AL
Analysis Analysis of the upper fraction was conducted
on UHPLC system 1 using two different mo- bile phases to aid retention of certain polar compounds such as adenine and nicotine. The mobile phases were alternated between pH 3.0 and pH 8.3; it should be noted that the recon- ditioning time for the basic pH was critical to ensure the system remained stable.
It was found that for highly polar compounds, high organic content or large injection vol- umes were detrimental to their peak shape. Conversely, for lipophilic compounds, losses were observed due to poor solubility in more aqueous solvent (e.g., 5% or 10% methanol). However, for other compounds, the results were consistent across the dilution factors.
The organic lower fraction analysis was separated on UHPLC system 2. Due to the au- tomated system, evaporation was achieved in less than 7 and 14 minutes for CHCl3
and MeOH,
respectively. It was determined that a volume of 150 µL was sufficiently concentrated to use for further testing.
Table 1 lists the different metabolites extracted from the algae cells. Only a few compounds with ampiphilic properties, such as metopro- lol, were retrieved in both fractions (pka values were calculated). These results are in agree- ment with comparative analyses of manually extracted samples. Analytical results obtained using a PAL RTC can be directly subjected to a library search using LC-MS/MS data in SWATH mode, eliminating the need for further targeted experiments to identify unknowns. This signifi- cantly reduced experimental time and resulted in faster processing of results.
Manual sample preparations were performed in parallel to assess the repeatability of the automated Bligh and Dyer extraction, which also allowed for comparison of any variation between the two methods. As shown in Figure 5, the automated method had lower variation and therefore better repeatability for both the aqueous and organic fractions. This is particu- larly noticeable for the aqueous fraction at pH 8.3 and the organic analyses.
Conclusion Fully integrated automation using a PAL
RTC system can transform even complex AMERICAN LABORATORY 31 MARCH 2016
chromatography protocols, such as experi- ments that contain multiple separations or lengthy extraction techniques. It is evident that increased automation can save time and reduce expenses while improving results. Streamlining workflow in this way increases consistency and allows scientists to devote more time to operations that require their unique skills and experience.
Emmanuel Varesio, Sandra Jahn, Sandrine Cudré and Gérard Hopfgartner are with Life Sciences Mass Spectrometry, School of Pharmaceutical Sciences, University of Geneva, University of Lau- sanne, Switzerland. Renzo Picenoni and Guenter Boehm are with CTC Analytics AG, Industriestrasse 20, 4222 Zwingen, Switzerland; tel.: +41 61 765 81 00; e-mail:
info@ctc.ch;
www.palsystem.com
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