# The Cell Membrane of _Sulfolobus_ spp.--Homeoviscous Adaption and Biotechnological Applications
## 4 Extraction and Lipid Analysis
For the visualization of lipids, methods such as the incubation in 8-anilino-1-naphtalene or sulfuric acid and afterwards heating can be used. Advantages are rapid and cheap analysis. Limited reproducibility and the lack of making a qualitative, but only a qualitative, analysis are the major disadvantages of this method [108; 109]. Quantitative analysis and exact identification of the lipid classes require mass spectrometric (MS) procedures. Analyte ionization occurs via electrospray ionization (ESI), electron impact (EI), matrix assisted laser desorption/ionization (MALDI), or by atmospheric pressure chemical ionization (APCI). ESI, MALDI and APCI are so-called soft ionization techniques, and in contrast to EI, can be used for non-volatile analytes. After analyte ionization, the mass dependent ion separation and ion detection takes place in the mass spectrometer. Different lipid classes are then identified by the mass to charge ratio (_m_/_z_). Lipid analysis can be separated into the shotgun lipidomics approach and using MS in tandem with other separation techniques such as chromatography. In shotgun lipidomics, the crude total lipid extract is directly inserted into the MS, with prior analyte ionization, but without prior separation. However, ion suppression is a major disadvantage of this shotgun procedure. A consequence of this phenomenon is that low abundant species might be missed. This drawback is partially overcome by chromatography methods. Liquid chromatography (LC) techniques can separate the lipid classes according to their carbon chain length and the presence of double bonds [110; 111; 112]. The accurate quantification of lipid classes requires internal standards, which have to exhibit very similar physicochemical properties as the analyte, but can be distinguished by different \(m\)/\(\!\!\!\!/\) values [113]. Labeled standards for archaeal lipids are not widely commercially available. Only one tetraether lipid standard is supplied by Matreya LLC (State College, PA, USA). It is a lipid extract of the main polar lipid (Hex-GDGT-PG) of _T. acidophilium_. This was used for quantification by [PERSON] et al. They remarked that it contains contaminating amounts of GDNT and Hex-GDNT, and therefore these lipid classes could not be quantified in their analysis [42]. Apart from that, the other discussed research articles either did not specify their used lipid standards [43; 46; 78], or used internal standards instead [39; 79; 86]. [PERSON] et al. used lipid extracts of _S. acidocaldarius_ for the quantification of the membrane lipids in _A. sulfurrieducens_[86]. [PERSON] et al. [39] and [PERSON] et al. [79] used synthesized C\({}_{46}\) GDGT for the determination of GDGT abundance. The synthesis was established by [PERSON] and [PERSON], and the quantification method by [PERSON] et al. [114; 115]. The recent publications all identified their lipid classes by MS approaches [42; 43; 78; 79]. However, the used quantification standards were either not the same, or not specified. Hence, the contradictory results could be a result of different extraction methods or quantifications methods, if not as a result of the applied cultivation conditions.