Performing Extractions and Sample Preparation for Quantitative NMR Analysis

DATE: August 2021
University of California, Riverside | Department of Chemical and Environmental Engineering
Justin Hoover

The Hamilton products of choice would be a set of microliter pipettes that will be used for improved accuracy when performing extractions and sample preparation for quantitative NMR analysis, both of which are typically performed at milliliter and milligram scales. The reliability and accuracy of Hamilton products, specifically the accuracy and precision to fractions of a percent for the 100-1000uL pipettes, will ensure that our analysis and sample preparation methods are robust and capable of properly quantifying numerous unknowns by minimizing volumetric variability. One specific example is during the preparation of samples containing acylated polysaccharides, where multiple extraction and purification steps are required and each step introduces a change in concentration from the original sample. These polysaccharides are a principal component in Aloe vera responsible for numerous aspects of the health and healing benefits that the plant offers, having a high molecular weight and existing in low concentrations naturally. Quantification of these compounds allows validation of new production methods in addition to verification of existing extraction and processing techniques from natural sources, such as from the inner leaf gel of the plant. In order to accurately calculate the unknown polysaccharide content within the original sample it is necessary to have accurate data on volumes added and removed throughout these steps. Preparation of these samples begins with a water extraction followed by centrifugation and separation of the supernatant, the volume of which is essential knowledge as it allows determination of residual polysaccharide left within the biomass due to retained solvent. This step is followed by the addition of a secondary solvent to induce precipitation of the polysaccharides and the precipitates are subsequently dried and solvated with deuterium oxide. Once the desired compounds are dissolved in the NMR solvent, an aliquot must be taken for analysis. The accuracy of this volume is critical as quantitative NMR analysis is dependent upon accurate knowledge of sample mass within the solution, which can be derived from the siphoned volume and corresponding bulk concentration. Finally, a molar comparison can be made and the concentration of polysaccharides within the original sample can be calculated after accounting for retention within the biomass, dilution through extraction steps, and volume of active solution added to the NMR tube. Currently, there exists error that originates with inconsistent pipette availability and is compounded though each of the aforementioned steps. Ultimately, these preparation steps are applicable to many of our analysis methods and this is just a single example, despite ongoing NMR analysis in other areas such as structural analysis and quantification of carotenoids and their intermediates for biosynthetic pathway identification and development in algae mutants of the genus Symbiodinium, lipid quantification for biofuel production based on the model organism Chlamydomonas reinhardtii, and quantification and identification of plant hormones to assess potential consumptive hazards that may be associated with alternative methods of polysaccharide production from aloe.

Currently, there are a number of pipettes in the lab, however, some of the brands and quality standards do not match in addition to the aforementioned inconsistent pipette availability due to numerous users and projects. Additionally, most of these devices are utilized for qualitative processes and liquid transfers elsewhere in the lab where volumetric accuracy is not critical as in quantitative processes. By obtaining these Hamilton Pipettes, we will be able to improve precision across numerous experiments as well as improve accuracy when applied to quantitative analysis. Overall, receiving this grant would revitalize our quantitative analytical capabilities.

The Jinkerson lab is helping to innovate the future in coordination with NASA to grow plants in space, as well as pioneering pathways for more sustainable and efficient production methods of numerous compounds on earth by optimizing natural processes found in plants and algae, such as polysaccharides from Aloe vera for human health benefits and lipids from algae for biofuels. Hamilton products will play a vital role in ensuring that research into these areas is reproducible and accurate, thereby allowing the lab to produce stronger research results and ensuring data is not misunderstood or misrepresentative, saving time and research funds. Overall, generation of quality data is a necessity for the Jinkerson lab as we are working toward innovations that benefit the environment and mankind as a whole, thus we need to be sure that results are reported with the utmost accuracy and precision.

The Jinkerson lab consists of more than 12 members across a range of collegiate levels, including; PhD, MS, and a diverse set of undergraduates. In pursuit of inspiring upcoming generations in the scientific community, the Jinkerson lab trains and provides research opportunities to ~7 undergraduates and high school students at any time. Meanwhile, as a whole, UC Riverside is a Hispanic-Serving Institution with over 57% of undergraduates being first-generation college students. In combination, this diverse pool of students is drawn upon to be researchers in our lab and involving them in research-based projects will promote Hamilton’s goals to reach a diverse and broad range of individuals. Overall, these Hamilton products will be used by graduates and undergraduates alike, so that undergraduate student researchers can develop strong exploratory and validation skills with confidence while graduate students can assertively validate and report new discoveries.

Website: http://jinkersonlab.engr.ucr.edu

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