A general protocol for optimizing peak capacity for the separation of low molecular weight molecules under gradient elution conditions has not yet been developed. By studying the effects of gradient time, flow rate, temperature, final eluent composition, and column length on peak capacity, a protocol has been developed for the optimization of a separation of small molecules such as those seen in metabolomic studies. The strategy developed employs the Linear-Solvent-Strength Theory (LSS Theory)  to predict retention, building on an approach for the optimization of the peak capacity of large molecules (peptides) in fixed column format separations .
Bibliographical noteFunding Information:
The insightful discussions with Dr. Xiaoli Wang of Agilent Technolgies are gratefully acknowledged. We thank Prof. W.E Noland of the University of Minnesota and his research group for the generous gift of the hydrophobic indoles. The authors would also acknowledge financial support from National Institutes of Health ( 5R01GM54585-15 ) and Agilent Technologies for the donation of the Zorbax SB-C3 columns and Medtronix Inc. for donation of the LC equipment.
- Gradient elution
- Linear-Solvent-Strength Theory
- Method development
- Peak capacity
- Reversed phase