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NEWS 03.2014 GB:Layout 1 CONTENT APPLICATION Changing forest – what happens during Autumn? 2 About knackwursts and sausages – The big breakfast test using the EZ-Test-X Texture Analyzer (part 3) 7 Partial oxidation of isobutane – New analysis method using GC 8 Plastics – Water content as indicator for ageing processes 10 CH2 CH2 The nightmare of doping offenders – R1 R1 Analysis of doping agents using the 2 2 new LCMS-8050 Triple Quadrupole H3C R H3C R Mass Spectrometer 12 NN NN Espresso in Lake Constance – Five Mg Mg application examples of simultaneous NN NN elemental analysis sensitivity 15 H3C CH3 H3C CH3 Bilirubin determination in cerebro - spinal fluid following a subarachnoid hemorrhage – UV spectroscopy sup- H3COOC O H3COOC O 3 ports fast response 17 R OOC HOOC New: TOC Application Handbook, Chlorophyll a Chlorophyll b Chlorophyll c Version 2 19 1 1 1 2 R = CH3 R = CHO c1 R = CH3 R = C2H5 2 2 1 2 R = C2H5 R = C2H5 c2 R = CH3 R = CH = CH2 3 3 1 2 PRODUCTS R = Phytyl R = Phytyl c3 R = COOCH3 R = CH = CH2 „What can i do for you?” The new i-series – Nexera-i / Prominence-i – CH new integrated (U)HPLC systems 4 Phytyl: 3 H CHC 3 3 3 LATEST NEWS Precise temperature control of sample Figure 1: Structure of chlorophyll and its various naturally occurring species [3] cells – New CPS-100 accessory for UV-VIS spectrophotometers 6 Changing forest – what happens during Autumn? uring Autumn leaves lose rophylls are bound to apoproteins and can lead to the destruction of their green coloring, a pro - whereby the absorbed energy can vital plant organelles. This is why D cess also known as senes- enter metabolism. chlorophyll must be degraded cence. This is a generic term for very rapidly after separation from the process of ageing in living With increased ageing, this pro - the protein. This process is accom - organisms. It is assumed that se - cess becomes less stable and the panied by the color change [2]. nescence is associated with the proteins are gradually removed. Each chlorophyll molecule also chlorophyll contained in plants Should chlorophylls, at this point, contains four nitrogen atoms, (see infobox). These interrelation- not decompose as well, the energy which can be recycled for new ships and processes have been well produced would need to flow into processes in the plant. investigated scientifically and can other processes. In this case, oxy- be described as follows: gen molecules would be available Approach to absorb the energy. Oxygen mol - NEWS – Shimadzu’s customer maga - During photosynthesis, chloro- ecules in the ground state (O2) For a more detailed investigation 3 zine is also available as WebApp phylls absorb energy from sun- and in the triplet state ( O2) will of senescence, the chlorophyll (for iOS and Android) via: light to convert carbon dioxide yield activated (O2*) and singlet content of the leaves of an oak 1 www.shimadzu-webapp.eu and water into carbohydrates and oxygen ( O2) [1]. Such an oxygen (Quercus) was determined. The or as app for iPhone in the AppStore. oxygen. During this process, chlo - molecule is much more reactive oak leaves were also analyzed with SHIMADZU NEWS 3/2014 APPLICATION respect to their other naturally cules contain magnesium as the methanol, so that the sample ma - based magnesium standards in - occurring chlorophyll species (see central atom (see figure 1). As trix and the standard matrix are stead of expensive chlorophyll figure 1). Aim of the investigation before, the chlorophyll species identical. The water content of the standards makes the coupling of was direct comparison of green were separated via HPLC Mg-standard does not cause any HPLC and AAS extremely lucra- and yellow, already withered (se - (Pro minence Modular HPLC). problems due to the high dilution tive. The possibility to study nesced) leaves. In order to assure For a more specific detection, factor and can be neglected. other chlorophyll species and the direct comparability of sam- their degradation is also interest- ples with different extent of age- ing. Chlorophyll c, for instance, is ing, a tree was sampled in autumn. »Chlorophyll and photosynthesis« mainly present in algae. Although At that time, the tree bore green algae do not age depending on as well as yellow, already withered The term ‘chlorophyll’ is derived from ancient Greek and means ‘green season, they are however limited leaves. leaf.’ Chlorophyll is responsible for the green coloring of leaves. But it is in their life expectancy by ecologi- not only present in plants since other organisms also contain chlorophyll. cal changes. Methanol was used to extract the chlorophylls from the leaves. Pre - It is necessary for photosynthesis, the photochemical process that pro- Authors liminary experiments showed that duces glucose, oxygen and energy from water and carbon dioxide. This Florian Pahlke, Prof. Dr. Jürgen Schram an influence on the different chlo - is, however not always the case. Oxygen production only occurs in oxy- Laboratory for Element and Species Analysis rophyll species could be largely genic phototrophs. Faculty of Chemistry excluded. University of Applied Sciences Photosynthesis can also generate compounds other than oxygen and Krefeld, Germany For detection purposes, the UV- glucose and this type of photosynthesis is carried out by anoxygenic VIS activity of chlorophyll is gen- phototrophs. Both the products generated and the chlorophylls involved Literature erally taken advantage of. The in these processes differ. [1] T. Masuda und Y. Fujita, „Regulation and chlorophyll species are separated evolution of chlorophyll metabolism“, using HPLC and subsequently Photochemical & Photobiological Sciences, detected using a diode-array de - Bd. 7, Nr. 10, p. 1131, 2008. tector (DAD). This method would flame AAS (AA-7000) was subse- Result [2] S. Hörtensteiner, „The loss of green color however require the use of refer- quently used. The absorption during chlorophyll degradation – a prereq- ence standards which are only (output as current in μA) can be Except for one intermediate in the uisite to prevent cell death?“, Planta, available for chlorophyll a and b, tapped off via the analog port of chlorophyll degradation process Bd. 219, Nr. 2, pp. 191-194, 2004. to ensure an accurate analysis. A the AA-7000 and can be time-de - (chlorophyllide), none of the de - [3] Chlorophylle. In: Römpp Online. Georg reliable statement on other chlo - pendently re corded. gradation products contain the Thieme Verlag, (May, 19. 2014). rophyll species (see figure 1) bound magnesium. This is why would not be possible. As chlorophyll is ashed in the the measurement of senescence flame and the magnesium is then was limited to the decrease in in - A different approach was there- present in its atomic form, a con- tensity of chlorophyll a and b. fore used, in which the specific ventional water-based magnesium structure of chlorophyll was taken standard can be used. This stan- The concentration of chlorophyll Further information advantage of, as each of the mole- dard is diluted accordingly using a decreased by a factor of 8.5 due on this article: to senescence. In chlorophyll b, • Application Note this decrease was even more dra- Analysis of Mg by HPLC 30 matic. The samples only contained FAAS (PDF) 6.1 % chlorophyll b in compari- 25 son to the sample with the non- www.shimadzu.eu/shimadzu-news-2014 withered leaves. Before senes- a 20 cence, the ratio between chloro- phyll a and b was 4.4 :1. The natu- 15 ral ratio is 3 :1. This indicates that the green leaves were also at the 10 initial stage of senescence, which could be confirmed by the time of 30 sampling (autumn). After ageing, the ratio increased to 8.4 :1. Chlo - Intensity [mV] 25 rophyll b was therefore degraded faster than chlorophyll a. Chloro - 20 phyll degradation is accompanied b by the change of color when other 15 plant substances such as carot - enoids (yellow/orange) and tannin 10 (brown) are emerging. 5 6 7 8 9 10 11 This problem can be addressed Time [min] using the AA-7000 coupled to the Figure 2: Determination of chlorophyll a and b in oak leaf extracts using HPLC-F-AAS. modular Prominence HPLC. In a: an oak leaf still green in autumn, b: a withered oak leaf. particular, the possibility to cali- brate using cost-effective water- SHIMADZU NEWS 3/2014 3.
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