An Introduction to the Capabilities of Microscale 2D-RP/RP Peptide Chromatography with an ACQUITY UPLC M-Class System Matthew A Lauber, Stephan M Koza, and Kenneth J Fountain 2D-RP/RP with the ACQUITY M-Class System To demonstrate the performance capabilities and reproducibility of microscale 2D-RP/RP peptide chromatography with an ACQUITY and Columns reliably generates the high peak UPLC M-Class System and Column. peptide samples. capacity separations needed to study complex High peak capacity peptide separations can be obtained through multidimensional chromatography wherein the combination of orthogonal separations results in greater resolving power. Two-dimensional reversedphase (2D-RP/RP) chromatography is a unique example. The utility of 2D-RP/RP involving high pH fractionation with a highly stable, organo-silica hybrid stationary phase (BEH Technology™) followed by gradient separation on a sub-2-µm particle analytical column has been reported previously. ACQUITY UPLC M-Class HSS T3 C18 300 µm x 150 mm, 1.8 µm Sample Loop 10 µL/min A: 20 mM Ammonium Formate, pH 10 B: Acetonitrile Microscale LC-MS methods have been used extensively in the field of proteomics. Recently, these techniques have become increasingly attractive as orthogonal methods alongside immunoassays for the analysis of host cell protein impurities in biotherapeutics products. Narrow (300 µm ID) columns can be employed in these and other such applications as a means to derive an abundance of information from a relatively minimal amount of sample. Obtaining high peak capacity peptide separations is desirable in such work, because better separation efficiency means trace analytes can be more easily resolved. 8 µL/min A: 0.1% Formic Acid (v/v) in Water B: 0.1% Formic Acid (v/v) in Acetonitrile Figure 1. Microscale two dimensional reversed phase (2D-RP/RP). (A) Fluidics configuration. (B) Base peak intensity chromatograms of MassPREP Digestion Standard Mix 1 as obtained using an ACQUITY UPLC M-Class, SYNAPT® G2-S, 5 high pH fractionation steps, ACQUITY UPLC M-Class HSS T3, 300 µm x 150 mm Column, and a sample load

Here, we demonstrate that 2D-RP/RP chromatography operated at ≥10K psi with an ACQUITY UPLC M-Class and an ACQUITY UPLC M-Class 300 µm analytical column can be a robust solution for obtaining high peak capacity. It will be shown that such a system affords high resolving power and outstanding chromatographic reproducibility and performance with extended use. T H E S O LU T I O N A tryptic digest mixture derived from 4 different proteins (MassPREP™ Digestion Standard Mix 1, p/n 186002865) was studied by 2D-RP/RP using the configuration outlined in Figure 1A. Peptides from 400 fmoles of sample were...

Based on peak widths observed in the extracted ion chromatograms of 15 different peptides in this mixture (Figure 2A), the average peak capacity (10% peak height) for the 2nd dimension separations was estimated to be 277. As these analyses were completed with 5 step fractionation, the multiplicative resolving power of the two dimensions indicates that the apparatus is capable of producing a theoretical peak capacity of 1385. This demonstrates that remarkably high peak capacity peptide separations can be achieved by means of 2D-RP/RP with an ACQUITY UPLC M-Class System. The reproducibility of...