.

0.0

0.0

0.5

1.0

1.5

2.0

5.0

10.0

15.0

20.0

0.0

10.0

20.0

30.0

40.0

50.0

25.0

30.0

35.0

Standard value [%]

Mea

sure

d in

tens

ity

[cps

/μA

]

Standard value [%]

Mea

sure

d in

tens

ity

[cps

/μA

]

0.0

10.0

20.0

30.0

40.0

50.0

60.0

70.0

Mea

sure

d in

tens

ity

[cps

/μA

]M

easu

red

inte

nsit

y [c

ps/μ

A]

0.0

0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0

Standard value [%]

0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0

5.0 10.0 15.0 20.0 25.0 30.0 35.0

Standard value [%]

0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 35.0

Element

none

Si

Ca

Ti

Fe

Coeff.

none

-0.003

-0.054

0.137

-0.007

Mean deviation

4.80 %

4.94 %

15.63 %

3.71 %

3.54 %

Matrix correction for Al2O3

Table 3: Elements und coefficients for the dj matrix correction from table 2 with

corresponding mean deviation from ICPE values

ICPE

FP

Cal. (no corr.)

Cal. (Si corr.)

Cal. (no corr.)

Cal. (Ti corr.)

Cal. (Fe corr.)

78.25

84.02

74.22

75.49

77.45

77.43

77.14

59.58

68.75

59.90

57.60

58.85

56.43

61.29

60.77

67.34

65.03

64.66

65.33

60.44

61.40

62.63

68.69

67.08

67.09

39.84

61.29

65.13

66.78

67.63

63.71

63.64

59.80

61.16

61.49

Baux 1 Baux 2 Baux 3 Baux 4 Baux 5

Concentration of Al2O3 in %wgt.

Table 2: Aluminum content in five different samples of bauxite, measured with different

methods. The calibrated EDX methods differ only in the element used for calculation in

the dj matrix correction.

Table 1: Comparison of the results for Al2O3, SiO2, Na2O and K2O for different zeolite samples and methods. All elements are defined as oxides for a better description of the

mineral sample.

Method

Al2O3

SiO2

Na2O

K2O

SampleICPE

33.06

41.90

15.09

9.59

FP

33.34

40.78

13.86

11.98

Cal.

34.05

40.24

15.38

9.68

ICPE

33.77

41.90

12.05

12.57

FP

32.32

40.70

10.85

16.02

Cal.

34.00

41.20

11.89

13.04

ICPE

34.67

40.34

11.27

13.39

FP

32.49

39.57

10.81

17.03

Cal.

34.38

40.33

11.69

13.72

ICPE

33.18

40.44

10.50

15.42

FP

32.33

39.55

9.67

18.29

Cal.

34.68

40.71

10.36

14.86

Zeolite 3A sample 1 Zeolite 3A sample 2 Zeolite 3A sample 3 Zeolite 3A sample 4

Concentration in %wgt

EDX(Baux1, FE) – ICPE(Baux1)Dev (Baux1, FE) = · 100 %

ICPE(Baux1)

Dev(Baux1, FE) + Dev(Baux2, FE) + ... Dev (FE) =

5

APPLICATION

Formula 2: Average deviation – calculated for each methodFormula 1: Sample deviation

Delivering confidence

Automotive

full range of analytical and testing solutions Visit Shimadzu at

May 21-23, 2019Stuttgart, GermanyStand 8530

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pounds to be recovered as freebases which are generally easier topowderize, typically providingbetter quality results when used indrug screening and pharmacoki-netic studies. All eluted com-pounds are finally collected in ahighly volatile organic solvent,which reduces fraction dry-downtime by up to 90 % compared toconventional LC fractions.

Preparative purification ofIbuprofen and its analogs

Ibuprofen is one type of nonste-roidal anti-inflammatory drug(NSAID) used as a fever-reducingdrug and analgesic. The UnitedStates Pharmacopeia (USP) pro-vides analytical methods for Ibu-profen and its analog 4-isobutyl-acetophenone, using valerophe-none as an internal standard [2].This article describes preparativepurification of these three compo-nents using the Shimadzu UFPLCsystem (figure 3). Preparative LCand purification conditions aredisplayed in table 1.

Figure 4 shows the preparative LCchromatogram of the mixed solu-tion of Ibuprofen and relatedcompounds. It was prepared bydissolving the three target com-

Purification made easyPreparative purification of Ibuprofen and its related substances by Prominence UFPLC

P harmaceutical companies are developing compoundsof increased complexity –

drugs with numerous functionalgroups in a single molecule, poly-meric compounds, biopharmaceu-ticals such as peptides, proteinsand many others – that must bepurified, in production quantities.Regulatory agencies continue topress for more stringent require-ments in purity of pharmaceuticalproducts and, especially, drugsubstances. An acute need existsfor other tools in addition to crys-tallization, the classic techniquefor purification, to address agrowing number of purificationproblems.

Currently, preparative HPLC isthe most powerful and versatilemethod for purification tasks inthe pharmaceutical industry. TheProminence Ultra Fast Preparativeand Purification Liquid Chroma-tograph (UFPLC) enables sub-stantial labor savings in prepara-tive purification.

This happens by automation notonly of fractionation of the targetcompound but also the relatedprocesses of concentration, purifi-cation and recovery. This articleintroduces an example of prepara-tive purification of a mixed sampleof the pharmaceutical compoundIbuprofen and its analogs usingShimadzu’s UFPLC AdvancedSystem (figure 1) [1].

Procedures of preparativepurification using the ShimadzuUFPLC system

UFPLC automatically performsthe various processes related topreparative isolation of targetcompounds using a combinationof preparative LC and trappingcolumns. Details of these process-es are as follows:1. separation of target compounds

in complex sample by prepara-tive LC and introduction intotrapping columns

2. replacement of solvent in trap-ping columns with ultrapurewater

3. elution of target compoundsfrom trapping columns byorganic solvent.

An outline of the respective pro-cesses is shown in figure 2.

The system integrates preparativeLC with fraction trapping for upto five compounds of interest. It iscontrolled by a dedicated walk-upsoftware designed to simplify theworkflow also for non-expertusers. It allows to easily set condi-tions for chromatographic separa-tion and isolation of target com-pounds, trapping, eluting and col-lecting highly purified compoundsin as little as 90 minutes. Forapplications involving the isola-tion of low concentration targets,replicate injection and collectionto the same trapping column to

increase the amount of compoundtrapped on column prior to elu-tion is easily accomplished.

High purity compounds, optionally recovered as a free base

The Prominence UFPLC elimi-nates some of the problems asso-ciated with conventional prep LC,especially poor purity of collectedcompounds due to mobile phaseadditives, which become contami-nants in the final collected fractionand inhibit powderization.Shimadzu’s “Shim-pack C2P-H“trapping column strongly retainstarget compounds, allowing un-wanted organic solvents, waterand additives to be flushed away.Additionally, rinsing the columnwith an aqueous ammonia solu-tion after trapping allows com-

Figure 1: Prominence UFPLCTM

Figure 2: Prominence UFPLCTM workflow of fractionation, concentration, purification and elution

APPLICATION


pounds with mobile phases to aconcentration of 5,000 mg/L.

Verification of purity ofIbuprofen and its analogs

The fractions of Ibuprofen, vale-rophenone and 4-isobutylaceto-phenone collected by UFPLCwere analyzed by standard HPLCto verify the purity of the com-pounds. Table 2 shows the analyti-cal conditions and figure 5 showsthe chromatograms obtained foreach fraction. Purities of the targetcompounds contained in eachfraction determined by area nor-malization at UV 230 are listed intable 3.

Conclusion

Prominence UFPLC Ultra-FastPreparative and PurificationLiquid Chromatograph wasshown to enable fast recovery of

highly purified target compoundsin a simple automated workflow.Ultrahigh weight purity com-pounds can be obtained by imple-menting preparative separationsand fraction purification. Forpowderization of the pure sample,an extremely long drying time isnecessary using conventionalreversed-phase preparative LC,due to a high amount of water inthe mobile phase. Moreover, incases where a nonvolatile buffersolution is used, the salt can pre-cipitate after drying. In prepara-tive purification using UFPLC, it is possible to remove the non-volatile salt used in the separationprocess, as desalting is performedin the trapping columns. Dryingtime is substantially reduced byuse of organic solvent for samplerecovery from the trapping col-umns, contributing greatly toimproved efficiency in any appli-cation requiring preparativepurification.

Literature[1] N. Kosuke, Shimadzu Application News

No. L526, Preparative Purification of

Ibuprofen and Its Related Substances

by Prominence UFPLC

[2] USP Monograph for Ibuprofen

Figure 3: Chemical structures of Ibuprofen

and related compounds

Figure 4: Preparative LC chromatogram of Ibuprofen and its analogs

(UFPLC)

Figure 5: HPLC analysis of Ibuprofen and related compounds in fractions obtained

by UFPLC

Table 1: Conditions of preparative LC separation and purification by

UFPLC system

Table 3: Purities of target compounds contained in collected fractions

(area percentage, UV 230 nm)

Table 2: Conditions of analytical HPLC separation of purified fractions

0

mAU

Minutes

0.0

100

200

300

400

2.5 5.0 7.5

0

mAU

Minutes

0.0

500

1,000

1,500

2.5 5.0 7.5

Column

Mobile phase

Flow rate

Column Temperature

Injection volume

Detection

Shim-packTM VP-ODS (250 × 10 mm, 5 μm)

A: 1 % (wt/v) chloroacetic acid (pH 3.0)

B: Acetonitrile

A/B = 2/3 (v/v)

9.0 mL/min

Ambient

100 μL

UV 230 nm

Column

Rinse solvent

Time program

Shim-packTM C2P-H (30 × 20 mm, 25 μm)

A: 2 % (v/v) acetonitrile aq. sol., B: water

A: 15 mL/min (0 - 2 min) → A: 8 mL/min

(2.01 - 4 min) → B: 8 mL/min (4.01-8 min)

Preparative LC conditions

Rinse conditions

Eluent

Flow rate

Detection

Acetonitrile

4.5 mL/min

UV 230 nm

Elution conditions

Column

Mobile phase

Flow rate

Column Temperature

Injection volume

Detection

Shim-packTM VP-ODS (250 × 4.6 mm, 5 μm)

A: 1 % (wt/v) chloroacetic acid (pH 3.0)

B: acetonitrile

A/B = 2/3 (v /v)

2.0 mL/min

30 °C

10 μL

UV 230 nm

Analytical LC conditions

Ibuprofen

Valerophenone

4-Isobutylacetophenone

99.2

99.6

99.8

Compounds Area %

APPLICATION


T he Institute of ToxicologicalResearch at Wroclaw Medi-cal University in Poland

routinely analyzes biological sam-ples and evidence material oncommission of the police, theprosecutor’s office and the courts.Research encompasses both tar-geted and screening analysesincluding pharmaceuticals, drugsof abuse, new psychoactive sub-stances, alcohols, volatile sub-stances, anabolics and many oth-ers. The Institute is also interestedin developing new methods ofanalysis for specific needs of cus-tomers from scientific fields,industries, veterinary medicineand medicine.

The Equine Internal MedicineUnit, part of the Department ofInternal Disease with Clinic for

limb localized pain. Dueto strong analgesic prop-erties and fast action,capsaicin has been placedon the Equine Prohibi-ted Substances List creat-ed by the InternationalFederation for Eques-trian Sports.

Y. You et al. [1] in 2013proved the possibility ofusing UHPLC-MS-MSfor the detection of cap-saicin in plasma samples.The method described isfast, selective, sensitive,reproducible, reliable andfully validated.

The aim of this papers’[2] research was to deter-mine the time limit forthe detection of capsaicinand dihydrocapsaicinafter long-term use of gelcontaining capsaicin onhorses. Of particularinterest was to determine

how soon after prolonged use ofointment or gel with capsaicin inpain treatment (following manu-facturer application recommenda-tions), horses would be able to

In the case of horses, both cap-saicin and dihydrocapsaicin areused as analgesic and warminggels or ointment. They are widelyused in treatment of lameness and

Horses, Dogs and Cats, is a scien-tific group focused on clinicalwork, teaching and research in thefield of internal diseases of horses.Main current research of groupmembers includes asthma, meta-bolic and endocrine diseases andkidney failure of horses. Appli-cation possibilities of innovativebiomarkers in diagnostics of inter-nal diseases of horses are alsoinvestigated.

Strong analgesic properties of Capsicum peppers

Capsaicin and dihydrocapsaicin,alkaloids from Capsicum peppers,are used in medicine and veteri-nary medicine due to their stronganalgesic properties. Local anal-gesic effect is related to defunc-tionalization of nerve endings.

New anti-doping method for equestrian sportsApplication of LCMS-8050 to quantify capsaicin and dihydrocapsaicin in horse serum

Members of research team

Members of research team


APPLICATION

take part in competition bearingin mind the anti-doping regula-tions applied in equestrian sports.

Sample preparation

A serum sample (200 μL) wastransferred into a 2 mL polypro-pylene tube. 20 μL of an internalstandard solution (methanol solu-tion of phenacetin, 100 ng/mL)was added. A liquid-liquid extrac-tion using dichloromethane (1 mL)was carried out for five minutes.After shaking, samples were cen-trifuged at 10,000 rpm for fiveminutes. The organic phase wastransferred into a clean 2 mL tubeand evaporated to dryness under a stream of nitrogen at 40 °C. Theextract was dissolved in 25 μL ofmethanol, transferred to an inertglass insert and analyzed usingUHPLC-QqQ-MS/MS.

UHPLC-MS/MS analysis

Analysis was performed using aNexera X2 UHPLC system cou-pled with an LCMS-8050 triplequadrupole mass spectrometerwith ESI in positive ionization.The analytes were then quantifiedby multiple reaction monitoring(MRM). MRM transitions werem/z 180.30 → 110.05; 180.30 →93.10; 180.30 → 65.05 for phena-cetin, m/z 306.10 → 137.05;

Y. You et al. research from 2013 oncapsaicin and dihydrocapsaicinconcentrations in equine plasma[1] has proven that after applica-tion of paste containing 0.025 %of capsaicin, both capsaicin anddihydrocapsaicin occurred in plas-ma, with a concentration of nearly242 and 155 pg/mL respectively intwo hours, but after 24 hours theconcentrations declined to nearly5 and 3 pg/mL, respectively. Byreaching LOQ at 0,5 pg/mL and 1 pg/mL level, the method pre-sented therefore enables effectivedetection of capsaicin even after24 hours following application ofgels containing this compound.

Very good linearity was achievedin the concentration range from0.5 to 1,000 pg/mL (capsaicin) and1 to 1,000 pg/mL (dihydrocapsai-cin). Calibration curves and linearregression coefficients are shownin figure 2.

Real samples analysis

The method described was suc-cessfully applied for the detectionof capsaicin and dihydrocapsaicinin horse serum following long-term local administration [2].

Conclusions

The undoubted advantages of themethod presented are its simplici-ty, high sensitivity and the fastsample preparation procedure.The possibility of detection ofcapsaicin and its metabolite inconcentrations from 0,5 pg/mLrespectively 1 pg/mL enables theeffective fight against this kind of doping in equestrian sports.Capsaicin also has more applica-tions, so after small modifications,

306.10 → 94.10; 306.10 → 122.05for capsaicin and m/z 308.40 →137.10; 308.40 → 94.10; 308.40 →122.10 for dihydrocapsaicin. Theanalytes were separated using aC18 1.7 μm, 2.1 x 50 mm columnat 40 °C. A combination of 10mM ammonium formate / 0.1 %formic acid in water (A) and 0.1 % formic acid in acetonitrile(B) was used as mobile phase.Injection volume was 1 μL.

Results

The limits of quantification of theanalysis were 0.5 pg/mL for cap-saicin and 1 pg/mL for dihydro-capsaicin. Figure 1 shows the chro-matogram of pretreated horse se-rum containing capsaicin (1 pg/mL)and dihydrocapsaicin (1 pg/mL).

0.00

0.25

0.50

0.75

1.00

1.25

1.50

1.75

2.00(x 10,000)

Minutes

2.65 2.70 2.75 2.80 2.85 2.90 2.95 3.00 3.05 3.10 3.15 3.20 3.25 3.30 3.35 3.40 3.45 3.50 3.55 3.60 3.65 3.70 3.75 3.80

Figure 1: MRM of capsaicin and dihydrocapsaicin in blank equine serum at concentration of 1 pg/mL

0.0

0.5

1.0

1.5

2.0

2.5

Concentration

Are

a

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0

0.00

0.25

0.50

0.75

1.00

Concentration

Are

a

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0

Figure 2: Calibration curves of capsaicin (above) and dihydrocapsaicin (below)

the method described can be usedfor determination of capsaicin anddihydrocapsaicin in chili peppers,self-defense weapons or biologicalsamples collected from individualsexposed to these substances.

AuthorsPaweł Szpot, Marcin Zawadzki, Marta

Siczek, Agnieszka Zak, Natalia Siwinska,

Malwina Słowikowska, Artur Niedzwiedz

The Equine Internal Medicine Unit, part of

the Department of Internal Disease with

Clinic for Horses, Dogs and Cats, Wrocław,

Poland

Literature1. Y. You, C. E. Uboh, L. R. Soma, F. Guan,

D. Taylor, X. Li, Y. Liu, J. Chen: Validated

UHPLC–MS-MS Method for Rapid

Analysis of Capsaicin and Dihydrocapsa -

icin in Equine Plasma for Doping

Control, Journal of Analytical Toxicology

(2013) 37(2) 122-132. DOI 10.1093/jat/

bks098.

2. A. Zak, N. Siwinska, M. Slowikowska,

H. Borowicz, P. Szpot, M. Zawadzki and

A. Niedzwiedz: The detection of cap-

saicin and dihydrocapsaicin in horse

serum following long-term local adminis-

tration BMC Veterinary Research (2018)

14:193. DOI 10.1186/s12917-018-1518-9

(x 1,000) (x 1,000) (x 1,000)

(x 1,000) (x 1,000) (x 1,000)

(x 1,000) (x 1,000) (x 1,000)

0.51.01.52.02.53.0

0.5

1.0

1.5

2.0

2.5

3.0

0.5

1.0

1.5

2.0

2.5

3.0

3.54.04.55.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

0.5

1.0

1.5

2.0

2.5

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

9.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

1.0

2.0

3.0

4.0

5.0

6.0

17.0 17.5 18.0 18.5

36.5 37.0 37.5 38.0

27.0 27.5 28.0 28.5 31.0 31.5 32.0 32.5 36.5 37.0 37.5 38.0

16.5 17.0 17.5 18.0 20.5 21.0 21.5 22.0

22.0 22.5 23.0 23.5 27.5 28.0 28.5 29.0

1 2 3

4 5 6

7 8 9

APPLICATION

P ersistent organic pollutants(POPs) are semi-volatile andcan be detected worldwide,

even in remote regions. They bio-accumulate with potential negativeinfluence on environment and hu-man health. To a varying degree,these organic compounds resistphotolytic, biological and chemi-cal degradation.

POPs in foods and feeds can beanalyzed with several methods.Dioxins are particularly toxic evenfor POPs, so quantitative analysisis required down to low concen-trations. Until recently, the analy-sis of dioxins was performed usinghigh-resolution GC-HRMS,which provides highly accuratequantitation. However, triplequadrupole GC-MS/MS is lessexpensive and easier to handlethan GC-HRMS, so its use isincreasingly being investigated.

In recent years, the quantitativeaccuracy of GC-MS/MS has im-proved significantly. Accordingly,the use of this analysis method hasbeen officially recognized in theEU (EU589/2014, 644/2017).However, to change from GC-HRMS to GC-MS/MS it is neces-sary to compare their respectivequantitative abilities.

The Shimadzu GCMS-TQ8050NX triple quadrupole mass spec-


Less expensive with easierhandlingAnalysis of dioxins in foods and feeds using GC-MS/MS

Figure 1: GCMS chromatograms for a concentration of 0.050 pg/uL

GCMS-TQ8050 NX

TEQ (pg/μL) TEQ (pg/μL)

TEQ (pg/μL) TEQ (pg/μL)

TEQ (pg/μL)

TEQ (pg/μL)

TEQ (pg/μL)

0

0

0.1

0.2

0.3

0.4

0.5

0

0.1

0.2

0.3

0.4

0.5

0

0

0.1

0.2

0.3

0.4

0.5

0.20.40.60.8

11.2

0

0.1

0.2

0.3

0.4

0.5

0.02

0.04

0.06

0.08

0.1

0

0.02

0.04

0.06

0.08

0.1

1

GC-HRMS

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45

Sample 1 Sample 2 Sample 3 Sample 4 Sample 5

Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Sample 6 Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Sample 6

Sample 1 Sample 2 Sample 3 Sample 4 Sample 5

2 3 4 5 6 7 8 0 10 11 12Sample

1Sample

2Sample

3Sample

4Sample

5Sample

6Sample

7

Pork (Fat) Raw milk

Equine (Liver) Oil

Hen (Egg)

Grass

Leek

GC-MS/MS

GC-HRMS GC-MS/MS

GC-HRMS GC-MS/MS

GC-HRMS GC-MS/MS

GC-HRMS GC-MS/MS

GC-HRMS GC-MS/MS

GC-HRMS GC-MS/MS

APPLICATION


automatic pretreatment unit (ex-traction: SpeedExtractor [BUCHI];purification: GO-xHT [Miura Co.,Ltd.]). 10 uL of Nonane was usedas final solvent for the samples.For the standard, a mixture of DF-

ST and DF-LCS from Wel lingtonLaboratories was used. In terms of the analytical conditions forGC-MS/MS, the conditions regis-tered in the method package wereused.

Analytical conditions in detail areshown in table 1 (page 19). �

Figure 2: Comparison of the TEQ results for each food and feed

trometer uses a high-sensitivitydetector, capable of detection atfemtogram order concentrations,enabling the analysis of dioxins infoods and feeds. Additionally, the“GC-MS/MS Method Package forDioxins in Foods” consists ofmethod files registered with opti-mal conditions for the analysis ofdioxins, as well as a report creationtool which documents the itemsrequired by EU regulations.

In this article, dioxins (polychlori-nated dibenzo-p-dioxin (PCDD)and polychlorinated dibenzofuran(PCDF) only) were analyzed in 44types and 200 samples of foods andfeeds using the GCMS-TQ8050 NXin combination with the methodpackage. Additionally, GC-MS/MSanalysis results were comparedwith those from GC-HRMS inorder to evaluate the quantitativecapabilities of both techniques.

Method files for the analysis of dioxins

The features of the “EU Regula-tion Compliant GC-MS/MSMethod Package for Dioxins inFoods” are shown below.

• Method files registered withoptimal conditions for the ana-lysis of dioxins.

• Retention times and time pro-grams can be adjusted automati-cally, even when the retentiontimes for the measured com-pounds change, such as whenconducting maintenance of thecolumn tip.

• A report creation tool is includ-ed in this product. It can auto-matically create reports showingitems required by EU regula-tions.

Experiment

For the various food samples, pre-treatment was performed using an

100

10

11 10 1000.1

0.1

0.01

0.001

0.010.001

GC-HRMS TEQ (pg/μL)

GC

-MS/

MS

TEQ

(pg

/μL)

Boar (Muscular)

Carp

Rape oil

Fish

Honey

Mixed milk

Oysters

Raw milk

Tuna

Boar (Liver)

Cereal

Deer (Muscular)

Pork fat

Leek

Moules

Pigment Egg Yolk

Salad

Veal (Grease)

Bovine (Grease)

Chicken (Egg)

Deer (Liver)

Grass

Cheese

Oil

Pike

Sediment

Veal (Muscular)

Cabbage

Corn

Equine (Liver)

Heifer (Milk)

Mackerel

Ovine (Liver)

Powder

Soil

Water

Canola seed

Dairy product

Feed mixes

Hen (Egg)

Milk

Ovine (Fat)

Quail (Muscular)

Straw

APPLICATION

Analysis results for the standards

In the analysis of dioxins in foods,Maximum Levels (ML) are pre-scribed for each sample. With thefood and feed samples in thisinvestigation, ML for pig fat andpig meat were the lowest at 1 pg/gof fat. Additionally, the limit ofquantitation (LOQ) required foreach compound in the analysisdepends on the sample’s ML, the

S/N Ratio (hereinafter »Method 1«)

The concentration of an analyte inthe extract of a sample which pro-duces an instrumental response attwo different ions to be moni-tored with an S/N (signal/noise)ratio of 3 :1 for the less intensiveraw data signal.

Lowest concentration point on the calibration curve(hereinafter »Method 2«)

The lowest concentration point ona calibration curve that gives anacceptable (≤ 30 %) and consistent(measured at least at the start andat the end of an analytical series ofsamples) deviation from the aver-age relative response factor calcu-lated for all points on the calibra-tion curve in each series of sam-ples. In this technical report, forthe purposes of confirmation, anevaluation was performed usingboth criteria.

As noted above, for 2,3,7,8-Tetra-chlorodibenzo-p-dioxin it is necessary to set the LOQ to 0.060 pg/uL or less. Accordingly,the STD was prepared so that theconcentration of each compoundwas 0.050 pg/uL (for Octachloro-dibenzo-p-dioxin and Octachlo-rodibenzofuran 0.100 pg/uL).From the results of the analysis, itwas evident that the criteria formethod 1 were satisfied for allcompounds. S/N ratios for eachcompound are shown in figure 1and table 2.

Additionally, with method 2, acalibration curve was created with all six levels used, including0.025 pg/uL, 0.050 pg/uL, 0.100pg/uL, 0.250 pg/uL, 0.500 pg/uLand 1 pg/uL. The concentrationsfor each compound at each cali-bration curve point (level) areshown in table 3. For each com-pound, when the level 1 RRF andaverage RRF were compared, itwas found that all compounds sat-isfied the criteria for method 2.From the above-mentioned re-sults, it was evident that at theLOQ, the criteria were satisfiedfor all compounds.

pretreatment method and the TEF(toxic equivalence factor) of eachcompound.

The compounds 2,3,7,8-Tetrachlo-rodibenzo-p-dioxin and 1,2,3,7,8-Pentachlorodibenzo-p-dioxin havethe highest TEF (TEF = 1), re-quiring lower LOQs than othercompounds. In this investigation,the LOQ for both dioxins in pigfat and pig meat was 0.060 pg/uLat the concentration in the final vial.


Figure 3: Summarized comparison of the TEQ results for TQ and HR MS

APPLICATION


Analysis results for the test samples

As previously noted, the level oftoxicity differs for each dioxincompound. The TEF, calculatedfor each compound by taking thetoxicity of 2,3,7,8-Tetrachlorodi-benzo-p-dioxin as 1, is used as anindex of strength. The TEF valuesfor each compound are shown intable 3. The ML for the dioxins infoods and feeds are prescribed bytheir toxic equivalents (TEQ). TheTEQ is calculated by multiplyingthe concentration of each com-pound by the TEF and then calcu-lating the total TEQ for all com-pounds.

Figure 2 (page 17) shows a com-parison of the TEQ values forGC-MS/MS and GC-HRMS forfood samples. A TEQ of 0.060 pg/uL (red line) and a TEQ of 0.025pg/uL (green line) are marked asindicators for the samples.

Conclusion

In this technical report, dioxinswere analyzed in 44 types and atleast 200 samples of foods andfeeds using the GCMS-TQ8050and the “EU Regulation Com-pliant GC-MS/MS Method Pack-age for Dioxins in Foods”. Addi-tionally, the GC-MS/MS analysisresults were compared with theanalysis results from GC-HRMSin order to assess the quantitativecapabilities of both methods. Be-fore analyzing, a STD was meas-ured using GC-MS/MS, and itwas confirmed that the criteriawere satisfied at the LOQ.

From the above-mentioned re-sults, it is evident that analysiswith GCMS-TQ8050 NX andmethod package provides a quan-titative capability equivalent tothat of GC-HRMS for samples atthe concentration levels requiredfor analysis. However, at concen-trations below the required level,differences in quantitative capabil-ity could arise. For this reason, itis necessary to be aware of thesystem status by confirming quan-titative capability at the LOQ,and evaluating whether there hasbeen a decrease in sensitivity.

Table 1: GC-MS/MS analytical conditions

Table 2: S/N results for standards according to method 1

Table 3: Each calibration point concentration and RRF for the measured compounds

Pretreatment Unit

(Extraction)

Pretreatment Unit

(Purification)

Autosampler

GC-MS/MS

Software

# of Rinses with

Solvent (Pre-run)

# of Rinses with

Solvent (Post-run)

# of Rinses with

Sample

Washing Volume

Injection Volume

Viscosity Comp. Time

Speed Extractor (BUCHI)

GO-xHT (Miura Co., Ltd.)

AOC-20i/S

GCMS-TQ-8050 NX

GCMSsolution™ Ver. 4.45 SP1

LabSolutions Insight™ Ver. 3.2 SP1

GC-MS/MS method package for

dioxins in foods

3

3

0

6 μL

2 μL

0.2 sec.

Insert Liner

Column

Injection Mode

Sampling Time

Injection Temp.

Column Oven Temp.

HP Injection

Flow Control Mode

Purge Flow

Carrier Gas

Ion Source Temp.

Interface Temp.

Detector Voltage

Topaz® single gooseneck liner,

with wool

SH-Rxi™-5Sil MS (60 m, 0.25 mm I.D.,

0.25 µm), SHIMADZU

Splittless

1.00 min.

280 °C

150 °C (1 min) →(20 °C/min) →220 °C →(2 °C/min) →260 °C (3 min)

→(5 °C/min) →320 °C (3.5 min)

450 kPa (1.5 min.)

Linear Velocity (45.6 cm/sec.)

20 mL/min.

Helium

230 °C

300 °C

1.8 kV (Absolute)

System configuration

Analytical conditions (AOC-20i/s)

Analytical conditions (MS)

Analytical conditions (GC)

Nr. of compound in figure 1

Compound name Calculated S/N

1

2

3

4

5

6

7

8

9

2,3,7,8-Tetrachlorodibenzo-p-dioxin

1,2,3,7,8-Pentachlorodibenzo-p-dioxin

1,2,3,4,7,8-Hexachlorodibenzo-p-dioxin

Octachlorodibenzo-p-dioxin

2,3,7,8-Tetrachlorodibenzofuran

1,2,3,7,8-Pentachlorodibenzofuran

1,2,3,7,8,9-Hexachlorodibenzofuran

1,2,3,4,6,7,8-Heptachlorodibenzofuran

Octachlorodibenzofuran

285

1,658

396

2,518

2,117

1,882

546

1,784

4,282

Compound name

2,3,7,8-Tetrachlorodibenzo-p-dioxin

1,2,3,7,8-Pentachlorodibenzo-p-dioxin




1,2,3,4,6,7,8-Heptachlorodibenzo-p-dioxin

Octachlorodibenzo-p-dioxin

2,3,7,8-Tetrachlorodibenzofuran









Octachlorodibenzofuran

TEF

1

1

0.1

0.1

0.1

0.01

0.0003

0.1

0.03

0.3

0.1

0.1

0.1

0.1

0.01

0.01

0.0003

Avg RRF

1.07

1.09

1.14

0.95

1.03

0.92

1.19

1.10

1.04

0.97

1.03

1.09

1.09

1.06

1.17

1.02

1.00

RRF (lvl 1)

1.15

0.97

1.39

0.92

1.25

0.82

1.04

1.05

1.00

0.89

0.82

1.36

1.39

1.23

1.05

0.97

0.84

RRF Dev (%)lvl 1

8.1

10.56

22.26

2.72

21.44

11.46

12.21

4.66

3.23

7.59

20.72

24.62

27.83

16.10

10.37

4.97

15.80

PRODUCTS


Speeding up with Velox Core ShellThe new “Velox Core Shell“ LC columns offer more application possibilities

Since system back pressure in-creases strongly with smaller par-ticles, another benefit of core shellparticles is a lower increase ofoverall pressure, especially whenthe HPLC system is not verypressure stable and a faster separa-tion is desired. The flow rate cantherefore be higher than for col-umns with fully porous materials.In the following text, these effectsare tested in an application.

Measurement parameters and methods

Instrument: LC-2040C 3D(Shimadzu)Column: Shim-pack Velox C18;(150 mm x 3.0 mm I.D., 2.7 μm);Shim-pack GIST C18; (150 mm x3.0 mm I.D., 3.0 μm)Mobile phase: 35 % H2O; 65 % ACNOven temperature: 40 °CFlow rate: 0.4 mL/minInjection volume: 0.5 μL

Results

The two chromatograms in figures2 and 3 demonstrate a comparisonof a fully porous column and aCore Shell column. The methodswere identical for both columns.The retention times are comparedin table 1.

When comparing the figures andthe table, it becomes apparent thatthe Velox-C18 column has muchshorter retention times than thefully porous GIST-C18. The peakwidth is also smaller, meaning that

S o far, the Shim-pack columnportfolio has exclusivelycovered fully porous col-

umns. The new ”Shim-pack VeloxCore Shell“ series closes the gapin the range of columns and thusserves a larger number of applica-tion requirements than before.The word velox derives fromLatin, meaning ”fast“ or ”quick“.It stands for columns with a fastseparation performance leading to shorter analysis time whencompared to conventional, fullyporous columns.

Core Shell technology

Core shell means that the singlefilling material particles of the col-umn are not fully porous as usualbut have a solid core covered withthe fully porous material, just likea shell (figure 1). Core shell liter-ally means wrapped core.

The solid core is impermeable tosolvents and analytes. This resultsin a considerably shorter contacttime between sample and station-

ary phase, thus leading to shorteranalysis time and higher efficiencyof the analysis. One of the charac-teristics is that the efficiency of a

smaller, fully porous particle size(e.g. 1.9 μm) is comparable to alarger particle size of a core shellparticle (2.7 μm).

Figure 1: Schematic structure of a core shell particle with a solid core (dark blue)

and the porous layer (light blue)

0

Minutes

mAU

0 1.0 2.0 3.0 4.0

25

50

75

Figure 2: Chromatogram of the GIST-C18 column; 3 μm, 150 x 3.0 mm Figure 3: Chromatogram of the Velox C18 column; 2.7 μm, 150 x 3.0 mm

0

Minutes

mAU

0 1.0 2.0 3.0 4.0

25

50

75

GIST C18

Peak width 50 %

Velox C18

Peak width 50 %

0.74

0.03

0.65

0.02

1.87

0.05

1.13

0.03

2.90

0.07

1.66

0.03

3.41

0.08

1.94

0.03

t R (min) Uracil

t R (min) Methyl benzoate

t R (min) Toluene

t R (min) Naphthalene

PRODUCTS

the Velox-C18 column has a con-siderably higher efficiency.

In the last peak (naphthalene),retention time and peak width dif-fered significantly. Besides theeffect of Core Shell Particles orfully porous particles, this is dueto the role of carbon content andthe surface of the columns in addi-tion to the column type, whichcontribute strongly to the reten-tion. The larger the surface areaand carbon content on the column,the higher are the interactionsbetween the analytes and the sta-tionary phase, and the higher is the retention. Carbon content is 7 % for the Velox C18 column and14 % for the GIST C18 column;surface area is 130 m2/g (Velox)and 350 m2/g (GIST). It is clearly

visible that the peaks in the Veloxcolumn method are much narrow-er and higher than with the fullyporous column.

Shim-pack Velox portfolio

The Core Shell Velox portfoliocovers a wide range of stationaryphases and dimensions. A suitableprecolumn is also available foreach stationary phase. Figure 4shows all phases with their specifi-cations.

In the following, the differenttypes are shown individually:

Shim-pack Velox SP-C18SP stands for “sterically protect-ed”. Due to the steric protection,the column can be used very effec-tively for mobile phases in thestrongly acidic pH range (pH 1-3).

The retention profile is balanced,and the column is also suitable forLC-MS/MS (mass spectrometry)analysis.

Shim-pack Velox C18This phase is the standard choicefor reversed phase chromatography.The Velox C18 has the highesthydrophobic retention in theVelox portfolio. It is compatiblewith slightly acidic to neutral pHvalues of the mobile phase.

Shim-pack Velox BiphenylThe biphenyl phase offers comple-mentary selectivity to the alkylphases. It is suitable for enhancedseparation of aromatic com-pounds. The biphenyl phase isideal for increasing the sensitivityand selectivity of LC-MS/MSanalyses.

Shim-pack Velox PFPPPFPP stands for pentafluorophe-nylpropyl. This group providesalternative selectivity for confor-mational isomers and halogenatedcompounds. In addition, chargedbases are retained more strongly.

Shim-pack Velox HILICWith Velox-HILIC (HydrophilicInteraction Chromatography),polar analytes can be retained. The chemical composition of thestationary phase is pure silica.

Table 1: Retention times and peak widths of GIST C18 and Velox C18

Figure 4: Various stationary phases of the Shim-pack Velox columns

MS sensitivity is increased, partlydue to the high organic contentthat can be used in HILIC mode.

Application

When does it make sense to investin a Core Shell column? As is of-ten the case, this depends on theapplication, but also on the exist-ing HPLC device to perform themeasurement. Many users useCore Shell columns to convert an

existing HPLC method into onewith UHPLC characteristics.Fully porous columns with largerparticles are replaced by CoreShell columns with smaller parti-cles. The user then benefits fromfaster analysis without too muchback pressure exceeding the limitsof the HPLC system. Furtheradvantages are large savings incosts and time.

Since the loading capacity of CoreShell columns is not as high asthat of fully porous columns dueto the solid core, there is lessmaterial in the column with whichthe sample can interact. If thesample is too highly concentrated,this has a negative effect on thepeak shape.

In general, a Core Shell column issuitable if:

• the analysis time should beshorter, without much invest-ment,

• a higher throughput is desired,

• the current HPLC system doesnot have a system volume that istoo high (narrow peaks arewidened again by higher systemvolumes),

• UHPLC columns have an excessively high back pressureor a short service life,

• problems with clogging occurwith the current method, or

• loadability is not important.


IMPRINT

Shimadzu NEWS, Customer Magazine of Shimadzu Europa GmbH, Duisburg

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

Editorial TeamUta SteegerPhone: +49 (0)203 76 87-410 Ralf Weber, Maximilian Schulze

Design and Productionm/e brand communication GmbH GWADuesseldorf

Circulation German: 5,360 · English: 3,975

CopyrightShimadzu Europa GmbH, Duisburg, Germany – February 2019.

Windows is a trademark of Microsoft Corporation. ©2019

Apple Inc. All rights reserved. Apple, the Apple logo, Mac OS and iOS are trademarks of Apple Inc.

0.00

0.25

0.50

0.75

1.00

4,000

Bottle, transparent, colorless, outside. Still water. QATR-S cm-1

Polyethylene terephthalate (PET) DuraSampeII-RII cm-1

3,800 3,600 3,400 3,200 3,000 2,800 2,600 2,400 2,200 2,000 1,900 1,800 1,700 1,600 1,500 1,400 1,300 1,200 1,100 1,000 900 800 700 600

4,000 3,800 3,600 3,400 3,200 3,000 2,800 2,600 2,400 2,200 2,000 1,900 1,800 1,700 1,600 1,500 1,400 1,300 1,200 1,100 1,000 900 800 700 600

Abs

.

0.00

0.25

0.50

0.75

1.00

Abs

.APPLICATION

are also used, such as PLA, a plas-tic based on polylactic acid. How-ever, it does not have the sameflexibility as PET in terms of itsphysical properties (stability, bot-tles, boxes and foils etc.) PLAused in food applications has upto now been rather stiff and brit-tle. It can be found in disposabledishes and disposable cups, but itis also used in 3D printers forprinting.

Can natural renewable raw materials be 100 % recycled?

It is questionable when about25 % of a beverage bottle is madefrom naturally renewable rawmaterials but is then declared tobe 100 % recyclable. To solve thispuzzle, FTIR spectroscopy wasused because a PET label wasfound on the beverage bottle.

FTIR spectroscopy allows thesubstances to be identified. A

P lastic packaging, plasticwaste, microplastic andenvironmental protection –

the pollution of nature and theseas by plastic is one of the topissues in the press. Plastic doesn’trot or decay; it takes decades orcenturies before it is decomposedby nature.

Among other things, PET, a versa-tile plastic, is used to make bever-age bottles. Despite strong recy-cling efforts, production has in-creased from 40 to 56 million tons(2008-16) [1]. That the sturdypolymer-based beverage bottlesare made of polyethylene tereph-thalate (PET) can be read on thebottles. The symbol for PET inrecycling is a clearly written ‘PET’in the recycling symbol or a ‘1’ inthe recycling triangle. This sub-stance can be recycled as waste.

In order to appear more environ-mentally friendly, alternative plas-tics from natural basic substances


Organic plastic in beveragebottlesFTIR analysis of beverage bottles, partly produced from naturally renewable raw materials

Even the PET bottles themselves,which are available on the market,show different ratios of propor-tions of A- and C-PET. They arealso unevenly distributed over theentire bottle body.

The PET variants are usually notthe same on the outside, inside orat the bottleneck. The reason isthat PET reacts to heat formingwith crystallization [2]. The in-frared spectrum is further influ-enced by the arrangement of themolecular structures in the PETand by the manufacturing processof PET as a polymer. This articlefocuses on PET as A/C PET.

A-PET and C-PET distributed differently

In a separate screening of packag-ing material, various PET bottlesfor beverages from Europe andJapan were analyzed. For compari-son, 9 samples were used (table 1).In all investigations of PET bot-tles, a higher proportion of A-PETwas found on the outer wall andmore of C-PET on the inner wall.

Pure A-PET is rarely found inbottles, which is probably in thenature of PET. Likewise pure C-PET does not exist either (max-imum proportion of crystallizationis about 75 % [2] [3]). A typicalinfrared spectrum of these bottlesis shown in figure 1. The focus ison the signals at about 1,410 and1,340 cm-1. Depending on the pro-portion, the signal at 1,340 cm-1

(closer to C-PET) is higher thanthe signal at 1,410 cm-1 (closer toA-PET). Furthermore, shifts ofthe -CH and CH2 groups takeplace, which is evident in the re-gion around 2,900 cm-1 (figure 2).

In addition to ‘standard bottles’, a bottle claiming to be partly made

Shimadzu IRSpirit-T equippedwith a diamond-based QATR-Ssingle-reflection unit was used forthe analysis. This is possible be-cause the polymers react to heatand the molecular groups of thesechemical compounds vibrate.

These vibrations are characteristicfor each substance, and they arerecorded wave-dependent on themolecular absorption in the rangeof about 400 to 4,000 cm-1 againstthe molecular absorption to re-ceive a so-called mid infraredspectrum. It can then be identifiedwith the help of libraries.

A/C-PET in the analysis

PET bottles are made of A/CPET. Depending on the wallthickness (soft or hard polymer)and application, PET can containmore amorphous (A-PET) andless crystalline (C-PET) PET orvice versa. The two versions havedifferent FTIR spectra.

Figure 1: PET spectrum of the plant based raw material bottle identified by the infrared spectrum search in polymer libraries;

best hit was found in the ATR polymer library.

Abs


0.00

0.05

0.10

0.15

0.20

0.25

0.30

1,430 1,420 1,410 1,400 1,390 1,380 1,370 1,360 1,350 1,340 1,350 1,3201,440

Abs

0.01

0.02

0.03

0.04

0.05

0.06

3,040 3,020 3,000 2,980 2,960 2,940 2,920 2,900 2,880 2,860 2,840 2,820


Abs

0.0

0.2

0.4

0.6

0.8

1.0

4,000 3,500 3,000 2,500 2,000 1,750 1,500 1,250 1,000 750



APPLICATION

of plant material was also investi-gated. The bottle wall was cutopen and analyzed from bothsides using the ATR technique. As the infrared beam enters thesurface only to a depth of about 2 μm, the wall was examined inthree different ways: inside, in themiddle and outside. PET could befound in all three layers. The ana-lysis results of the sample allowbetter assignment of the signalgroups to the PET variants.

The spectra clearly show the dif-ferent degrees of crystallization of PET. Inner wall and outer wallshow differences. The spectrum of the middle layer is the result of the outer and inner wall.

Figure 2: Infrared spectra of a PET-based bottle with the plant based raw materials

for still water: Inner wall (green line), outer wall (black line) and a spectrum from the

middle (red line) of the bottle wall.

Figure 3: Enlargement from figure 2, range 3,050 to 2,820 cm-1, range of vibration

of the aliphatic and aromatic -CH molecular groups [2]

Figure 4: Enlargement from figure 2- range 1,440-1,320 cm-1, range of vibration

attributed to ethylene glycol [2]

Table 1: Transparent PET bottles collected after use and examined using ATR

infrared spectroscopy. All but one bottle were labeled as polymers of recycling

category 01 for PET.

After superposition, addition ofthe two outer surfaces showsapproximately the nature of themiddle layer (figures 2 to 4).

Conclusion

The actual analysis was meant forthe bottle containing plant basedraw materials (counter 9 in table 1).Using the library search, it hasbeen identified as PET (figure 1).On the bottle, PET is declared inminiature figure, which was pro-duced up to 22.5 % from renew-able raw materials. Is this a para-dox on first glance? The solutionto the puzzle lies in the develop-ment of the starting products forthe production of PET, which is produced, for example, from the

monomers ethylene glycol andterephthalic acid. According tothe manufacturer and readable onthe Internet, the PET raw materialethylene glycol is obtained fromcane sugar. At this point, plantsfrom nature meet production of a technical material such as PET,which is one of the polymers thatcan be recycled easily and wellafter usage.

Bio-plastics are becoming moreand more important to the indus-try, and alternatives to sugar caneare being researched, such asplants that are available world-wide. Nevertheless, the way to100 percent organic plastic bottlesstill seems far away. [4]

Literature[1] https://de.wikipedia.org/wiki/

Polyethylenterephthalat

[2] „Plastic Additives Handbook“, H. Zweifel

et all, 6th edition, Hanser Verlag, 2009.

[3] FTIR spectroscopic analysis of poly(ethyl-

ene terephthalate) on crystallization,

Ziyu Chen, et all, European Polymer Jour -

nal 48(9):1586-1610 September 2012.

[4] https://www.weser-kurier.de/

deutschland-welt/deutschland-welt-

wirtschaft_artikel,-CocaCola-Bis-2020-

alle-Flaschen-aus-Zucker-_arid,

1286848.html

Mineral water with Cola

flavor sugar free

Mineral water with lemon

flavor

Green tea

Smoothie

Beer

Tasty Blue water

Mineral water conataining CO2

Lemonade

Still mineral water

GER

NL

JPN

GER

GER

NL

NL

JPN

GER

PET

PET

PET

PET

PET

PET

PET

PET

Bottle with plant based raw

materials and miniature symbol

for PET (not very apparent)

Product Origin Polymer declared

0

20,000

40,000

60,000

80,000

0

20,000

40,000

60,000

80,000

0 20 40 60 80 100 120 140 160 180 200

Forc

e (N

)Fo

rce

(N)

Maximum testforce

Time (s)

0 20 40 60 80 100 120 140 160 180 200

Time (μs)

Change in test force can bemeasured in detail duringfailure occurrence

The interval between datapoints on test force plot isapproximately 3.3 μs

a

b

APPLICATION

I mproving energy consumptionis one of the most importanttasks of the automotive market

in order to comply with environ-mental regulations in each coun-try. One of the solutions to solvethe problem of energy consump-tion is weight reduction. It is nec-essary to achieve it while stillsecuring the safety of the passen-gers. Various new materials arebeing developed in order to bal-ance these needs. Looking at met-als, these are high-tension steel,aluminium alloys, magnesiumalloys and others. With plasticsand composite materials such asCFRP (Carbon Fiber ReinforcedPlastic) and GFRP (Glass-FiberReinforced Plastic), attention isgiven to combining weight reduc-tion with high-strength proper-ties. Practical usage of severalmaterials has already started in themarket, but research is still ongo-ing.

In the case of composite materials,the internal structure is complicat-ed, so there is a possibility of afailure mode different from con-ventional materials. If the cause ofbreakdown can be demonstratedby means of structural analysissimulation, the design of automo-

Testing machine creates signal for camera

In this test, the change in loadoccuring during specimen fracturewas used to trigger the HPV-Xhigh-speed video camera. Specif-ically, the AG-X plus precisionuniversal testing machine wasconfigured to generate a signalwhen the test force on the speci-men reaches half the maximumtest force (referred to as Maxi-mum test force in figure 2), withthis signal being sent to the high-speed video camera. Static tensiletesting and fracture observationwere performed according to the

is extremely important from theperspective of safe application ofCFRP materials, e.g. in aircraftand other.

tive parts using composite materi-als will greatly advance. For this reason, it is important tounderstand the mechanism ofoccurrence and fracture. A newtest method confirming the struc-ture of composite materials is pro-posed here. In addition, a dedicat-ed example is presented, althoughseveral test methodologies existfor composite materials.

Universal testing machine andhigh-speed video camera

This article explains how to usethe precision universal testingmachine (Autograph AG-X plus250 kN) and the high-speed videocamera (Hyper Vision HPV-X)(figure 1) to evaluate the staticfracture behaviour of a CFRPbased on a test force attenuationgraph and images of material fail-ure. Information on specimens isshown in table 1. A 6 mm diame-ter hole is machined in the speci-men centre. Fractures are knownto propagate easily through com-posite materials from the initialdamage point, and when a crackor hole is present, the materialstrength is reduced markedly.Therefore, evaluation of thestrength of open-hole specimens


New testing methodology forweight reductionAutomotive industry – Case of tensile test with DIC strain analysis of composite material

Figure 1: Main unit combined with the HPV-X2 Camera

Figure 2: Test graphs

Note:

The CFRP laminate board used in theactual test was created by laying uppre-impregnated material with fibersoriented in a single direction.

The [+45/0/ -45/+90]2s shown as thelaminate structure in table 1 refers to the laying up of 16 layers of mate-rial with fibers oriented at +45°, 0°, -45°, and +90° in two-layer sets.

Laminate structureDimensions

L (mm) x W (mm) x T (mm),hole diameter (mm)

[+45/0/ -45+90]2s 150 x 36 x 2.9, Ø 6

Load cell capacity

Jig

Grip space

Loading speed

Test temperature

Software

Fracture observation

DIC analysis

Testing machine250 kN

Upper: 250 kN non-shift wedge type grips

(with trapezoidal file teeth on grip faces for

composite materials)

Lower: 250 kN high-speed trigger-capable grips

100 nm

1 mm/min

Room temperature

TRAPEZIUM X (single)

HPV-X high-speed video camera

(recording speed 600 kfps)

StrainMaster (LaVision GmbH)

AG-Xplus


APPLICATION

conditions shown in table 2. Atest force-displacement plot forthe open-hole quasi-isotropicCFRP (OH-CFRP) is shown infigure 2a. A test force-time plotduring the occurrence of materialfracture is also shown in figure 2b.

Figure 2a can be interpreted toshow that the specimen fracturedat the moment it reached maxi-mum test force, at which point theload on the specimen was sudden-ly released. This testing systemcan be used to perform high-speedsampling to measure in detail thechange in test force in the regionof maximum test force. The timeinterval between data points onthe test force plot in figure 2b is3.3 μs.

Images 1 through 8 in figure 3capture the behavior of the speci-men during fracture around thecircular hole. Image 1 shows themoment cracks occur in a surface+45° layer. In this image, the ten-sile load being applied is deform-ing the circular hole, with holediameter in the direction of theload of approximately 1.4 times

around the circular hole is focuseddiagonally toward the top-left (-45 °) and toward the bottom-left(+45 °) from the circular hole.Images 5 through 8 show thefocusing of strain diagonallytoward the bottom-right (-45 °)and toward the top-right (+45 °)from the circular hole in areaswhere it was not obvious inimages 1 through 4. This showsthat an event is occurring in thesurface layer of the specimen thatis similar to the process of frac-ture often seen during tensile test-ing of ductile metal materials, i.e.crack propagation in the directionof maximum shear stress.

that perpendicular to the load. Inimage 2, cracks occurring aroundthe circular hole are propagatingalong the surface +45° layer. Inimages 3 through 6, a substantialchange can be observed in theexternal appearance of the speci-men near the end of the crackpropagating to the bottom rightfrom the circular hole. This sug-gests that not only the surfacelayer, but internal layers are alsofracturing. Based on the images ofthe same area and the state of theinternal layers that can be slightlyobserved from the edges of thecircular hole in images 7 and 8,the internal fracture has propagat-ed quickly in the 18 μs periodbetween images 3 and 8.

Digital Image Correlation (DIC) analysis

DIC analysis performed on thefracture observation images of fig-ure 3. Black signifies areas of thesurface layer of the specimenunder little strain, and red signi-fies areas under substantial strain.Looking at images 1 through 4 (infigure 4), it can be seen that strain

Figure 3: Observation of fracture

Table 1: Test specimen information

Figure 4: Observation of fracture (DIC analysis)

Table 2: Test conditions (*fps stands for frames per second. This refers to the number

of frames that can be captured in one second).

APPLICATION

R esearchers have been experi-menting with alternativefuels due to steadily increas-

ing demand and decreasing supplyof petroleum crude oil. Ethanolhas been recognized as a possible

This gas chromatographic methodincludes the determination of atest portion with split injectionmode into a gas chromatograph(GC) system. Addition of pentan-3-ol to the sample is the only sam-ple pretreatment step. The sampleis then introduced to the gas chro-matograph (GC) system usingsplit injection mode. A FlameIonization Detector (FID) is usedfor the detection of the analytes.

Apparatus

Gas chromatograph: ShimadzuGC 2010 Plus, Split/Splitlessinjector, FID detectorCapillary column: DB-1701 (60 m × 0.25 mm ID, 1.00 μm filmthickness)

Researchers have used ethanol as acomponent in conventional petro-leum fuels because it has a higherthermal efficiency than ordinarypetroleum motor fuels. Anotherbenefit is its lower risk of fire dur-ing storage and transportation

compared to petroleum, whichhas greater volatility and lowerflash point than ethanol.

The EN 15721 EuropeanStandard relates to the determi-nation of several compoundsthat may be present in ethanolat different concentration levels.

The target compounds areseparated into the following

groups:

a) Higher alcohols:propan-1-ol, butan-1-ol, butan-2-ol, 2-methylpropan-1-ol(isobutanol), 2-methylbutan-1-ol, 3-methylbutan-1-ol. Thecompounds of the first groupcan be determined up to 3 %(m/m).

b) Impurities:ethanol (acetic aldehyde), ethyl-ethanoate (ethyl acetate), 1,1-diethoxy ethane (acetal) arespecified up to 2 % (m/m).

liquid fuel alternative which canbe available in significant quanti-ties throughout the remainder ofthis century [1].

Ethanol blending is a process ofmixing ethanol with petrol toenhance the octane content infuel. This procedure reducesengine carbon monoxide (CO)and carbon dioxide emissions byup to 30 %. Moreover, it improvesengine operation since it acts as ananti-knock agent [2].


Ethanol as a blending componentfor petrolDetermination of higher alcohols and volatile impurities by gas chromatographic method

Two calibration stock solutionsare used for the preparation ofcalibration standards and controlsamples. The first solution (cata-logue number EN 15721-A) con-tains the ten target-compounds at1 % (m/m) and the second (cata-logue number EN 15721-A-IS)contains the internal standard (1 % m/m pentan-3-ol).

Calibration solution and sample preparation

Preparation of Calibration solu-tion: 1 mL of ethanol is trans-ferred into a 2 mL vial and isweighed to the nearest 0.1 mg. 100 uL of calibration stock solu-tion (catalogue number EN 15721-A) is added and mass isrecorded to the nearest 0.1 mg. 80 uL of internal standard stocksolution (catalogue number EN15721-A-IS) is added and mass isrecorded to the nearest 0.1 mg [3].

Sample Preparation: 1 mL ofsample is transferred into a 2 mLvial and is weighed to the nearest 0.1 mg. 80 uL of internal standardstock solution (catalogue numberEN 15721-A-IS) is added and massis recorded to the nearest 0.1 mg [3].

GC-2010 Plus

Figure 1: Chromatogram of 3-pentanol (ISTD) Figure 2: Chromatogram of calibration stock solution

Ethanal

Methanol

1-propanol

Ethyl acetate

2-butanol

Isobutanol

Acetal

1-butanol

3-methyl-1-butanol

2-methyl-1-butanol

3-pentanol (ISTD)

Compound5.268

5.567

9.360

9.680

10.406

11.555

12.672

12.876

15.516

15.614

13.747

0.0009

0.0002

0.0006

0.0017

0.0002

0.0003

0.0006

0.0001

0.0003

0.0002

––

0.0028

0.0006

0.0019

0.0050

0.0006

0.0009

0.0017

0.0002

0.0010

0.0006

––

Retention time (min) LOD (% m/m) LOQ (% m/m)

Calibration compounds Description

Split injector temperature

Split ratio

FID temperature

FID air flow

FID hydrogen flow

FID make-up flow

Carrier gas

Linear velocity

Column flow

Oven temperature program

Part of GC200 °C

50

260 °C

400 mL/min

40 mL/min

30 mL/min

He

22 cm3/sec

1.4 mL/min

40 °C → 1 min

5 °C → 250 °C → 2 min

Values


APPLICATION

and response factor results ofthree calibration standard injec-tions.

Expression of results

Results were calculated throughthe GC solution software accord-ing to the following equations:

Resolution: R = 2 (t2 - t1) / 1,699(W1 + W2)Response Factor: RF = Area(ISTD) x C (compound) / Area(compound) x C (ISTD) Impurities: Q = ethanol + ethyl-ethanoate + 1,1-diethoxyethane +oxygenated compoundsMethanol: Cm = methanol con-tentHigher alcohols: Ch = propan-1-ol + butan-1-ol + butan-2-ol + 2-methylpropanol + 2-methylbutan-1ol +3-methylbutan-1-ol

Concentration is expressed ing/100 g, % (m/m).

Conclusion

The EN 15721 standard was successfully applied with the

Results and discussion

Chromatograms

The analysis was started with theinjection of internal standardstock solution (catalogue numberEN 15721-A-IS) and the injectionof calibration stock solution sinceretention times of all compoundsshould be determined.

The next step was the analysis ofCalibration Standard Solution toconfirm compliance with theacceptance criteria, as far as chro-matographic resolution is con-cerned. Baseline separation wasobtained for all componentsexcept for 2-methylbutan-1-ol and3-methyl-butan-1-ol (R = 1.3, R ≥1.0 is the EN 15721 specification).

Calibration curve

The Calibration Standard Solutionwas injected three times to obtaina two-point calibration curve (y =a x) for each analyte. The desiredrepeatability levels were achievedsince the % RSD was 0.0 - 0.5 %(% RSD ≤ 5.0 is the EN 15721specification) for both area ratio

Table 1: Method parameters

Table 2: Reagents and materials

Table 3: Compound table

Figure 3: Chromatogram of calibration solution

Finally, proper maintenance of theinjection port and column ovenbefore analysis helped to obtainchromatograms with low noiselevels and high S/N values for thetarget-compounds.

GC-2010 Plus. The appropriateuse of the instrument and theaccurate application of EN 15721resulted in chromatograms withan acceptable number of theoreti-cal plates while the presence of theAOC-20i auto-sampler contribut-ed to the excellent repeatability.

Selection of an appropriate col-umn was critical to achieve accept-able resolution (Rs > 1.0) between2-methyl-1-butanol and 3-methyl-1-butanol. Also, the chromato-graphic method parameters of EN15721 and the adoption of defaultvalues in gas flow rates for thedetector (FID) ensured that allacceptance criteria of the Standardwere fulfilled.

AuthorsFotis Fotiadis, Georgia Flessia,

Dr. Gerasimos Liapatas,

Dr. Manos Barbounis

N.Asteriadis S.A.,

Athens, Greece

Literature[1] G. Najafi, B. Chobadian, T. Tavakoli, D.R.

Buttsworth, T.F. Yusaf, M. Faizollhnejad,

Appl. Energy 86 (2009) 630-639.

[2] Y. Barakat, Ezis N. Awad, V. Ibrahim,

Egyptian Petroleum Research Institute

(EPRI), Egypt (2016), “Fuel consumption

of gasoline ethanol blends at different

engine rotational speeds”

[3] EUROPEAN STANDARD EN 15721

“Ethanol as a blending component for

petrol – Determination of higher alco-

hols, methanol and volatile impurities –

Gas chromatographic method”

Methanol

Propan-1-ol

Butan-1-ol

Butan-2-ol

2-methylpropanol

2-methylbutan-1-ol

3-methylbutan-1-ol

Ethanal (acetic aldehyde)

Ethyl-ethanoate (ethyl acetate)

1,1-diethoxy ethane (acetal)

Pentan-3-ol

Ethanol

––

Higher alcohols

Higher alcohols

Higher alcohols

Higher alcohols

Higher alcohols

Higher alcohols

Impurity

Impurity

Impurity

Internal standard

Solvent

28

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