Waters BioSuite Peptide Analysis Columns - Manual (Page 7)

[ Care and Use ManUal ]
BioSuite Peptide Analysis Columns 7
Note: Flow splitters after the column will introduce additional bandspread-
ing. System optimization, especially in a system that contains a flow split-
ter, can have dramatic effects on sensitivity and resolution. Optimization
includes using correct-depth ferrules and minimizing tubing diameter and
lengths. An example is given in Figure 9 where system optimization resulted
in a doubling of sensitivity and resolution of the metabolite in an LC/MS/MS
system.
Non-optimized LC/MS/MS System Optimized System
Figure 9: Chromatographic Performance on Non-Optimized vs. Optimized
LC/MS/MS System.
X. system modIFICatIon reCommendatIons For bIosuIte
2.1 mm I.d. peptIde analysIs Columns
1. Use a microbore detector flowcell with BioSuite 2.1 mm i.d. Peptide
Analysis Column.
Note: Detector sensitivity is reduced with the shorter flowcell pathlength in
order to achieve lower bandspread volume.
2. Minimize injector sample loop volume.
3. Use 0.009 inch (0.25 mm) tubing between pump and injector
4. Use 0.009 inch (0.25 mm) tubing for rest of connections in stan-
dard systems and 0.005 inch (0.12 mm) tubing for narrowbore
(>2.1 mm i.d.) systems.
XI. addItIonal ConsIderatIons when usIng bIosuIte C
18
peptIde analysIs Columns
Waters BioSuite Peptide Analysis Columns contain either 3 μm (i.e., BioSuite
C
18
3 μm PA-A) or 3.5 μm (i.e., BioSuite C
18
3.5 μm PA-B) particles that pro-
vide very efficient separations without sacrificing column lifetime.
This section describes five parameters to consider when performing peptide
separations with BioSuite Peptide Analysis Columns.
Note: Columns that contain 3 μm or 3.5 μm particles have smaller outlet
frits to retain packing material. These columns should not be backflushed.
1. Backpressure: Backpressures for columns containing either 3 μm or
3.5 μm particles are higher than seen using columns packed with 5 μm
or 10 μm material.
2. Temperature: As seen in Table 1, use of a column heater set at
elevated temperature (e.g., 40 °C) will significantly reduce the
observed system backpressure when using BioSuite Peptide Analysis
Columns.
3. Sampling Rate: Use a data sampling rate of 10 points per second or
greater.
4. Detector Time Constant: Use a time constant of 0.1 seconds for fast
analysis.
7.00 7.50
Non-optimized LC/MS/MS System Optimized System
8.00 7.00 7.50 8.00