# Quantifying Fermentable Sugars in Beer: Development and Validation of a Reliable HPLC-ELSD Method
## 3 Results and Discussion  
### Method Development and Optimization  
Initial chromatographic conditions for HPLC-ELSD were guided by values from the literature, setting baseline parameters for analyzing a mixture of five sugars. Using these initial settings, the chromatographic run was optimized to improve peak resolution and S/N ratios. Optimization of ELSD parameters, specifically Gain, Smoothing, and Data Rate, led to an enhanced chromatogram, demonstrating significant improvements in S/N by adjusting the data smoothing and acquisition rates to 5.0 s and 10 Hz, respectively.  
Following parameter adjustments, each sugar was injected individually to confirm elution order. The sugars eluted in the order of fructose, glucose, sucrose, maltose, and maltotriose. This elution sequence is attributed to the number of hydroxyl groups in each molecule, with fructose having fewer groups and thus weaker interactions with the stationary phase, and maltotriose, with three glucose residues, exhibiting stronger retention as a result of increased polarity and hydrogen bonding. Elution conditions were further optimized by adjusting the mobile phase composition (ACN:H\({}_{2}\)O ratio). Initial isocratic runs showed that lower water content decreased elution time but compromised separation of closely eluting peaks, such as fructose and glucose (Figure 1a).  
The study also demonstrated that the sensitivity of the ELSD response varied significantly with the composition of the mobile phase. For higher acetonitrile-to-water ratios (ACN:H\({}_{2}\)O), a substantial decrease in sensitivity was observed, while increased water content enhanced detection sensitivity (Figure 1c). This behavior is attributed to differences in viscosity between solvents: water (\(\mu\) = 1.00 cP) exhibits higher viscosity than acetonitrile (\(\mu\) = 0.37 cP). Considering that greater solvent viscosity results in larger mean particle diameters during nebulization, larger solute particles scatter more light, thereby improving detection sensitivity. This finding reveals that the composition of the mobile phase plays a crucial role in the sensitivity of the ELSD method, emphasizing the importance of carefully optimizing solvent ratios for improved analytical performance. A gradient starting with an ACN:H\({}_{2}\)O ratio of 85:15 followed by a shift to 75:25 improved separation of early peaks and shortened malotriose elution time. Final elution conditions of 86:14 balanced peak separation and detection sensitivity, enabling a 20 min runtime with minimal baseline drift (Figure 2).  
Figure 1: Variation in ACN:H\({}_{2}\)O ratios: (**a**) 70:30; (**b**) 85:15; and (**c**) 90:10. The peaks correspond to 1—fructose, 2—glucose, 3—sucrose, 4— maltose, and 5—maltotriose (note that in (**c**), only the first four peaks are present, as maltotriose has yet to elute).  
Further ELSD optimization included tuning the nebulizer gas flow rate and adjusting the evaporation temperature. Lowering the gas flow from the standard 1.6 SLM to 1.1 SLM enhanced detection sensitivity due to larger droplet formation, which improves light scattering (Figure S1, Supplementary Materials). The chosen evaporation temperature of 85 \({}^{\circ}\)C aligned with specifications for non-volatile compounds, balancing complete solvent evaporation and analyte stability. The nebulization temperature was set at 60 \({}^{\circ}\)C, a balance to prevent solvent boiling, which could lead to noise spikes in the baseline (Figure S2). The optimal conditions for all parameters were established, and these, along with the corresponding final chromatogram, are presented in Figure S3.