Effect of plant sample preparation and measuring methods on ATR-FTIR spectra results

Attenuated Total Reflectance-Fourier-transform infrared (ATR-FTIR) spectroscopy is an effective tool for analysing the chemical composition of biological material, as well as distinguishing plant species. However, the results obtained from ATR-FTIR spectroscopy for the same plant differ between other researchers. This is related to the methodology used by scientists in the preparation of the material and its measurement. Therefore, it is very important to investigate the influence of sample preparation, as well as measuring methods for the ATR-FTIR spectra. In this work we determined the influence of (i) the preparation methods of homogeneous plant material and (ii) the influence of sample measuring methods on the obtained ATR-FTIR spectra of angiosperms (flowering plants), which were measured by ATR-FTIR spectroscopy. Moreover, (iii) we investigated which studied factor has greater influence on the obtained ATR-FTIR spectra. For this purpose, we measured, using ATR-FTIR spectroscopy, six different plant species representing a wide range of clearly distinctive plant functional groups, leaf phenology, as well as phylogeny. They were prepared by five different methods, and measured by two different methods. ATR-FTIR spectra, as well as Principal Components Analysis (PCA) and Linear Discriminant Analysis (LDA) analyses of attained spectra, showed that both sample preparation, as well as measurement methodology, has an influence on the obtained ATR-FTIR results. Interestingly, the results achieved showed that the chemical composition of plants should be made using fresh plants, while the interaction between the plants should be made using any prepared method. We found surprisingly higher impact of measuring then sample preparation method on ATR-FTIR spectra results. It seems very important finding from researchers point of view because they very rare pay attention and discuss issues connected with the impact of used sample preparation and measuring methods on their results.