Waters BioSuite Peptide Analysis Columns - Manual (Page 6)

[ Care and Use ManUal ]
BioSuite Peptide Analysis Columns 6
a. Minimizing Peak Band Broadening
Figure 6 shows the influence of tubing internal diameter on system band
spreading and peak shape. As can be seen, the larger tubing diameter causes
excessive peak broadening and lower sensitivity for your peptide mapping
applications.
Figure 6: Effect of Connecting Tubing on System
VII. measurIng system bandspread Volume
This test should be performed on an HPLC system with a single wavelength
UV detector (not a Photodiode Array (PDA).
1. Disconnect column from system and replace with a zero dead volume
union.
2. Set flow rate to 1 mL/min.
3. Dilute a test mix in mobile phase to give a detector sensitivity
0.5-1.0 AUFS (system start up test mix can be used which contains
uracil, ethyl and propyl parabens; Waters part number WAT034544).
4. Inject 2 to 5 μL of this solution.
5. Using 5 sigma method measure the peak width at 4.4% of peak
height: Band Spread (μL) = Peak Width (min) x Flow Rate (μL/min)
= 0.1 min x 1000 μL/min
= 100 μL
Figure 7: Determination of System Bandspread Volume Using
5-Sigma Method
VIII. measurIng gradIent delay Volume
1. Replace the column with a zero dead volume union.
2. Prepare eluent A (pure solvent, such as methanol) and eluent B
(solvent plus sample, such as 5.6 mg/mL propylparaben in methanol).
3. Equilibrate the system with eluent A until a stable baseline is achieved.
4. Switch to 100% eluent B.
5. Record the half height of the step and determine dwell volume
(Figure 8).
Figure 8: Determination of Dwell Volume
The dwell volume should be less than 1 mL. If the dwell volume is
greater than 1 mL, then please refer to Section X: “System Modification
Recommendations for BioSuite 2.1 mm i.d. Peptide Analysis Columns” on
how to reduce system volume.
IX. use oF bIosuIte “narrow-bore” peptIde analysIs
Columns (I.e., 2.1 mm I.d.)
This section describes how to minimize extra column effects and provides
guidelines on maximizing the performance of BioSuite “Narrow-Bore”
Peptide Analysis Columns in an HPLC system in order to eliminate excessive
system bandspread volume. Without proper system modifications, excessive
system bandspread volume causes peak broadening and has a large impact
on peak width as peak volume decreases.
Impact of Bandspread Volume on BioSuite 2.1 mm i.d. Peptide Analysis
Column Performance
• System with 70 μL bandspread: 10,000 plates
• System with 130 μL bandspread: 8,000 plates (same column)
Diluted/Distorted Sample Band
0.005 inches
0.020 inches
0.040 inches
1.0
0.8
0.6
Height
Volume (μL)
0.4
0.2
0.0
Dwell
Volume
System Volume
4.4% h