With HPLC and UHPLC analysis of peptides and tryptic digests, there are several approaches to improve resolution between peaks, including the use of longer columns, shallow gradients, and different temperatures. In the example presented here, increasing temperature improves the resolution of amyloid β peptides and fragments. These compounds are important in the study of Alzheimer’s disease (AD). The dramatic effect of higher column temperatures on these amyloid peptides is the topic of this LabNote. At 50 °C, peaks 3 and 4 closely elute with a resolution of 0.53 (Fig. 1, top trace). By increasing the temperature to 70 °C, peaks 3 and 4 begin to pull apart with a resolution of 0.98 (Fig. 1, middle trace). Finally, with a column temperature of 90 °C, complete resolution (Rs 2.77) is achieved between peaks 3 and 4 (Fig. 1, bottom trace). As column temperature is increased, both mobile phase viscosity and peak retention decrease, and peaks widths become narrower. In addition, a temperature-dependent shift in selectivity occurs for peptides 3 and 4, which is beneficial in this case. However, increasing column temperature may not always improve resolution, although it is an easy parameter to change, because it does not require preparation of any additional mobile phases or samples.
Figure 1. Effect of increasing temperature on selectivity and resolution for peaks 3 and 4. Experiments were also run at 60 °C, 80 °C, and 100 °C. The changes in resolution between peaks 3 and 4 followed the same trend as shown here.
Another benefit of using higher temperatures for peptide analysis is their effect on recovery. The recovery for the Aβ(17-42) fragment (peak 9) increased substantially with higher column temperature as shown in Figure 2. At 80 °C, the area of peak 9 is 68 mAU·s (Fig. 2, top trace). When the temperature is raised to 90 °C (Fig. 2, middle trace), the Aβ(17-42) fragment peak area increases to 153 mAU·s (up 125% in relative recovery). Finally, when the column temperature is increased to 100 °C, the Aβ(17-42) fragment peak area increases to 208 mAU·s (Fig. 2, bottom trace)—a 206% recovery increase over that for the 80 °C trace.
Figure 2. Effect of increasing temperature on the recovery of Peak 9. Data from 50, 60 °C, and 70 °C not shown here. For peak identities, see Figure 1.
In Figure 3, a graph of peak area (effective recovery) vs. temperature shows the strong effect of temperature on the area of the Aβ(17-42) fragment (peak 9). The area for this peak increased more than 9-fold over the temperature range from 50–100 °C. Similar to the improvement in resolution, the increased column temperature results in decreased viscosity and faster diffusion for the Aβ(17-42) fragment, which leads to a narrower, taller peak. This apparent increase in recovery could possibly be due to improved solubility or disaggregation of the Aβ(17-42) fragment.
Figure 3. Graph of Peak 9 area vs. temperature.
To take advantage of higher column temperatures, it is important to use columns that can perform reproducibly using the combination of high temperature and low pH mobile phases described in this LabNote. HALO Peptide columns utilize steric-protection silane technology, which prevents the bonded phase from being removed by hydrolysis under the high temperature and acidic pH conditions typically used for peptide and tryptic digest separations.
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