Owner's manual

[ 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