Weight reduction Clear view in wet conditions Less expensive and easy

New testing methodology Polarizing sunglasses block the Analysis of dioxins in foods for composite material glare from wet surfaces and feeds using GC-MS/MS CONTENT

APPLICATION 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 with fluorescence spectroscopy 6 Quality control of zeolites for washing powder with EDX-8000P 9 Purification made easy – Prepara- tive purification of Ibuprofen and its 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 weight reduction 24 Ethanol as a blending component for petrol – Determination of higher alcohols and volatile impurities by gas chromatogra- phic method 26

PRODUCTS Speeding up with Velox Core Shell – The new “Velox Core Shell“ LC columns offer more application possibilities 20

LATEST NEWS Mission for a good cause – Social Day 2018: Shimadzu Europe plants 1,500 new trees 5 Pyrolysis GC-MS user meeting – March 28, 2019 at Shimadzu’s Laboratory World Clear view in wet Duisburg, Germany 28 conditions

MARKETS UV-Vis spectroscopy: Polarizing sunglasses block the Chemical, Petrochemical, Biofuel and Energy glare from wet surfaces

Clinical s the days become longer, sunglasses are a common Environment A sight again. Today, there are no limits to design and color Food, Beverages, Agriculture of frame and glasses.

Pharmaceutical But not all design choices are purely aesthetic: Yellow glasses Plastics and Rubber for example enhance contrast while the advantage of green Figure 1: Investigated sunglasses. Upper half: Models without polarizing filters, Automotive glass es is lower color distortion. lower half: Models with polarizing filters.

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Polarization on a surface

Light can be described as an elec- tromagnetic wave, as shown in figure 2, with propagation direc- tion C, wavelength ␭, electric field vector E and magnetic field vector B perpendicular to E. The direc- tion of the electric field vector is also described as polarization. A beam of light is composed of many such waves with the same direction of propagation. Depend - ing on the light source, the prop- erties of these photons can be very different (e.g. sunlight) or uniform Figure 2: Vector representation of an electromagnetic wave with propagation direction (e.g. laser). C, wavelength λ, electric field vector E and magnetic field vector B.

The beam of light is polarized when some polarization directions are filtered out and one polariza- tion direction becomes dominant. One example is reflection on a smooth surface, as shown in figure 3. The light with polarization hor- izontal to that surface is reflected to a higher degree [1], e.g. by snow or water, but also by the optical element of a UV-Vis spec- trophotometer, such as a mirror or grating. The degree of polarization depends on the angle of incidence in both cases. Figure 3: Polarization by reflection on a surface. Light with polarization vertical This polarization of the sample to the surface enters the surface, meaning that the horizontally polarized light is beam must be compensated for reflected. The degree of polarization depends on the angle of incidence. when a sample with polarizing properties is examined. A polariz- A depolarizer (figure 4, right side) spectrophotometer was used. The er (figure 4, left side) is transpar- converts polarized light into com- baseplate of the rotating film hold- ent only to light with one polar- pletely unpolarized light [2]. er accessory was used for mount- ization direction and is used to ing the polarizer and depolarizer. Important for the necessary pro- enforce a complete polarization of Setup and samples This setup is shown in figure 5 tective functions are the shading the sample beam. By this, polar- (page 4). To compare the effects of (attenuation of the transmitted ization effects (e.g. by reflection For the measurements described polarized and unpolarized light, a light) and the wavelength range from the sample) are eliminated. here, a Shimadzu UV-3600 Plus foil polarizer (spectral range 400 - covered. Both are optimized by 700 nm) and a Hanle depolarizer the choice of base material and (spectral range 180 - 2,500 nm) coating. Quality glasses block all were used. light of wavelength 400 nm and below for complete UV protec- Two different sunglasses with po - tion. lar izing filters and two different sunglasses without polarizing fil- A special case are polarizing sun- ters were investigated. The samples glasses, promoted for watersports were carefully positioned so that and car driving purposes, just to the frame didn’t block the sample name two examples. In this case, beam. Of each sample, the trans- polarizing filters are built into the mission spectrum was measured glasses which block horizontally in the wavelength range of 400 - polarized light. Sunlight is gener- 700 nm in four different configu- ally unpolarized, but the reflec- rations: tion on a smooth surface causes a polarization horizontal to that • without any additional optics in surface. Polarizing filters are sup- the light path posed to protect the user from Figure 4: Polarizer (left) and depolarizer (right). The line on the polarizer mount indicates • with unpolarized light (depolar- this glare. the polarization direction of the transmitted light. izer in position A) ‘

SHIMADZU NEWS 1/2019 3 APPLICATION

• with vertical polarized light influence on the measurements. (polarizer set to 0° in position This is different to the spectra of A) the black and multicolored sun- • with horizontal polarized light glasses. As expected from sun- (polarizer set to 90° in position glasses promoted for watersports, A). the transmission for horizontally polarized light is very low. The Polarizing filters for black sunglasses show transmis- selective absorption sion values of around 20 % for vertical polarized light in this Figure 6 shows the transmission measurement range, but only 10 % spectra of the sunglasses described for unpolarized light and less than above. The samples are labelled by 5 % for horizontal polarized light. their glass color as orange (upper left), green (upper right), black This effect is weaker with the (lower left) and multicolored (low - multicolored sunglasses. The spec- er right). The spectra are arranged tra measured with unpolarized analog to the pho tos of figure 1 and vertically polarized light are (page 2). nearly congruent. The attenuation of horizontally polarized light is Only the spectra of polarizing still remarkable though. The best glasses without additional optics attenuation is observed in the show peaks at 470, 580 and 665 nm. spectral range of 500 - 620 nm, Transmission in these measure- while the black sunglasses show ments is also below the values an even attenuation over the whole measured for the same glasses spectral range up to 650 nm. with a depolarizer in the light Figure 5: Experimental setup. A: Mounting position for polarizer or depolarizer, path. This shows that the sample B: Sunglasses, C: Sample beam, D: Reference beam. Conclusion beam is horizontally polarized to some degree. As the degree of rectly dependent on the wave- For the orange and green sun- Many projects in the optic indus- polarization depends on the angle length. The spectra measured glasses, all four spectra are con- try are concerned with polarized of incidence to the polarizing ele- with out depolarizer are influenced gruent. The polarization of the light. Using a depolarizer will ment, this absorption is only indi- by such polarization effects. sample beam does not have any ensure that the sample is irradiat- ed with completely unpolarized light. Measurement artifacts are Sunglasses Orange Sunglasses Green therefore avoided and the true 100 40 properties of polarizing samples are elucidated. 80

60 Literature 20 40 [1] https://en.wikipedia.org/wiki/ Polarization_(waves) 20

Transmission [%T] Transmission [%T] Transmission [2] https://en.wikipedia.org/wiki/Depolarizer_ 0 0 (optics) 400 500 600 700 400 500 600 700 Wavelength [nm] Wavelength [nm] Further information on this article: Sunglasses Black Sunglasses Multicolored • Application: 100 40 shimnet.shimadzu. local/product/Spectro/ 40 Spectro_UV/Application_Notes/SCA- 100-022_Sunglasses.pdf 20 20 Transmission [%] Transmission [%] Transmission 0 0 400 500 600 700 400 500 600 700 Wavelength [nm] Wavelength [nm]

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

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.

4 SHIMADZU NEWS 1/2019 LATEST NEWS Mission for a good cause Social Day 2018: Shimadzu Europe plants 1,500 new trees

he social day, at which land is part of the Duisburg them grows best in the first 20 to which secure the best spot in the Shimadzu employees sup- Sechs-Seen-Platte (Six Lakes Re- 30 years. This replicates nature sun over the longest period, T port social projects, has gion), a recreational area with a that produces an unimaginably receiving the most sunlight in the become a tradition within the network of hiking trails of 18 kilo- large number of seedling offspring long term. company since 2013. Once again, meters. by natural seeding from the moth- volunteers have been actively er trees, which evolve over the Social Responsibility involved in their community and The two-three-year-old trees with years so that only a few trees in December 2018 helped to refor- a size of about 1.50 m were plant- remain and their number contin- ‘Realizing wishes for the well- est an area in the city forest of ed relatively densely, so they can ues to reduce further as they being of mankind and the Earth’ Duisburg with 1,500 new trees. decide among themselves which of grow. Usually the trees prevail is one of Shimadzu’s established This will contribute to the long- corporate principles. Engaging in lasting woodlands development. community, society and the envi- So far, the heavily thinned out ronment expresses this claim at area was covered only with birch the local level. and bracken. In the year of its 50th anniversary The 1,200 young European oak in Europe, Shimadzu gladly sup- and 300 sweet chestnut, littleleaf ported this long-term project and linden and hornbeam have been thanks all 30 participating employ - selected to fit the natural tree ees of Shimadzu Germany and species spectrum, growing well in Shimadzu Europa for their indi- the sandy soil. Forests with these vidual social commitment. indigenous trees serve as a habitat for many rare and endangered ani- mals, plants and fungus species. Additionally, oak and linden trees are particularly long-lived, so future generations will also enjoy this biotope. The planted wood-

SHIMADZU NEWS 1/2019 5 APPLICATION What’s in the water? Water analysis of humic acid with fluorescence spectroscopy

into the ground and water. Humic acid is not a stable then into water molecule. It oxidizes through bodies or deeper oxygen and decomposes chemical- wells. It absorbs soil ly into tryptophan and L-tyro- components and sine. It could be deduced from carries them in solu- this reaction that if no more tion or as suspended humic acid is present, the neces- particles. There are sary amount of oxygen in the both visible and water is also consumed [1], [2], [3]. invisible portions. In the following application, it Visible particles was interesting to determine the usually give the change in humic acid content by water an earthy fluorescence spectroscopy. Humic color, an appearance acid, tryptophan and tyrosine that is normal for show a fluorescence spectrum outdoor ponds or under excitation in the ultraviolet lakes (figure 1). However, if a slight tint in a bottle of drinking water is found, it is per- ceived purely sub- jectively as deviat- ing from norm and experience. Drin- king water is ex- pected to be color- less and clear.

The warm summer of 2018 brought a supply of still min- eral water with a slight tint (figure 2). This had to be investigated with fluorescence spec- troscopy. Figure 1: Autumn view of a city park with a pond supplied with fresh water It is known from the literature ater is the basis of all life, portant task in chemistry since it that water can contain humic acid, all of nature and its habi- is a good solvent and absorbs a natural degradation product of W tats. When processed many substances. organic sources such as leaves and into food as drinking water, it’s a grasses. This occurs naturally with Figure 2: Still mineral water with substance that is strictly tested. The most common way of water open water, and the acid serves as a slight coloration from a delivery in However, water also has an im- to enter the natural cycle is as rain food for living organisms in the summer 2018

Fluorophores Samples and references Humic acid Tyrosine Tryptophan Bi-distilled Mineral Well water, Well water, Pond water Pond water with 20 mg/L 1 mg/L 1 mg/L water standing, freshly pumped, standing fresh water exchange garden hose garden hose A 0 0 976 0 0 34 0 86.1 38.6 B 0 583 0 0 0 44.5 0 45.7 32 C1 100 0 0 0 27 34.9 36.6 24.9 82.6 C2 51 0 0 0 17 30 26.5 17.3 40

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

6 SHIMADZU NEWS 1/2019 APPLICATION

range. Due to their chemical struc- bidest_water_180814_114003.fs3f - CorrectionData humic_acid_20mg/L_180814_134403.fs3f - CorrectionData EM Wavelenght (nm ) / EX Wavelenght (nm) EM Wavelenght (nm ) / EX Wavelenght (nm) ture, the complex molecules have 400 400 400 400 many ring systems, and individual electrons can be excited to ener- 300 300 getically higher states. The energy absorbed in this way is released 350 350 again via radiation of photons 200 200 when the electron returns to its energetic ground state. 300 100 300 100

For this fluorescence analysis a 0 0 rapid screening was used, ending 250 250 in an EEM view (Excitation 250 300 350 400 450 500 550 600 250 300 350 400 450 500 550 600 Emission Matrix). Fluorescence EM: 600.0/EX:250/-11.5 EM: 600.0/EX:250/43.9 spectroscopy is a very sensitive Tryptophan_1mg/L_180814_130607.fs3f - CorrectionData Tyrosine_1mg/L_180814_115700.fs3f - CorrectionData and selective measurement tech- EM Wavelenght (nm ) / EX Wavelenght (nm) EM Wavelenght (nm ) / EX Wavelenght (nm) 400 400 400 400 nique. It can make traces of fluo- rescent-active substances visible in a mixture. 300 300 350 350

Samples 200 200

Various samples were collected for 300 100 300 100 this study:

0 0 • A chemically pure water – double distilled, 250 250 250 300 350 400 450 500 550 600 250 300 350 400 450 500 550 600 • Well water, standing, from a EM: 600.0/EX:250/-1.9 EM: 600.0/EX:250/6.5 garden hose, • Well water, freshly pumped, Figure 3: Representation of the EEM matrix of double distilled water (top left), humic acid (top right), tryptophan (bottom left) from a garden hose, and L-tyrosine (bottom right), the scaling of the representation was set to 0 - 400 intensities (red corresponding to very intense, • Pond water standing, black up to no intensity). • Pond water with fresh water exchange, and • Mineral water without carbon fresh_water_source_1_180817_101228.fs3f - CorrectionData fresh_water_source_2_180817_103733.fs3f - CorrectionData EM Wavelenght (nm ) / EX Wavelenght (nm) EM Wavelenght (nm ) / EX Wavelenght (nm) dioxide (still). 400 100 400 100

Reference materials were solutions 80 80 of tyrosine (1 mg/L), tryptophan (1 mg/L) and humic acid (20 mg/L, 350 60 350 60 pH 8 to 9). 40 40 All liquids used were transferred 300 20 300 20 into a fluorescence cell. In the standard version, the cuvettes have 0 0 four polished windows and a layer 250 250 thickness of 10 x 10 mm. The 250 300 350 400 450 500 550 600 250 300 350 400 450 500 550 600 quartz in this cuvette is fluores- EM: 600.0/EX:250/-5.1 EM: 600.0/EX:250/-1.2 cence-free. The windows are need- waste_water_source_1_180817_105907.fs3f - CorrectionData waste_water_source_2_180817_111956.fs3f - CorrectionData ed because fluorescence is a scat- EM Wavelenght (nm ) / EX Wavelenght (nm) EM Wavelenght (nm ) / EX Wavelenght (nm) 400 100 400 100 tered light which is detected at an angle of 90 degrees from the irra- 80 80 diation direction of the excitation source. The reference material was 350 60 350 60 balanced and diluted with double distilled water. This water was also 40 40 tested to exclude possible contami- 300 300 nation (see figure 3). 20 20

0 0 Analysis of DOM in natural water 250 250 250 300 350 400 450 500 550 600 250 300 350 400 450 500 550 600 EM: 600.0/EX:250/-1.2 EM: 600.0/EX:250/5 “Dissolved Organic Matter” was investigated with the Shimadzu Figure 4: Representation of the EEM matrices of standing well water in a hose (top left), flowing well water from a hose (top right), RF-6000 fluorescence spectropho- standing pond water (bottom left) and flowing pond water (bottom right). tometer. ‘

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In a quick screening, EEM matri- Mineral_water_180814_140456.fs3f - CorrectionData EM Wavelenght (nm ) / EX Wavelenght (nm) ces of the samples and references 400 100 were created. The EEM matrix is formed when the excitation wave- 80 lengths are increased slowly in small steps and the respective flu- 350 60 orescence spectrum is applied. Depending on the recording 40 speed, it can take approx. 2 min- 300 20 utes (60,000 nm/s) or approx. 13 minutes (2,000 nm/s) to create the 0 matrix. 250 250 300 350 400 450 500 550 600 From the reference measurements, EM: 600.0/EX:250/-0.1 it was possible to obtain the ana- bidest_water_180817_095419.fs3f - CorrectionData EM Wavelenght (nm ) / EX Wavelenght (nm) lytical wavelengths for the identi- 400 100 fication of the proteins involved. Distilled water shows no presence 80 of a fluorophore, as was to be 350 60 expected.

40 The excitation wavelength (EX) at

275 nm and the emission wave- 300 20 length range (EM) at 340 - 381 nm were found for tryptophan. For 0 L-tyrosine the EX are at 275 nm 250 and EM at 310 - 320 nm. The 250 300 350 400 450 500 550 600 humic acid has two active surfaces EM: 600.0/EX:250/-8.3 in the EEM at EX 300 - 370 nm Figure 5: Comparison of the EEM matrices of still mineral water from the bottle (summer 2018) and double-distilled water from the and EM 400 - 500 nm, as well as laboratory. The mineral water exhibits fluorescence activity in the area of humic acid-like proteins. EX 240 - 260 nm and an EM of 450 - 500 nm. to humic acid. Table 1 (page 6) ment processes and also for con- Further information In the same way, the five samples shows the detected intensities of tamination of natural water. on this article: from different sources were exam- all samples and references in- • Application: ined. All samples had lower con- volved to make the quantitative Literature AD-0133: Excitation- centrations than the reference aspect visible. [1] Hudson, N., Baker, A. and Reynolds, D. Emission Matrix (EEM) material. It became apparent (2007). Fluorescence analysis of dis- Fluorescence Spectroscopy for Analysis (figures 4 [page 7] and 5), that the Conclusion solved organic matter in natural, waste of Dissolved Organic Matter (DOM) in mineral water contained a low and polluted waters – a review. River Natural Water and Wastewaters concentration of a molecule simi- The Excitation Emission Matrix Research and Applications 23: 631-649. • Application: lar to humic acid. In the standing (EEM) of fluorescence spectros- [2] Yan, Y., Li, H. and Myrick, M. L. (2000). SCA_105_010: Dissolved organic matter waters, the signal group of humic copy is a very fast technique for Fluorescence fingerprint of waters: analysis (DOM) and its appearance acid has decreased strongly. obtaining an overview of dis- excitation-emission matrix spectroscopy under different environmental condi- Tryptophan and L-tyrosine-simi- solved organic components (DOM) as a tracking tool. Applied Spectroscopy tions – fluorescence EEM matrices of lar substances predominate. Both in water. With this option, the 54(10): 1539-1542. different sources fresh waters, on the other hand, water quality can be monitored, [3] AD-0133, Shimadzu Asia Pacific, 2016 contain mostly proteins similar for example, in wastewater treat-

8 SHIMADZU NEWS 1/2019 APPLICATION Hard x-ray for soft water Quality control of zeolites for washing powder with EDX-8000P

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

the scale of 0.3 - 0.5 nm and de- A fast alternative for routine qual- pends on the cations bound to the ity checks is energy-dispersive anionic cage, since they block the x-ray fluorescence spectroscopy pores to some degree depending (EDX), e.g. with the EDX-8000P, on their size, charge and binding a system penetrating samples with force. so-called hard x-rays of up to 50 keV. Here, the zeolite powder Figure 1: Structure of Zeolite A. Al and Si atoms (nodes) are connected by shared O For the application as water soften- is filled in a special sample cup atoms (lines), leading to a cage of alternating AlO4 and SiO4 tetrahedrons. Only some er, only sodium and potassium are without pretreatment. Digestion atoms are labelled to indicate the binding motif. valid cations. During the laundry, of the sample is not required, and they are exchanged easily by calci- measurement time is on the scale any washing powders um or magnesium from the hard of a few minutes including prepa- contain zeolites for water water, which have a higher binding ration. M softening. This role was force to the zeolite cage. Without fulfilled in the past by phosphates, free Ca2+ or Mg2+, the formation The EDX-8000P detector is opti- leading to a notorious growth of of lime is prevented. To ensure the mized for light elements such as algae and severe damage of aquatic best product quality for each target sodium, aluminum and silicon. ecosystems. Even though natural application, the elemental composi- For this measurement under vacu- zeolites exist, they are specifically tion (aluminum (Al), silicon (Si), um condition, the cups are closed synthesized with defined stoi- sodium (Na) and possibly other by a seal permeable to air to pre- chiometry and structure [1]. One elements) of these zeolites must be vent bursting of the cup by the widely used example is Zeolite A controlled. sudden pressure change and spill- with an equal content of alumina ing of the sample. and silicone. The advantage for Easy sample preparation technical applications is a well- Quick measurements defined anionic cage structure A common method for precise ele- 12- made from ([AlO2]12[SiO2]12) mental analysis is inductively cou- The EDX-8000P has another ad- blocks as shown in figure 1. pled plasma optical emission spec- vantage for this application: the troscopy (ICP-OES or short ICPE). sample composition can be deter- Molecules such as water that are For mineral samples, such as zeo- mined by fundamental parameters small enough to enter the pores lites, a thorough digestion is need- (FP) methods without the need for are bound there until the zeolite is ed. Sample preparation takes some standards. ‘ heated. The effective pore diame- time and requires the use of chemi- ter for use as molecular sieve is on cals such as hydrofluoric acid.

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6 The FP values for aluminum and For the mean deviation shown in silicon show a good match with table 3, relative deviation of each 5 the ICPE values, while the values EDX value from the ICPE value 4 for sodium and especially potassi- of the same sample was calculated 3 um show a bigger deviation. With for each method and sample (see the calibration curve method, only formula 1 page 11). 2 a small deviation from the ICPE Intensity [cps/μA] 1 values was found for all oxides. The average of these deviations 0 over all samples was then calculat- 0.5 1 1.5 2 2.5 3 3.5 4 To calibrate the EDX method, ed for each method (see formula 2 Energy [keV] ICPE results of the same samples page 11). were used as standards. Calibra- Figure 3: EDX spectrum of Zeolite A3 sample 1 at 15 kV tube voltage (C - Sc channel), tion curves are shown in figure 4. When the interfering element is 100 s live time and vacuum. The rhodium lines are scattered radiation from the x-ray While quantitation of aluminum hard to identify, testing different tube. and silicon by this calibration corrections as in this example and curve method seems to be inferior finding the method with the small- to the FP method at first glance, est deviation is a good starting Two such fundamental parameters FP method and a quantitative all standards had nearly identical point. The correcting element are the order number and concen- method calibrated with ICPE contents of these elements, so should have a small coefficient tration of the elements included, results. there was not enough difference in (±0.001) to avoid overcompensa- which are given as results from the intensity for a linear fit. tion. intensities of characteristic lines. Figure 3 shows a typical EDX- Other fundamental parameters, like spectrum of a synthetic zeolite Matrix corrections Conclusion the sample shape, density or fixed after 100 seconds measurement concentrations, are either added time. All peaks detected over the One important raw material for The EDX-8000P is the ideal in- manually or calculated from scatter defined measurement range are zeolite is the aluminum or baux- strument for quick quality checks. peaks to support the algorithm. fitted and analyzed by an FP ite, where the content of alumi- The fundamental parameters method. The rhodium lines are num and undesired oxides must be method can be enhanced by addi- FP methods are used for quick background from the x-ray tube monitored to optimize the process tional data, e.g. from loss of igni- screening and depend strongly on and not used for the quantitative parameters. The analytical meth- tion measurements to measure how well the sample is described analysis. ods used for artificial zeolites are unknown samples without the within the FP model. A higher easily adopted for the quality need for calibration. Using matrix quality quantitation of the elements Table 1 compares ICPE results check of natural bauxite. The pos- corrections, analysis of more com- is possible using calibration curves. given by the manufacturer of the sible influence of other oxides on plicated minerals is possible. This Here, four samples of Zeolite 3A samples to semi-quantitative and the aluminum intensity must way, the EDX method applying with different ratios of sodium to quantitative EDX measurements however be considered. hard x-rays supports applications potassium were analyzed with an for all Zeolite 3A samples. for water softening. To show the effect of different matrix corrections, table 2 lists the 35.0 results for five different samples Acknowledgements 50.0 30.0 of bauxite measured with different We gratefully acknowledge the assistance methods. The same samples were and supply of samples from our colleagues 25.0 40.0 used as standards. Iron was found in Shimadzu d.o.o. Sarajevo, Bosnia and 20.0 30.0 to be the interfering element in Herzegovina. 15.0 the case of aluminum in bauxite. 20.0 10.0 This was derived from the obser- Literature 10.0 vation that the change of the alu- [1] Zeolithe – Eigenschaften und technische Measured intensity [cps/μA] Measured Measured intensity [cps/μA] Measured 5.0 minum line intensities in samples Anwendungen, Lothar Puppe, Chemie in 0.0 0.0 0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 0.0 5.0 10.015.020.025.030.035.035.0 with constant concentration of unserer Zeit 1986, https://doi.org/ 10.1002/ciuz.19860200404 Standard value [%] Standard value [%] aluminum correlated to a change of the iron concentration. [2] X‐ray fluorescence analysis applying the- oretical matrix corrections. Stainless 70.0 2.0 To test the matrix correction, the steel, Willy K. De Jongh, X-Ray Spectro- 60.0 De Jongh (dj) method [2] was metry 1973, https://doi.org/10.1002/xrs. 1.5 50.0 used with one element for calcula- 1300020404 40.0 tion of the correction factor. Each 1.0 element was applied once for the 30.0 correction and the aluminum con- 20.0 0.5 centration in each sample was re- Measured intensity [cps/μA] Measured Measured intensity [cps/μA] Measured 10.0 calculated once with each method. 0.0 0.0 Table 3 shows the coefficient and 0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 average deviation from the ICPE Standard value [%] Standard value [%] value for each correcting element. As expected, the best results are Figure 4: Calibration curves for the EDX method from table 1 found with iron as element for the correction.

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Concentration in %wgt Sample Zeolite 3A sample 1 Zeolite 3A sample 2 Zeolite 3A sample 3 Zeolite 3A sample 4 Method ICPE FP Cal. ICPE FP Cal. ICPE FP Cal. ICPE FP Cal.

Al2O3 33.06 33.34 34.05 33.77 32.32 34.00 34.67 32.49 34.38 33.18 32.33 34.68 SiO2 41.90 40.78 40.24 41.90 40.70 41.20 40.34 39.57 40.33 40.44 39.55 40.71 Na2O 15.09 13.86 15.38 12.05 10.85 11.89 11.27 10.81 11.69 10.50 9.67 10.36 K2O 9.59 11.98 9.68 12.57 16.02 13.04 13.39 17.03 13.72 15.42 18.29 14.86

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.

Concentration of Al2O3 in %wgt. Matrix correction for Al2O3 Baux 1 Baux 2 Baux 3 Baux 4 Baux 5 Element Coeff. Mean deviation ICPE 78.25 59.58 60.77 62.63 66.78 none none 4.80 % FP 84.02 68.75 67.34 68.69 67.63 Si -0.003 4.94 % Cal. (no corr.) 74.22 59.90 65.03 67.08 63.71 Ca -0.054 15.63 % Cal. (Si corr.) 75.49 57.60 64.66 67.09 63.64 Ti 0.137 3.71 % Cal. (no corr.) 77.45 58.85 65.33 39.84 59.80 Fe -0.007 3.54 % Cal. (Ti corr.) 77.43 56.43 60.44 61.29 61.16 Table 3: Elements und coefficients for the dj matrix correction from table 2 with Cal. (Fe corr.) 77.14 61.29 61.40 65.13 61.49 corresponding mean deviation from ICPE values 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.

EDX(Baux1, FE) – ICPE(Baux1) Dev(Baux1, FE) + Dev(Baux2, FE) + ... Dev (Baux1, FE) = · 100 % Dev (FE) = ICPE(Baux1) 5

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

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May 21-23, 2019 Stuttgart, Germany Stand 8530 APPLICATION Purification made easy Preparative purification of Ibuprofen and its related substances by Prominence UFPLC

harmaceutical companies Procedures of preparative increase the amount of compound are developing compounds purification using the Shimadzu trapped on column prior to elu- P of increased complexity – UFPLC system tion is easily accomplished. drugs with numerous functional groups in a single molecule, poly- UFPLC automatically performs meric compounds, biopharmaceu- the various processes related to ticals such as peptides, proteins preparative isolation of target and many others – that must be compounds using a combination purified, in production quantities. of preparative LC and trapping Regulatory agencies continue to columns. Details of these process- press for more stringent require- es are as follows: ments in purity of pharmaceutical 1. separation of target compounds products and, especially, drug in complex sample by prepara- substances. An acute need exists tive LC and introduction into for other tools in addition to crys- trapping columns tallization, the classic technique 2. replacement of solvent in trap- for purification, to address a ping columns with ultrapure growing number of purification water problems. 3. elution of target compounds from trapping columns by Figure 1: Prominence UFPLCTM Currently, preparative HPLC is organic solvent. the most powerful and versatile High purity compounds, pounds to be recovered as free method for purification tasks in An outline of the respective pro- optionally recovered as bases which are generally easier to the pharmaceutical industry. The cesses is shown in figure 2. a free base powderize, typically providing Prominence Ultra Fast Preparative better quality results when used in and Purification Liquid Chroma- The system integrates preparative The Prominence UFPLC elimi- drug screening and pharmacoki- tograph (UFPLC) enables sub- LC with fraction trapping for up nates some of the problems asso- netic studies. All eluted com- stantial labor savings in prepara- to five compounds of interest. It is ciated with conventional prep LC, pounds are finally collected in a tive purification. controlled by a dedicated walk-up especially poor purity of collected highly volatile organic solvent, software designed to simplify the compounds due to mobile phase which reduces fraction dry-down This happens by automation not workflow also for non-expert additives, which become contami- time by up to 90 % compared to only of fractionation of the target users. It allows to easily set condi- nants in the final collected fraction conventional LC fractions. compound but also the related tions for chromatographic separa- and inhibit powderization. processes of concentration, purifi- tion and isolation of target com- Shimadzu’s “Shim-pack C2P-H“ Preparative purification of cation and recovery. This article pounds, trapping, eluting and col- trapping column strongly retains Ibuprofen and its analogs introduces an example of prepara- lecting highly purified compounds target compounds, allowing un- tive purification of a mixed sample in as little as 90 minutes. For wanted organic solvents, water Ibuprofen is one type of nonste- of the pharmaceutical compound applications involving the isola- and additives to be flushed away. roidal anti-inflammatory drug Ibuprofen and its analogs using tion of low concentration targets, Additionally, rinsing the column (NSAID) used as a fever-reducing Shimadzu’s UFPLC Advanced replicate injection and collection with an aqueous ammonia solu- drug and analgesic. The United System (figure 1) [1]. to the same trapping column to tion after trapping allows com- States 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 preparative purification of these three compo- nents using the Shimadzu UFPLC system (figure 3). Preparative LC and purification conditions are displayed in table 1.

Figure 4 shows the preparative LC chromatogram of the mixed solu- tion of Ibuprofen and related compounds. It was prepared by Figure 2: Prominence UFPLCTM workflow of fractionation, concentration, purification and elution dissolving the three target com-

12 SHIMADZU NEWS 1/2019 APPLICATION

Preparative LC conditions Column Shim-packTM VP-ODS (250 × 10 mm, 5 μm) Mobile phase A: 1 % (wt/v) chloroacetic acid (pH 3.0) B: Acetonitrile A/B = 2/3 (v/v) Flow rate 9.0 mL/min Column Temperature Ambient Injection volume 100 μL Detection UV 230 nm highly purified target compounds Rinse conditions in a simple automated workflow. Column Shim-packTM C2P-H (30 × 20 mm, 25 μm) Ultrahigh weight purity com- Rinse solvent A: 2 % (v/v) acetonitrile aq. sol., B: water pounds can be obtained by imple- Time program A: 15 mL/min (0 - 2 min) → A: 8 mL/min menting preparative separations (2.01 - 4 min) → B: 8 mL/min (4.01-8 min) and fraction purification. For Elution conditions Figure 3: Chemical structures of Ibuprofen powderization of the pure sample, Eluent Acetonitrile and related compounds an extremely long drying time is Flow rate 4.5 mL/min necessary using conventional Detection UV 230 nm pounds with mobile phases to a reversed-phase preparative LC, concentration of 5,000 mg/L. due to a high amount of water in Table 1: Conditions of preparative LC separation and purification by the mobile phase. Moreover, in UFPLC system Verification of purity of cases where a nonvolatile buffer Ibuprofen and its analogs solution is used, the salt can pre- Analytical LC conditions cipitate after drying. In prepara- Column Shim-packTM VP-ODS (250 × 4.6 mm, 5 μm) The fractions of Ibuprofen, vale- tive purification using UFPLC, Mobile phase A: 1 % (wt/v) chloroacetic acid (pH 3.0) rophenone and 4-isobutylaceto- it is possible to remove the non- B: acetonitrile phenone collected by UFPLC volatile salt used in the separation A/B = 2/3 (v/v) were analyzed by standard HPLC process, as desalting is performed Flow rate 2.0 mL/min to verify the purity of the com- in the trapping columns. Drying Column Temperature 30 °C pounds. Table 2 shows the analyti- time is substantially reduced by Injection volume 10 μL cal conditions and figure 5 shows use of organic solvent for sample Detection UV 230 nm the chromatograms obtained for recovery from the trapping col- each fraction. Purities of the target umns, contributing greatly to Table 2: Conditions of analytical HPLC separation of purified fractions compounds contained in each improved efficiency in any appli- fraction determined by area nor- cation requiring preparative Compounds Area % malization at UV 230 are listed in purification. Ibuprofen 99.2 table 3. Valerophenone 99.6 4-Isobutylacetophenone 99.8 Conclusion Literature [1] N. Kosuke, Shimadzu Application News Table 3: Purities of target compounds contained in collected fractions Prominence UFPLC Ultra-Fast No. L526, Preparative Purification of (area percentage, UV 230 nm) Preparative and Purification Ibuprofen and Its Related Substances Liquid Chromatograph was by Prominence UFPLC shown to enable fast recovery of [2] USP Monograph for Ibuprofen

mAU mAU 400

1,500 300

1,000 200

500

100 0

0 0.0 2.5 5.0 7.5

0.0 2.5 5.0 7.5 Minutes Minutes

Figure 4: Preparative LC chromatogram of Ibuprofen and its analogs Figure 5: HPLC analysis of Ibuprofen and related compounds in fractions obtained (UFPLC) by UFPLC

SHIMADZU NEWS 1/2019 13 APPLICATION New anti-doping method for equestrian sports Application of LCMS-8050 to quantify capsaicin and dihydrocapsaicin in horse serum

limb localized pain. Due to strong analgesic prop- erties and fast action, capsaicin has been placed on the Equine Prohibi- ted Substances List creat- ed by the International Federation for Eques- trian Sports.

Y. You et al. [1] in 2013 proved the possibility of using UHPLC-MS-MS for the detection of cap- saicin in plasma samples. The method described is fast, selective, sensitive, reproducible, reliable and fully validated.

The aim of this papers’ [2] research was to deter- mine the time limit for the detection of capsaicin and dihydrocapsaicin after long-term use of gel containing capsaicin on Members of research team horses. Of particular interest was to determine he Institute of Toxicological Horses, Dogs and Cats, is a scien- In the case of horses, both cap- how soon after prolonged use of Research at Wroclaw Medi- tific group focused on clinical saicin and dihydrocapsaicin are ointment or gel with capsaicin in T cal University in Poland work, teaching and research in the used as analgesic and warming pain treatment (following manu- routinely analyzes biological sam- field of internal diseases of horses. gels or ointment. They are widely facturer application recommenda- ples and evidence material on Main current research of group used in treatment of lameness and tions), horses would be able to commission of the police, the members includes asthma, meta- prosecutor’s office and the courts. bolic and endocrine diseases and Research encompasses both tar- kidney failure of horses. Appli- geted and screening analyses cation possibilities of innovative including pharmaceuticals, drugs biomarkers in diagnostics of inter- of abuse, new psychoactive sub- nal diseases of horses are also stances, alcohols, volatile sub- investigated. stances, anabolics and many oth- ers. The Institute is also interested Strong analgesic properties in developing new methods of of Capsicum peppers analysis for specific needs of cus- tomers from scientific fields, Capsaicin and dihydrocapsaicin, industries, veterinary medicine alkaloids from Capsicum peppers, and medicine. are used in medicine and veteri- nary medicine due to their strong The Equine Internal Medicine analgesic properties. Local anal- Unit, part of the Department of gesic effect is related to defunc- Internal Disease with Clinic for tionalization of nerve endings. Members of research team

14 SHIMADZU NEWS 1/2019 APPLICATION

take part in competition bearing (x 10,000) in mind the anti-doping regula- 2.00 tions applied in equestrian sports. 1.75 1.50 Sample preparation 1.25 1.00 A serum sample (200 μL) was 0.75 transferred into a 2 mL polypro- 0.50 pylene tube. 20 μL of an internal 0.25 standard solution (methanol solu- 0.00 tion of phenacetin, 100 ng/mL) 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 was added. A liquid-liquid extrac- Minutes tion using dichloromethane (1 mL) was carried out for five minutes. Figure 1: MRM of capsaicin and dihydrocapsaicin in blank equine serum at concentration of 1 pg/mL After shaking, samples were cen- trifuged at 10,000 rpm for five minutes. The organic phase was transferred into a clean 2 mL tube 306.10 → 94.10; 306.10 → 122.05 Y. You et al. research from 2013 on the method described can be used and evaporated to dryness under for capsaicin and m/z 308.40 → capsaicin and dihydrocapsaicin for determination of capsaicin and a stream of nitrogen at 40 °C. The 137.10; 308.40 → 94.10; 308.40 → concentrations in equine plasma dihydrocapsaicin in chili peppers, extract was dissolved in 25 μL of 122.10 for dihydrocapsaicin. The [1] has proven that after applica- self-defense weapons or biological methanol, transferred to an inert analytes were separated using a tion of paste containing 0.025 % samples collected from individuals glass insert and analyzed using C18 1.7 μm, 2.1 x 50 mm column of capsaicin, both capsaicin and exposed to these substances. UHPLC-QqQ-MS/MS. at 40 °C. A combination of 10 dihydrocapsaicin occurred in plas- mM ammonium formate / 0.1 % ma, with a concentration of nearly Authors UHPLC-MS/MS formic acid in water (A) and 242 and 155 pg/mL respectively in Paweł Szpot, Marcin Zawadzki, Marta analysis 0.1 % formic acid in acetonitrile two hours, but after 24 hours the Siczek, Agnieszka Zak, Natalia Siwinska, (B) was used as mobile phase. concentrations declined to nearly Malwina Słowikowska, Artur Niedzwiedz Analysis was performed using a Injection volume was 1 μL. 5 and 3 pg/mL, respectively. By The Equine Internal Medicine Unit, part of Nexera X2 UHPLC system cou- reaching LOQ at 0,5 pg/mL and the Department of Internal Disease with pled with an LCMS-8050 triple Results 1 pg/mL level, the method pre- Clinic for Horses, Dogs and Cats, Wrocław, quadrupole mass spectrometer sented therefore enables effective Poland with ESI in positive ionization. The limits of quantification of the detection of capsaicin even after The analytes were then quantified analysis were 0.5 pg/mL for cap- 24 hours following application of Literature by multiple reaction monitoring saicin and 1 pg/mL for dihydro- gels containing this compound. 1. Y. You, C. E. Uboh, L. R. Soma, F. Guan, (MRM). MRM transitions were capsaicin. Figure 1 shows the chro- D. Taylor, X. Li, Y. Liu, J. Chen: Validated m/z 180.30 → 110.05; 180.30 → matogram of pretreated horse se- Very good linearity was achieved UHPLC–MS-MS Method for Rapid 93.10; 180.30 → 65.05 for phena- rum containing capsaicin (1 pg/mL) in the concentration range from Analysis of Capsaicin and Dihydrocapsa - cetin, m/z 306.10 → 137.05; and dihydrocapsaicin (1 pg/mL). 0.5 to 1,000 pg/mL (capsaicin) and icin in Equine Plasma for Doping 1 to 1,000 pg/mL (dihydrocapsai- Control, Journal of Analytical Toxicology cin). Calibration curves and linear (2013) 37(2) 122-132. DOI 10.1093/jat/ 2.5 regression coefficients are shown bks098. in figure 2. 2. A. Zak, N. Siwinska, M. Slowikowska, 2.0 H. Borowicz, P. Szpot, M. Zawadzki and 1.5 Real samples analysis A. Niedzwiedz: The detection of cap-

Area saicin and dihydrocapsaicin in horse 1.0 The method described was suc- serum following long-term local adminis- 0.5 cessfully applied for the detection tration BMC Veterinary Research (2018) 14:193. DOI 10.1186/s12917-018-1518-9 0.0 of capsaicin and dihydrocapsaicin 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 in horse serum following long- Concentration term local administration [2].

1.00 Conclusions

0.75 The undoubted advantages of the method presented are its simplici- Area 0.50 ty, high sensitivity and the fast 0.25 sample preparation procedure. The possibility of detection of 0.00 capsaicin and its metabolite in 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 concentrations from 0,5 pg/mL Concentration respectively 1 pg/mL enables the effective fight against this kind of doping in equestrian sports. Figure 2: Calibration curves of capsaicin (above) and dihydrocapsaicin (below) Capsaicin also has more applica- tions, so after small modifications,

SHIMADZU NEWS 1/2019 15 APPLICATION

GCMS-TQ8050 NX Less expensive with easier handling Analysis of dioxins in foods and feeds using GC-MS/MS

ersistent organic pollutants (POPs) are semi-volatile and (x 1,000) (x 1,000) (x 1,000) 3.0 P can be detected worldwide, 5.0 3.0 4.5 even in remote regions. They bio- 2.5 2.5 accumulate with potential negative 4.0 123 3.5 influence on environment and hu- 2.0 2.0 3.0 man health. To a varying degree, 2.5 1.5 1.5 these organic compounds resist 2.0 1.0 1.0 photolytic, biological and chemi- 1.5 cal degradation. 1.0 0.5 0.5 0.5

POPs in foods and feeds can be 17.0 17.5 18.0 18.5 22.0 22.5 23.0 23.5 27.5 28.0 28.5 29.0 analyzed with several methods. Dioxins are particularly toxic even (x 1,000) (x 1,000) (x 1,000) 7.0 8.0 for POPs, so quantitative analysis 6.0 is required down to low concen- 6.0 7.0 455.0 6 6.0 trations. Until recently, the analy- 5.0 sis of dioxins was performed using 5.0 4.0 4.0 high-resolution GC-HRMS, 4.0 3.0 3.0 which provides highly accurate 3.0 2.0 quantitation. However, triple 2.0 2.0 quadrupole GC-MS/MS is less 1.0 1.0 1.0 expensive and easier to handle than GC-HRMS, so its use is 36.5 37.0 37.5 38.0 16.5 17.0 17.5 18.0 20.5 21.0 21.5 22.0 increasingly being investigated. (x 1,000) (x 1,000) (x 1,000) 9.0 In recent years, the quantitative 2.5 4.0 8.0 accuracy of GC-MS/MS has im- 7893.5 7.0 2.0 proved significantly. Accordingly, 3.0 6.0 2.5 the use of this analysis method has 1.5 5.0 been officially recognized in the 2.0 4.0 EU (EU589/2014, 644/2017). 1.0 1.5 3.0 However, to change from GC- 1.0 2.0 0.5 HRMS to GC-MS/MS it is neces- 0.5 1.0 sary to compare their respective quantitative abilities. 27.0 27.5 28.0 28.5 31.0 31.5 32.0 32.5 36.5 37.0 37.5 38.0

The Shimadzu GCMS-TQ8050 Figure 1: GCMS chromatograms for a concentration of 0.050 pg/uL NX triple quadrupole mass spec-

16 SHIMADZU NEWS 1/2019 APPLICATION

trometer uses a high-sensitivity detector, capable of detection at TEQ (pg/μL) Pork (Fat) TEQ (pg/μL) Raw milk femtogram order concentrations, 0.1 0.1 enabling the analysis of dioxins in 0.08 foods and feeds. Additionally, the 0.08 0.06 “GC-MS/MS Method Package for 0.06 0.04 Dioxins in Foods” consists of 0.04 0.02 method files registered with opti- 0.02 0 mal conditions for the analysis of 0 Sample Sample Sample Sample Sample Sample Sample dioxins, as well as a report creation 1 23456780101112 1 2 3 4 5 6 7 tool which documents the items GC-HRMS GC-MS/MS GC-HRMS GC-MS/MS required by EU regulations.

TEQ (pg/μL) Equine (Liver) TEQ (pg/μL) Oil In this article, dioxins (polychlori- 0.5 0.5 nated dibenzo-p-dioxin (PCDD) 0.4 0.4 and polychlorinated dibenzofuran 0.3 0.3 (PCDF) only) were analyzed in 44 types and 200 samples of foods and 0.2 0.2 feeds using the GCMS-TQ8050 NX 0.1 0.1 in combination with the method 0 0 package. Additionally, GC-MS/MS Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 analysis results were compared GC-HRMS GC-MS/MS GC-HRMS GC-MS/MS with those from GC-HRMS in order to evaluate the quantitative capabilities of both techniques. TEQ (pg/μL) Hen (Egg) TEQ (pg/μL) Leek 1.2 0.5 1 0.4 Method files for the 0.8 0.3 analysis of dioxins 0.6 0.2 0.4 The features of the “EU Regula- 0.2 0.1 tion Compliant GC-MS/MS 0 0 Method Package for Dioxins in Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Sample 6 Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Sample 6 Foods” are shown below. GC-HRMS GC-MS/MS GC-HRMS GC-MS/MS

• Method files registered with TEQ (pg/μL) Grass optimal conditions for the ana- 0.5 lysis of dioxins. 0.4 • Retention times and time pro- 0.3 grams can be adjusted automati- cally, even when the retention 0.2 times for the measured com- 0.1 0 pounds change, such as when 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 conducting maintenance of the column tip. GC-HRMS GC-MS/MS • A report creation tool is includ- ed in this product. It can auto- Figure 2: Comparison of the TEQ results for each food and feed matically create reports showing items required by EU regula- tions. automatic pretreatment unit (ex- ST and DF-LCS from Wel lington Analytical conditions in detail are traction: SpeedExtractor [BUCHI]; Laboratories was used. In terms shown in table 1 (page 19). ‘ Experiment purification: GO-xHT [Miura Co., of the analytical conditions for Ltd.]). 10 uL of Nonane was used GC-MS/MS, the conditions regis- For the various food samples, pre- as final solvent for the samples. tered in the method package were treatment was performed using an For the standard, a mixture of DF- used.

SHIMADZU NEWS 1/2019 17 APPLICATION

S/N Ratio (hereinafter 100 »Method 1«)

The concentration of an analyte in the extract of a sample which pro- duces an instrumental response at two different ions to be moni- 10 tored with an S/N (signal/noise) ratio of 3:1 for the less intensive raw data signal.

Lowest concentration point on the calibration curve (hereinafter »Method 2«) 1 0.001 0.01 0.1 1 10 100 The lowest concentration point on a calibration curve that gives an acceptable (≤ 30 %) and consistent (measured at least at the start and at the end of an analytical series of 0.1 samples) deviation from the aver- GC-MS/MS TEQ (pg/μL) age relative response factor calcu- lated for all points on the calibra- tion curve in each series of sam- ples. In this technical report, for the purposes of confirmation, an evaluation was performed using 0.01 both 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, 0.001 the STD was prepared so that the GC-HRMS TEQ (pg/μL) concentration of each compound was 0.050 pg/uL (for Octachloro- Boar (Muscular) Boar (Liver) Bovine (Grease) Cabbage Canola seed dibenzo-p-dioxin and Octachlo- Carp Cereal Chicken (Egg) Corn Dairy product rodibenzofuran 0.100 pg/uL). Rape oil Deer (Muscular) Deer (Liver) Equine (Liver) Feed mixes From the results of the analysis, it Fish Pork fat Grass Heifer (Milk) Hen (Egg) Honey Leek Cheese Mackerel Milk was evident that the criteria for Mixed milk Moules Oil Ovine (Liver) Ovine (Fat) method 1 were satisfied for all Oysters Pigment Egg Yolk Pike Powder Quail (Muscular) compounds. S/N ratios for each Raw milk Salad Sediment Soil Straw compound are shown in figure 1 Tuna Veal (Grease) Veal (Muscular) Water and table 2.

Additionally, with method 2, a Figure 3: Summarized comparison of the TEQ results for TQ and HR MS calibration curve was created with all six levels used, including 0.025 pg/uL, 0.050 pg/uL, 0.100 Analysis results for pretreatment method and the TEF pg/uL, 0.250 pg/uL, 0.500 pg/uL the standards (toxic equivalence factor) of each and 1 pg/uL. The concentrations compound. for each compound at each cali- In the analysis of dioxins in foods, bration curve point (level) are Maximum Levels (ML) are pre- The compounds 2,3,7,8-Tetrachlo- shown in table 3. For each com- scribed for each sample. With the rodibenzo-p-dioxin and 1,2,3,7,8- pound, when the level 1 RRF and food and feed samples in this Pentachlorodibenzo-p-dioxin have average RRF were compared, it investigation, ML for pig fat and the highest TEF (TEF = 1), re- was found that all compounds sat- pig meat were the lowest at 1 pg/g quiring lower LOQs than other isfied the criteria for method 2. of fat. Additionally, the limit of compounds. In this investigation, From the above-mentioned re- quantitation (LOQ) required for the LOQ for both dioxins in pig sults, it was evident that at the each compound in the analysis fat and pig meat was 0.060 pg/uL LOQ, the criteria were satisfied depends on the sample’s ML, the at the concentration in the final vial. for all compounds.

18 SHIMADZU NEWS 1/2019 APPLICATION

Analysis results for System configuration Analytical conditions (GC) the test samples Pretreatment Unit Speed Extractor (BUCHI) Insert Liner Topaz® single gooseneck liner, (Extraction) with wool As previously noted, the level of Pretreatment Unit GO-xHT (Miura Co., Ltd.) Column SH-Rxi™-5Sil MS (60 m, 0.25 mm I.D., toxicity differs for each dioxin (Purification) 0.25 µm), SHIMADZU compound. The TEF, calculated Autosampler AOC-20i/S Injection Mode Splittless for each compound by taking the GC-MS/MS GCMS-TQ-8050 NX Sampling Time 1.00 min. toxicity of 2,3,7,8-Tetrachlorodi- Software GCMSsolution™ Ver. 4.45 SP1 Injection Temp. 280 °C benzo-p-dioxin as 1, is used as an LabSolutions Insight™ Ver. 3.2 SP1 Column Oven Temp. 150 °C (1 min) →(20 °C/min) → index of strength. The TEF values GC-MS/MS method package for 220 °C →(2 °C/min) →260 °C (3 min) for each compound are shown in dioxins in foods →(5 °C/min) →320 °C (3.5 min) table 3. The ML for the dioxins in Analytical conditions (AOC-20i/s) HP Injection 450 kPa (1.5 min.) foods and feeds are prescribed by # of Rinses with 3 Flow Control Mode Linear Velocity (45.6 cm/sec.) their toxic equivalents (TEQ). The Solvent (Pre-run) TEQ is calculated by multiplying # of Rinses with 3 Purge Flow 20 mL/min. the concentration of each com- Solvent (Post-run) pound by the TEF and then calcu- # of Rinses with 0 Carrier Gas Helium lating the total TEQ for all com- Sample Analytical conditions (MS) pounds. Washing Volume 6 μL Ion Source Temp. 230 °C Injection Volume 2 μL Interface Temp. 300 °C Figure 2 (page 17) shows a com- Viscosity Comp. Time 0.2 sec. Detector Voltage 1.8 kV (Absolute) parison of the TEQ values for GC-MS/MS and GC-HRMS for Table 1: GC-MS/MS analytical conditions food samples. A TEQ of 0.060 pg/ uL (red line) and a TEQ of 0.025 Nr. of compound Compound name Calculated S/N pg/uL (green line) are marked as in figure 1 indicators for the samples. 1 2,3,7,8-Tetrachlorodibenzo-p-dioxin 285 2 1,2,3,7,8-Pentachlorodibenzo-p-dioxin 1,658 Conclusion 3 1,2,3,4,7,8-Hexachlorodibenzo-p-dioxin 396 4 Octachlorodibenzo-p-dioxin 2,518 In this technical report, dioxins 5 2,3,7,8-Tetrachlorodibenzofuran 2,117 were analyzed in 44 types and at 6 1,2,3,7,8-Pentachlorodibenzofuran 1,882 least 200 samples of foods and 7 1,2,3,7,8,9-Hexachlorodibenzofuran 546 feeds using the GCMS-TQ8050 8 1,2,3,4,6,7,8-Heptachlorodibenzofuran 1,784 and the “EU Regulation Com- 9 Octachlorodibenzofuran 4,282 pliant GC-MS/MS Method Pack- age for Dioxins in Foods”. Addi- Table 2: S/N results for standards according to method 1 tionally, the GC-MS/MS analysis RRF Dev (%) results were compared with the Compound name TEF Avg RRF RRF (lvl 1) analysis results from GC-HRMS lvl 1 in order to assess the quantitative 2,3,7,8-Tetrachlorodibenzo-p-dioxin 1 1.07 1.15 8.1 capabilities of both methods. Be- 1,2,3,7,8-Pentachlorodibenzo-p-dioxin 1 1.09 0.97 10.56 fore analyzing, a STD was meas- 1,2,3,4,7,8-Hexachlorodibenzo-p-dioxin 0.1 1.14 1.39 22.26 ured using GC-MS/MS, and it 1,2,3,6,7,8-Hexachlorodibenzo-p-dioxin 0.1 0.95 0.92 2.72 was confirmed that the criteria 1,2,3,7,8,9-Hexachlorodibenzo-p-dioxin 0.1 1.03 1.25 21.44 were satisfied at the LOQ. 1,2,3,4,6,7,8-Heptachlorodibenzo-p-dioxin 0.01 0.92 0.82 11.46 Octachlorodibenzo-p-dioxin 0.0003 1.19 1.04 12.21 From the above-mentioned re- 2,3,7,8-Tetrachlorodibenzofuran 0.1 1.10 1.05 4.66 sults, it is evident that analysis 1,2,3,7,8-Pentachlorodibenzofuran 0.03 1.04 1.00 3.23 with GCMS-TQ8050 NX and 2,3,4,7,8-Pentachlorodibenzofuran 0.3 0.97 0.89 7.59 method package provides a quan- 1,2,3,4,7,8-Hexachlorodibenzofuran 0.1 1.03 0.82 20.72 titative capability equivalent to 1,2,3,6,7,8-Hexachlorodibenzofuran 0.1 1.09 1.36 24.62 that of GC-HRMS for samples at 2,3,4,6,7,8-Hexachlorodibenzofuran 0.1 1.09 1.39 27.83 the concentration levels required 1,2,3,7,8,9-Hexachlorodibenzofuran 0.1 1.06 1.23 16.10 for analysis. However, at concen- 1,2,3,4,6,7,8-Heptachlorodibenzofuran 0.01 1.17 1.05 10.37 trations below the required level, 1,2,3,4,7,8,9-Heptachlorodibenzofuran 0.01 1.02 0.97 4.97 differences in quantitative capabil- Octachlorodibenzofuran 0.0003 1.00 0.84 15.80 ity could arise. For this reason, it is necessary to be aware of the Table 3: Each calibration point concentration and RRF for the measured compounds system status by confirming quan- titative capability at the LOQ, and evaluating whether there has been a decrease in sensitivity.

SHIMADZU NEWS 1/2019 19 PRODUCTS

Since system back pressure in- creases strongly with smaller par- Speeding up with ticles, another benefit of core shell particles is a lower increase of overall pressure, especially when the HPLC system is not very Velox Core Shell pressure stable and a faster separa- tion is desired. The flow rate can therefore be higher than for col- The new “Velox Core Shell“ LC columns offer more umns with fully porous materials. In the following text, these effects application possibilities are tested in an application.

Measurement parameters o far, the Shim-pack column and methods portfolio has exclusively S covered fully porous col- Instrument: LC-2040C 3D umns. The new ”Shim-pack Velox (Shimadzu) Core Shell“ series closes the gap Column: Shim-pack Velox C18 ; in the range of columns and thus (150 mm x 3.0 mm I.D., 2.7 μm); serves a larger number of applica- Shim-pack GIST C18 ; (150 mm x tion requirements than before. 3.0 mm I.D., 3.0 μm) The word velox derives from Mobile phase: 35 % H2O; Latin, meaning ”fast“ or ”quick“. 65 % ACN It stands for columns with a fast Oven temperature: 40 °C separation performance leading Flow rate: 0.4 mL/min to shorter analysis time when Injection volume: 0.5 μL compared to conventional, fully porous columns. Results

Core Shell technology The two chromatograms in figures 2 and 3 demonstrate a comparison Core shell means that the single of a fully porous column and a filling material particles of the col- Core Shell column. The methods umn are not fully porous as usual were identical for both columns. but have a solid core covered with The retention times are compared the fully porous material, just like in table 1. a shell (figure 1). Core shell liter- Figure 1: Schematic structure of a core shell particle with a solid core (dark blue) ally means wrapped core. and the porous layer (light blue) When comparing the figures and the table, it becomes apparent that The solid core is impermeable to ary phase, thus leading to shorter smaller, fully porous particle size the Velox-C18 column has much solvents and analytes. This results analysis time and higher efficiency (e.g. 1.9 μm) is comparable to a shorter retention times than the in a considerably shorter contact of the analysis. One of the charac- larger particle size of a core shell fully porous GIST-C18. The peak time between sample and station- teristics is that the efficiency of a particle (2.7 μm). width is also smaller, meaning that

mAU mAU 75 75

50 50

25 25

0 0 0 1.0 2.0 3.0 4.0 0 1.0 2.0 3.0 4.0 Minutes Minutes

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

20 SHIMADZU NEWS 1/2019 PRODUCTS

the Velox-C column has a con- • the analysis time should be 18 t R (min) t R (min) t R (min) t R (min) siderably higher efficiency. Uracil Methyl benzoate Toluene Naphthalene shorter, without much invest- ment, GIST C18 0.74 1.87 2.90 3.41 In the last peak (naphthalene), Peak width 50 % 0.03 0.05 0.07 0.08 retention time and peak width dif- • a higher throughput is desired, Velox C18 0.65 1.13 1.66 1.94 fered significantly. Besides the Peak width 50 % 0.02 0.03 0.03 0.03 effect of Core Shell Particles or • the current HPLC system does fully porous particles, this is due Table 1: Retention times and peak widths of GIST C18 and Velox C18 not have a system volume that is to the role of carbon content and too high (narrow peaks are the surface of the columns in addi- The retention profile is balanced, MS sensitivity is increased, partly widened again by higher system tion to the column type, which and the column is also suitable for due to the high organic content volumes), contribute strongly to the reten- LC-MS/MS (mass spectrometry) that can be used in HILIC mode. tion. The larger the surface area analysis. • UHPLC columns have an and carbon content on the column, Application excessively high back pressure the higher are the interactions Shim-pack Velox C18 or a short service life, between the analytes and the sta- This phase is the standard choice When does it make sense to invest tionary phase, and the higher is for reversed phase chromatography. in a Core Shell column? As is of- • problems with clogging occur the retention. Carbon content is The Velox C18 has the highest ten the case, this depends on the with the current method, or 7 % for the Velox C18 column and hydrophobic retention in the application, but also on the exist- 14 % for the GIST C18 column; Velox portfolio. It is compatible ing HPLC device to perform the • loadability is not important. surface area is 130 m2/g (Velox) with slightly acidic to neutral pH measurement. Many users use and 350 m2/g (GIST). It is clearly values of the mobile phase. Core Shell columns to convert an

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Shimadzu NEWS, Customer Magazine of Shimadzu Europa GmbH, Duisburg Figure 4: Various stationary phases of the Shim-pack Velox columns Publisher Shimadzu Europa GmbH visible that the peaks in the Velox Shim-pack Velox Biphenyl existing HPLC method into one Albert-Hahn-Str. 6 -10 · D-47269 Duisburg column method are much narrow- The biphenyl phase offers comple- with UHPLC characteristics. Phone: +49 - 203 - 76 87-0 er and higher than with the fully mentary selectivity to the alkyl Fully porous columns with larger Fax: +49 - 203 - 76 66 25 porous column. phases. It is suitable for enhanced particles are replaced by Core [email protected] separation of aromatic com- Shell columns with smaller parti- www.shimadzu.eu Shim-pack Velox portfolio pounds. The biphenyl phase is cles. The user then benefits from Editorial Team ideal for increasing the sensitivity faster analysis without too much Uta Steeger The Core Shell Velox portfolio and selectivity of LC-MS/MS back pressure exceeding the limits Phone: +49 (0)203 76 87-410 covers a wide range of stationary analyses. of the HPLC system. Further Ralf Weber, Maximilian Schulze phases and dimensions. A suitable advantages are large savings in Design and Production precolumn is also available for Shim-pack Velox PFPP costs and time. m/e brand communication GmbH GWA each stationary phase. Figure 4 PFPP stands for pentafluorophe- Duesseldorf shows all phases with their specifi- nylpropyl. This group provides Since the loading capacity of Core Circulation cations. alternative selectivity for confor- Shell columns is not as high as German: 5,360 · English: 3,975 mational isomers and halogenated that of fully porous columns due Copyright In the following, the different compounds. In addition, charged to the solid core, there is less Shimadzu Europa GmbH, Duisburg, types are shown individually: bases are retained more strongly. material in the column with which Germany – February 2019. the sample can interact. If the Shim-pack Velox SP-C18 Shim-pack Velox HILIC sample is too highly concentrated, Windows is a trademark of Microsoft SP stands for “sterically protect- With Velox-HILIC (Hydrophilic this has a negative effect on the Corporation. ©2019 ed”. Due to the steric protection, Interaction Chromatography), peak shape. Apple Inc. All rights reserved. Apple, the column can be used very effec- polar analytes can be retained. the Apple logo, Mac OS and iOS are tively for mobile phases in the The chemical composition of the In general, a Core Shell column is trademarks of Apple Inc. strongly acidic pH range (pH 1-3). stationary phase is pure silica. suitable if:

SHIMADZU NEWS 1/2019 21 APPLICATION Organic plastic in beverage bottles FTIR analysis of beverage bottles, partly produced from naturally renewable raw materials

1.00 Even the PET bottles themselves, which are available on the market, 0.75 show different ratios of propor- 0.50 tions of A- and C-PET. They are Abs. also unevenly distributed over the 0.25 entire bottle body. 0.00 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 The PET variants are usually not Bottle, transparent, colorless, outside. Still water. QATR-S cm-1 the same on the outside, inside or 1.00 at the bottleneck. The reason is

0.75 that PET reacts to heat forming with crystallization [2]. The in- 0.50 frared spectrum is further influ- Abs. 0.25 enced by the arrangement of the molecular structures in the PET 0.00 and by the manufacturing process 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 of PET as a polymer. This article Polyethylene terephthalate (PET) DuraSampeII-RII cm-1 focuses on PET as A/C PET.

A-PET and C-PET distributed Figure 1: PET spectrum of the plant based raw material bottle identified by the infrared spectrum search in polymer libraries; differently best hit was found in the ATR polymer library. In a separate screening of packag- ing material, various PET bottles lastic packaging, plastic are also used, such as PLA, a plas- Shimadzu IRSpirit-T equipped for beverages from Europe and waste, microplastic and tic based on polylactic acid. How- with a diamond-based QATR-S Japan were analyzed. For compari- P environmental protection – ever, it does not have the same single-reflection unit was used for son, 9 samples were used (table 1). the pollution of nature and the flexibility as PET in terms of its the analysis. This is possible be- In all investigations of PET bot- seas by plastic is one of the top physical properties (stability, bot- cause the polymers react to heat tles, a higher proportion of A-PET issues in the press. Plastic doesn’t tles, boxes and foils etc.) PLA and the molecular groups of these was found on the outer wall and rot or decay; it takes decades or used in food applications has up chemical compounds vibrate. more of C-PET on the inner wall. centuries before it is decomposed to now been rather stiff and brit- by nature. tle. It can be found in disposable These vibrations are characteristic Pure A-PET is rarely found in dishes and disposable cups, but it for each substance, and they are bottles, which is probably in the Among other things, PET, a versa- is also used in 3D printers for recorded wave-dependent on the nature of PET. Likewise pure tile plastic, is used to make bever- printing. molecular absorption in the range C-PET does not exist either (max- age bottles. Despite strong recy- of about 400 to 4,000 cm-1 against imum proportion of crystallization cling efforts, production has in- Can natural renewable the molecular absorption to re- is about 75 % [2] [3]). A typical creased from 40 to 56 million tons raw materials be 100 % ceive a so-called mid infrared infrared spectrum of these bottles (2008-16) [1]. That the sturdy recycled? spectrum. It can then be identified is shown in figure 1. The focus is polymer-based beverage bottles with the help of libraries. on the signals at about 1,410 and are made of polyethylene tereph- It is questionable when about 1,340 cm-1. Depending on the pro- thalate (PET) can be read on the 25 % of a beverage bottle is made A/C-PET in the analysis portion, the signal at 1,340 cm-1 bottles. The symbol for PET in from naturally renewable raw (closer to C-PET) is higher than recycling is a clearly written ‘PET’ materials but is then declared to PET bottles are made of A/C the signal at 1,410 cm-1 (closer to in the recycling symbol or a ‘1’ in be 100 % recyclable. To solve this PET. Depending on the wall A-PET). Furthermore, shifts of the recycling triangle. This sub- puzzle, FTIR spectroscopy was thickness (soft or hard polymer) the -CH and CH2 groups take stance can be recycled as waste. used because a PET label was and application, PET can contain place, which is evident in the re- found on the beverage bottle. more amorphous (A-PET) and gion around 2,900 cm-1 (figure 2). In order to appear more environ- less crystalline (C-PET) PET or mentally friendly, alternative plas- FTIR spectroscopy allows the vice versa. The two versions have In addition to ‘standard bottles’, tics from natural basic substances substances to be identified. A different FTIR spectra. a bottle claiming to be partly made

22 SHIMADZU NEWS 1/2019 SHIMADZU NEWS1/2019 of theouterandinnerwall. of themiddlelayerisresult show differences.Thespectrum of PET. Innerwallandouter ferent degreesofcrystallization The spectraclearlyshowthedif- groups tothePETvariants. better assignmentofthesignal lysis resultsofthesampleallow found inallthreelayers.Theana- middle andoutside.PETcouldbe three differentways:inside,inthe 2 μm,thewallwasexaminedin surface onlytoadepthofabout As theinfraredbeamenters sides usingtheATR technique. open andanalyzedfromboth gated. Thebottlewallwascut of plantmaterialwasalsoinvesti- middle (redline)ofthebottlewall. for stillwater: Innerwall (greenline), outerwall (blackline)andaspectrumfromthe Figure 2: InfraredspectraofaPET-based bottlewiththeplantbasedrawmaterials Figure 4: Enlargementfromfigure2-range1,440-1,320 cm attributed toethyleneglycol[2] Abs Abs 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.0 0.2 0.4 0.6 0.8 1.0 4,000 1,440 Bottle, transparent, colorless, outside. Stillwater. QATR-S Bottle, transparent, colorless, outside. Stillwater. QATR-S 3,500 1,430 1,420 3,000 1,410 2,500 1,400 2,000 1,390 1,380 1,750 h rdcino E,wihi produced,forexample,fromthe the productionofPET, whichis ment ofthestartingproductsfor to thepuzzleliesindevelop- dox onfirstglance?Thesolution able rawmaterials.Isthisapara- duced upto22.5%fromrenew- miniature figure,whichwaspro- On thebottle,PETisdeclaredin been identifiedasPET(figure1). Using thelibrarysearch,ithas 9intable1). (counter materials raw the bottlecontainingplantbased The actualanalysiswasmeantfor Conclusion middle layer(figures2to4). approximately thenatureof the twooutersurfacesshows After superposition,additionof 1,370 1,500 1,360 -1 1,250 , rangeofvibration 1,350 1,000 1,340 1,350 750 1,320 cm cm -1 -1 still seemsfaraway. [4] 100 percentorganic plasticbottles wide. Nevertheless,thewayto plants thatareavailableworld- are beingresearched,suchas try, andalternativestosugarcane and moreimportanttotheindus- Bio-plastics arebecomingmore after usage. can berecycledeasilyandwell which isoneofthepolymersthat a technicalmaterialsuchasPET, from naturemeetproductionof cane sugar. Atthispoint, plants ethylene glycolisobtainedfrom the Internet,PETrawmaterial the manufacturerandreadableon terephthalic acid.Accordingto monomers ethyleneglycoland category 01forPET. infrared spectroscopy. All butonebottle werelabeledaspolymersofrecycling Table 1: Transparent PETbottlescollectedafteruseandexaminedusing ATR of thealiphaticandaromatic-CHmoleculargroups[2] Figure 3: Enlargementfromfigure2, range3,050to2,820cm Mineral water withlemon flavor sugarfree Mineral water withCola flavor Still mineralwater Lemonade Mineral water conatainingCO Tasty Bluewater Beer Smoothie Green tea Abs 0.01 0.02 0.03 0.04 0.05 0.06 3,040 Bottle, transparent, colorless, outside. Stillwater. QATR-S Product 3,020 3,000 2,980 2 2,960 rgnPolymer declared Origin NL GER JPN NL NL GER GER JPN GER 2,940 Literature [2] „Plastic Additives Handbook“, H. Zweifel [1] https://de.wikipedia.org/wiki/ [4] https://www.weser-kurier.de/ [3] FTIR spectroscopicanalysisofpoly(ethyl- 2,920 et all, 6 Polyethylenterephthalat 1286848.html alle-Flaschen-aus-Zucker-_arid, wirtschaft_artikel,-CocaCola-Bis-2020- deutschland-welt/deutschland-welt- nal 48(9):1586-1610September2012. Ziyu Chen, etall, EuropeanPolymer Jour - ene terephthalate)oncrystallization, for PET(notveryapparent) materials andminiaturesymbol Bottle withplantbasedraw PET PET PET PET PET PET PET PET 2,900 th APPLICATION edition, Hanser Verlag, 2009. -1 2,880 , rangeofvibration 2,860 2,840 cm 2,820 -1 23 APPLICATION New testing methodology for weight reduction Automotive industry – Case of tensile test with DIC strain analysis of composite material

is extremely important from the Testing machine creates perspective of safe application of signal for camera CFRP materials, e.g. in aircraft and other. In this test, the change in load occuring during specimen fracture was used to trigger the HPV-X Note: high-speed video camera. Specif- The CFRP laminate board used in the ically, the AG-X plus precision actual test was created by laying up universal testing machine was pre-impregnated material with fibers configured to generate a signal oriented in a single direction. when the test force on the speci- men reaches half the maximum The [+45/0/-45/+90]2s shown as the test force (referred to as Maxi- laminate structure in table 1 refers mum test force in figure 2), with to the laying up of 16 layers of mate- Figure 1: Main unit combined with the HPV-X2 Camera this signal being sent to the high- rial with fibers oriented at +45°, 0°, speed video camera. Static tensile -45°, and +90° in two-layer sets. mproving energy consumption tive parts using composite materi- testing and fracture observation is one of the most important als will greatly advance. were performed according to the I tasks of the automotive market For this reason, it is important to in order to comply with environ- understand the mechanism of mental regulations in each coun- occurrence and fracture. A new Maximum test try. One of the solutions to solve test method confirming the struc- force the problem of energy consump- ture of composite materials is pro- 80,000 tion is weight reduction. It is nec- posed here. In addition, a dedicat- a essary to achieve it while still ed example is presented, although 60,000 securing the safety of the passen- several test methodologies exist gers. Various new materials are for composite materials. 40,000 being developed in order to bal- (N) Force ance these needs. Looking at met- Universal testing machine and 20,000 als, these are high-tension steel, high-speed video camera aluminium alloys, magnesium 0 alloys and others. With plastics This article explains how to use 0 20 40 60 80 100 120 140 160 180 200 and composite materials such as the precision universal testing Time (s) CFRP (Carbon Fiber Reinforced machine (Autograph AG-X plus Plastic) and GFRP (Glass-Fiber 250 kN) and the high-speed video

Reinforced Plastic), attention is camera (Hyper Vision HPV-X) 80,000 Change in test force can be given to combining weight reduc- (figure 1) to evaluate the static measured in detail during tion with high-strength proper- fracture behaviour of a CFRP failure occurrence ties. Practical usage of several based on a test force attenuation 60,000 b The interval between data materials has already started in the graph and images of material fail- points on test force plot is market, but research is still ongo- ure. Information on specimens is 40,000 approximately 3.3 μs

ing. shown in table 1. A 6 mm diame- (N) Force ter hole is machined in the speci- 20,000 In the case of composite materials, men centre. Fractures are known the internal structure is complicat- to propagate easily through com- ed, so there is a possibility of a posite materials from the initial 0 failure mode different from con- damage point, and when a crack 0 20 40 60 80 100 120 140 160 180 200 ventional materials. If the cause of or hole is present, the material Time (μs) breakdown can be demonstrated strength is reduced markedly. by means of structural analysis Therefore, evaluation of the Figure 2: Test graphs simulation, the design of automo- strength of open-hole specimens

24 SHIMADZU NEWS 1/2019 APPLICATION

Figure 3: Observation of fracture Figure 4: Observation of fracture (DIC analysis)

Dimensions Testing machine AG-Xplus Laminate structure L (mm) x W (mm) x T (mm), Load cell capacity 250 kN hole diameter (mm) Jig Upper: 250 kN non-shift wedge type grips (with trapezoidal file teeth on grip faces for [+45/0/-45+90]2s 150 x 36 x 2.9, Ø6 composite materials) Table 1: Test specimen information Lower: 250 kN high-speed trigger-capable grips Grip space 100 nm conditions shown in table 2. A that perpendicular to the load. In Loading speed 1 mm/min test force-displacement plot for image 2, cracks occurring around Test temperature Room temperature the open-hole quasi-isotropic the circular hole are propagating Software TRAPEZIUM X (single) CFRP (OH-CFRP) is shown in along the surface +45° layer. In Fracture observation HPV-X high-speed video camera figure 2a. A test force-time plot images 3 through 6, a substantial (recording speed 600 kfps) during the occurrence of material change can be observed in the DIC analysis StrainMaster (LaVision GmbH) fracture is also shown in figure 2b. external appearance of the speci- men near the end of the crack Table 2: Test conditions (*fps stands for frames per second. This refers to the number Figure 2a can be interpreted to propagating to the bottom right of frames that can be captured in one second). show that the specimen fractured from the circular hole. This sug- at the moment it reached maxi- gests that not only the surface around the circular hole is focused mum test force, at which point the layer, but internal layers are also diagonally toward the top-left load on the specimen was sudden- fracturing. Based on the images of (-45 °) and toward the bottom-left ly released. This testing system the same area and the state of the (+45 °) from the circular hole. can be used to perform high-speed internal layers that can be slightly Images 5 through 8 show the sampling to measure in detail the observed from the edges of the focusing of strain diagonally change in test force in the region circular hole in images 7 and 8, toward the bottom-right (-45 °) of maximum test force. The time the internal fracture has propagat- and toward the top-right (+45 °) interval between data points on ed quickly in the 18 μs period from the circular hole in areas the test force plot in figure 2b is between images 3 and 8. where it was not obvious in 3.3 μs. images 1 through 4. This shows Digital Image Correlation that an event is occurring in the Images 1 through 8 in figure 3 (DIC) analysis surface layer of the specimen that capture the behavior of the speci- is similar to the process of frac- men during fracture around the DIC analysis performed on the ture often seen during tensile test- circular hole. Image 1 shows the fracture observation images of fig- ing of ductile metal materials, i.e. moment cracks occur in a surface ure 3. Black signifies areas of the crack propagation in the direction +45° layer. In this image, the ten- surface layer of the specimen of maximum shear stress. sile load being applied is deform- under little strain, and red signi- ing the circular hole, with hole fies areas under substantial strain. diameter in the direction of the Looking at images 1 through 4 (in load of approximately 1.4 times figure 4), it can be seen that strain

SHIMADZU NEWS 1/2019 25 APPLICATION Ethanol as a blending component for petrol Determination of higher alcohols and volatile impurities by gas chromatographic method

esearchers have been experi- Researchers have used ethanol as a Two calibration stock solutions menting with alternative component in conventional petro- are used for the preparation of R fuels due to steadily increas- leum fuels because it has a higher calibration standards and control ing demand and decreasing supply thermal efficiency than ordinary samples. The first solution (cata- of petroleum crude oil. Ethanol petroleum motor fuels. Another logue number EN 15721-A) con- has been recognized as a possible benefit is its lower risk of fire dur- tains the ten target-compounds at ing storage and transportation 1 % (m/m) and the second (cata- compared to petroleum, which logue number EN 15721-A-IS) has greater volatility and lower contains the internal standard flash point than ethanol. (1 % m/m pentan-3-ol).

The EN 15721 European Calibration solution and Standard relates to the determi- sample preparation nation of several compounds This gas chromatographic method that may be present in ethanol includes the determination of a Preparation of Calibration solu- at different concentration levels. test portion with split injection tion: 1 mL of ethanol is trans- The target compounds are mode into a gas chromatograph ferred into a 2 mL vial and is separated into the following (GC) system. Addition of pentan- weighed to the nearest 0.1 mg. GC-2010 Plus groups: 3-ol to the sample is the only sam- 100 uL of calibration stock solu- ple pretreatment step. The sample tion (catalogue number EN liquid fuel alternative which can a) Higher alcohols: is then introduced to the gas chro- 15721-A) is added and mass is be available in significant quanti- propan-1-ol, butan-1-ol, butan- matograph (GC) system using recorded to the nearest 0.1 mg. ties throughout the remainder of 2-ol, 2-methylpropan-1-ol split injection mode. A Flame 80 uL of internal standard stock this century [1]. (isobutanol), 2-methylbutan-1- Ionization Detector (FID) is used solution (catalogue number EN ol, 3-methylbutan-1-ol. The for the detection of the analytes. 15721-A-IS) is added and mass is Ethanol blending is a process of compounds of the first group recorded to the nearest 0.1 mg [3]. mixing ethanol with petrol to can be determined up to 3 % Apparatus enhance the octane content in (m/m). Sample Preparation: 1 mL of fuel. This procedure reduces Gas chromatograph: Shimadzu sample is transferred into a 2 mL engine carbon monoxide (CO) b) Impurities: GC 2010 Plus, Split/Splitless vial and is weighed to the nearest and carbon dioxide emissions by ethanol (acetic aldehyde), ethyl- injector, FID detector 0.1 mg. 80 uL of internal standard up to 30 %. Moreover, it improves ethanoate (ethyl acetate), Capillary column: DB-1701 stock solution (catalogue number engine operation since it acts as an 1,1-diethoxy ethane (acetal) are (60 m × 0.25 mm ID, 1.00 μm film EN 15721-A-IS) is added and mass anti-knock agent [2]. specified up to 2 % (m/m). thickness) is recorded to the nearest 0.1mg [3].

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

26 SHIMADZU NEWS 1/2019 APPLICATION

Part of GC Values Split injector temperature 200 °C Split ratio 50 FID temperature 260 °C FID air flow 400 mL/min FID hydrogen flow 40 mL/min FID make-up flow 30 mL/min Carrier gas He Linear velocity 22 cm3/sec Column flow 1.4 mL/min Oven temperature program 40 °C→ 1 min 5 °C→ 250 °C→ 2 min

Table 1: Method parameters

Figure 3: Chromatogram of calibration solution Calibration compounds Description Methanol –– Propan-1-ol Higher alcohols Results and discussion and response factor results of Butan-1-ol Higher alcohols three calibration standard injec- Butan-2-ol Higher alcohols Chromatograms tions. 2-methylpropanol Higher alcohols 2-methylbutan-1-ol Higher alcohols The analysis was started with the Expression of 3-methylbutan-1-ol Higher alcohols injection of internal standard results Ethanal (acetic aldehyde) Impurity stock solution (catalogue number Ethyl-ethanoate (ethyl acetate) Impurity EN 15721-A-IS) and the injection Results were calculated through 1,1-diethoxy ethane (acetal) Impurity of calibration stock solution since the GC solution software accord- Pentan-3-ol Internal standard retention times of all compounds ing to the following equations: Ethanol Solvent should be determined. Resolution: R = 2 (t2 - t1) / 1,699 Table 2: Reagents and materials The next step was the analysis of (W1 + W2) Calibration Standard Solution to Response Factor: RF = Area GC-2010 Plus. The appropriate Finally, proper maintenance of the confirm compliance with the (ISTD) x C (compound) / Area use of the instrument and the injection port and column oven acceptance criteria, as far as chro- (compound) x C (ISTD) accurate application of EN 15721 before analysis helped to obtain matographic resolution is con- Impurities: Q = ethanol + ethyl- resulted in chromatograms with chromatograms with low noise cerned. Baseline separation was ethanoate + 1,1-diethoxyethane + an acceptable number of theoreti- levels and high S/N values for the obtained for all components oxygenated compounds cal plates while the presence of the target-compounds. except for 2-methylbutan-1-ol and Methanol: Cm = methanol con- AOC-20i auto-sampler contribut- 3-methyl-butan-1-ol (R = 1.3, R ≥ tent ed to the excellent repeatability. 1.0 is the EN 15721 specification). Higher alcohols: Ch = propan-1- ol + butan-1-ol + butan-2-ol + 2- Selection of an appropriate col- Calibration curve methylpropanol + 2-methylbutan- umn was critical to achieve accept- 1ol +3-methylbutan-1-ol able resolution (Rs > 1.0) between The Calibration Standard Solution 2-methyl-1-butanol and 3-methyl- was injected three times to obtain Concentration is expressed in 1-butanol. Also, the chromato- Authors a two-point calibration curve (y = g/100 g, % (m/m). graphic method parameters of EN Fotis Fotiadis, Georgia Flessia, a x) for each analyte. The desired 15721 and the adoption of default Dr. Gerasimos Liapatas, repeatability levels were achieved Conclusion values in gas flow rates for the Dr. Manos Barbounis since the % RSD was 0.0 - 0.5 % detector (FID) ensured that all N.Asteriadis S.A., (% RSD ≤ 5.0 is the EN 15721 The EN 15721 standard was acceptance criteria of the Standard Athens, Greece specification) for both area ratio successfully applied with the were fulfilled. Literature Compound Retention time (min) LOD (% m/m) LOQ (% m/m) [1] G. Najafi, B. Chobadian, T. Tavakoli, D.R. Ethanal 5.268 0.0009 0.0028 Buttsworth, T.F. Yusaf, M. Faizollhnejad, Methanol 5.567 0.0002 0.0006 Appl. Energy 86 (2009) 630-639. 1-propanol 9.360 0.0006 0.0019 [2] Y. Barakat, Ezis N. Awad, V. Ibrahim, Ethyl acetate 9.680 0.0017 0.0050 Egyptian Petroleum Research Institute 2-butanol 10.406 0.0002 0.0006 (EPRI), Egypt (2016), “Fuel consumption Isobutanol 11.555 0.0003 0.0009 of gasoline ethanol blends at different Acetal 12.672 0.0006 0.0017 engine rotational speeds” 1-butanol 12.876 0.0001 0.0002 [3] EUROPEAN STANDARD EN 15721 3-methyl-1-butanol 15.516 0.0003 0.0010 “Ethanol as a blending component for petrol – Determination of higher alco- 2-methyl-1-butanol 15.614 0.0002 0.0006 hols, methanol and volatile impurities – 3-pentanol (ISTD) 13.747 –– –– Gas chromatographic method” Table 3: Compound table

SHIMADZU NEWS 1/2019 27 ··· Don’t miss this! Limited number of participants ··· Don’t miss this! Limited number of participants ··· Pyrolysis GC-MS user meeting March 28, 20 19, at Shimadzu’s Laboratory World, Duisburg, Germany

ake this opportunity for However, there will be sufficient detailed discussions with time for sharing experiences and T experts from research and for discussions during the breaks. industry on the numerous pyrol - ysis GC-MS applications. We look forward to welcoming you to this special event in Duis - The all-day event offers a varied burg! program with lectures and practi - cal examples as well as cutting- edge information on instrument For further information, please technology and applications. use the link below: In addition, the tour around Shimadzu’s Laboratory World www.shimadzu.eu/Pyrolysis-User- provides insights into this high- Meeting end testing facility for the entire analytical instrumentation prod - uct range on over 1,500 m 2. GCMS-QP2020 NX

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