Table 1. Precolumn Pressure and Dilution Comparison^a.

precolumn	ACN [%]	flow. [mL/min]	temp. [°C]	ID [mm]	length [mm]	p. size [μm]
bpC1	1.0	2.20	30	4.6	5	2.7
cpC2	1.2	1.70	30	3.0	5	1.9
dpC3	1.5	1.35	30	2.1	5	1.7
epC4	2.3	0.80	60	4.6	30	2.7
fpC5	4.5	0.25	60	3.0	30	1.7
gpC6	6.1	0.11	60	2.1	100	3.5
hpC7	8.3	0.00	60	2.1	50	1.7

^aComparison of different precolumns used for peptide trapping with the mD-UPLC-MS/MS system. The pressure was measured by the ³D-pump during the analysis of the main peak fraction of Herceptin (trastuzumab, 50 μg injection). The mD-UPLC-MS/MS system was operating in the single-enzyme digestion mode with a trypsin IMER installed. The listed flow rates (Flow.) represent the ⁴D-pump flow rate for dilution excluding the ²D-pump 0.05 mL/min (50% ACN) and ³D-pump 0.25 mL/min (digestion buffer) flow rates. For each column, the highest ⁴D-pump flow rate is listed before the ³D-pump exceeds the pressure limit of the ³D-trypsin column (<170 bar). In addition, the calculated acetonitrile concentration while peptide trapping on the ⁴D precolumn is listed (ACN). For the small trapping columns with a length of 5 mm, the temperature was set to 30 °C for optimal trapping performance. For longer columns, the temperature (temp.) was set to 60 °C because of the high backpressure at low temperatures. Additionally, the inner diameter (ID), length, and particle size (p. size) are listed for each column.

^bpC1: Agilent InfinityLab Poroshell 120 SB-C18 Fast Guards.

^cpC2: Agilent InfinityLab Poroshell 120 SB-C18 Fast Guards.

^dpC3: Waters ACQUITY UPLC BEH C18 Precolumn.

^epC4: Agilent InfinityLab Poroshell 120 SB-C18 Column.

^fpC5: Waters ACQUITY UPLC Peptide BEH C18 Column.

^gpC6: Waters XSelect Peptide CSH C18 Column.

^hpC7: Waters ACQUITY UPLC Peptide BEH C18 Column.