Ultimate 3000: Preconcentration on a 75-μm i.d. x 15 cm ... - Dionex
Ultimate 3000: Preconcentration on a 75-μm i.d. x 15 cm ... - Dionex
Ultimate 3000: Preconcentration on a 75-μm i.d. x 15 cm ... - Dionex
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Technical Note 6<br />
UltiMate <str<strong>on</strong>g>3000</str<strong>on</strong>g> — <str<strong>on</strong>g>Prec<strong>on</strong>centrati<strong>on</strong></str<strong>on</strong>g> <strong>on</strong> a<br />
<strong>75</strong>-µm i.d. x 5 <strong>cm</strong> PepMap 00 (C 8) Nanocolumn<br />
IntroductI<strong>on</strong><br />
A strategy comm<strong>on</strong>ly used for the enrichment of<br />
trace comp<strong>on</strong>ents from complex mixtures in liquid<br />
chromatography is to prec<strong>on</strong>centrate the sample <strong>on</strong> a<br />
trap column prior to separati<strong>on</strong> (i.e., when large volume<br />
injecti<strong>on</strong>s need to be performed). This technique can<br />
also be applied for the desalting of compound mixtures<br />
isolated from biological matrices that often c<strong>on</strong>tain high<br />
amounts of n<strong>on</strong>volatile buffer salts. The presence of such<br />
buffers may interfere with the operati<strong>on</strong> of electrospray<br />
i<strong>on</strong> sources by suppressing i<strong>on</strong>izati<strong>on</strong>. In this technical<br />
note, the UltiMate <str<strong>on</strong>g>3000</str<strong>on</strong>g> setup for prec<strong>on</strong>centrati<strong>on</strong> and<br />
desalting of samples is described.<br />
ExpErImEntal<br />
A prec<strong>on</strong>centrati<strong>on</strong> LC experiment was carried out<br />
<strong>on</strong> the UltiMate <str<strong>on</strong>g>3000</str<strong>on</strong>g> system, using PepMap 100 (C18)<br />
nanocolumns for peptide trapping and separati<strong>on</strong>. The<br />
C18 trap and separati<strong>on</strong> columns were mounted in the<br />
FLM module of the UltiMate <str<strong>on</strong>g>3000</str<strong>on</strong>g> in a column-<br />
switching setup, as illustrated in Figure 1. The sample<br />
soluti<strong>on</strong> was loaded <strong>on</strong>to the trap column at a flow rate of<br />
30 µL/min. After loading, the sample comp<strong>on</strong>ents were<br />
eluted from the trap column in a counter flow directi<strong>on</strong><br />
with a linear gradient and separated <strong>on</strong> the nanocolumn.<br />
The method was applied for a cytochrome c tryptic digest.<br />
P/N 6720.0031 or P/N 6720.0032<br />
P/N 6720.0038<br />
waste<br />
4<br />
3<br />
5<br />
2<br />
1<br />
L<br />
6<br />
waste<br />
P/N 6720.0038<br />
10<br />
7<br />
9<br />
8<br />
P/N 160454<br />
2<br />
3<br />
WPS <str<strong>on</strong>g>3000</str<strong>on</strong>g><br />
SRD 3600<br />
DPG 3600<br />
P/N 6035.2550 or P/N 6035.2553<br />
1<br />
loop<br />
20 µL<br />
4<br />
P/N 6720.0033<br />
6<br />
5<br />
P/N 160321<br />
Flow Splitter<br />
FLM 3300<br />
Figure 1. Fluidic c<strong>on</strong>necti<strong>on</strong>s for a prec<strong>on</strong>centrati<strong>on</strong> experiment<br />
<strong>on</strong> a PepMap 100 (C18) nano column.<br />
Applicati<strong>on</strong> Update <strong>15</strong>7<br />
UV<br />
23276
The LC c<strong>on</strong>diti<strong>on</strong>s were as follows:<br />
LC system: UltiMate <str<strong>on</strong>g>3000</str<strong>on</strong>g><br />
Separati<strong>on</strong> Column: PepMap 100, C18, 3 µm,<br />
100 Å, <strong>75</strong>-µm i.d. x <strong>15</strong> <strong>cm</strong><br />
(P/N 1603211)<br />
Eluent Mobile Phase: A) Water, 0.05% TFA<br />
B) 20:80, Water:MeCN,<br />
0.04% TFA<br />
Gradient: 4% to 55% B in 30 min,<br />
90% B for 5 min, 25 min<br />
equilibrati<strong>on</strong><br />
Flow Rate: 300 nL/min<br />
Trap Column: PepMap 100, C18, 5 µm,<br />
100 Å, 300-µm i.d. x 5 mm,<br />
Loading Solvent: 98:2, Water:MeCN,<br />
0.05% TFA<br />
Loading Flow: 30 µL/min<br />
Loading Time: 3 min<br />
Sample: Cytochrome c digest,<br />
100 fmol/µL<br />
Inj. Volume: 10 µL<br />
Detecti<strong>on</strong>: UV, 214 nm<br />
Oven Temperature: 25 °C<br />
Sample Cooling: 5 °C<br />
rEsults<br />
A typical separati<strong>on</strong> performed <strong>on</strong> a prec<strong>on</strong>centrati<strong>on</strong><br />
LC setup is shown in Figure 2.<br />
Peptides are c<strong>on</strong>centrated <strong>on</strong> the 300-µm i.d.<br />
PepMap 100 (C18) trap column using a sec<strong>on</strong>d loading<br />
pump that facilitates the injecti<strong>on</strong> of large volumes in<br />
nano LC. In additi<strong>on</strong>, <strong>on</strong>-line desalting of samples can<br />
be realized.<br />
Passi<strong>on</strong>. Power. Productivity.<br />
Di<strong>on</strong>ex Corporati<strong>on</strong><br />
1228 Titan Way<br />
P.O. Box 3603<br />
Sunnyvale, CA<br />
94088-3603<br />
(408) 737-0700<br />
North America<br />
Sunnyvale, CA (408) 737-8522 Bannockburn, IL (847) 295-<strong>75</strong>00<br />
Houst<strong>on</strong>, TX (281) 847-5652 Atlanta, GA (770) 432-8100<br />
Marlt<strong>on</strong>, NJ (856) 596-0600 Canada (905) 844-9650<br />
www.di<strong>on</strong>ex.com<br />
The separati<strong>on</strong> <strong>on</strong> the <strong>75</strong>-µm i.d. PepMap 100<br />
(C18) nanocolumn in Figure 2 dem<strong>on</strong>strates the high<br />
chromatographic resoluti<strong>on</strong>. Using the prec<strong>on</strong>centrati<strong>on</strong><br />
setup, the chromatographic resoluti<strong>on</strong> is identical to that<br />
obtained with the direct sample injecti<strong>on</strong> setup. 1<br />
Using a Reagent-Free I<strong>on</strong> Chromatography (RFIC) System to M<strong>on</strong>itor Trace Ani<strong>on</strong> C<strong>on</strong>taminati<strong>on</strong><br />
in the Extracts of Electr<strong>on</strong>ic Comp<strong>on</strong>ents<br />
10<br />
mAU<br />
0<br />
0 10 20 30<br />
Minutes<br />
23277<br />
Figure 2. Typical chromatogram for a prec<strong>on</strong>centrati<strong>on</strong> experiment<br />
<strong>on</strong> a PepMap 100 (C18) nanocolumn.<br />
c<strong>on</strong>clusI<strong>on</strong>s<br />
The UltiMate <str<strong>on</strong>g>3000</str<strong>on</strong>g> system in prec<strong>on</strong>centrati<strong>on</strong><br />
mode forms an ideal instrument for the prec<strong>on</strong>centrati<strong>on</strong><br />
and <strong>on</strong>-line desalting of samples. The system combines<br />
high chromatographic resoluti<strong>on</strong> separati<strong>on</strong>s and flexibility<br />
towards sample injecti<strong>on</strong>. In additi<strong>on</strong>, with minor<br />
changes the system can be extended to a 2-D LC system.<br />
rEfErEncEs<br />
1. UltiMate <str<strong>on</strong>g>3000</str<strong>on</strong>g>—Direct Sample Injecti<strong>on</strong> <strong>on</strong>to a<br />
<strong>75</strong>-µm i.d. PepMap 100 (C18) Nanocolumn.<br />
Technical Note 60, LPN 1869, in press.<br />
Di<strong>on</strong>ex Corporati<strong>on</strong>, Sunnyvale, CA.<br />
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PepMap and UltiMate are trademarks of Di<strong>on</strong>ex Corporati<strong>on</strong>.<br />
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LPN 1870 PDF 09/06<br />
©2006 Di<strong>on</strong>ex Corporati<strong>on</strong>