UltiMate 3000 RSLCnano – Standard Applications
44
Table 9: UltiMate 3000 RSLCnano Automated Off-line RP-RP peptides kit (P/N 6720.340) contents - Continued
# Item Replacement P/N
PTFE tubing, 500 µm I.D. 100 cm, used as waste tubing 6720.0077
1/16" Universal Fingertight Fitting, one-piece design, extra long thread, 4 pcs. 6720.0015
Protein mixture digest, 100 pmol, Lyophilized 161088
Cytochrome C digest, 1.6 nmol, Lyophilized 161089
Polypropylene vials for WPS with glass insert, 250 µL, 25 pcs. 6820.0027
Polypropylene caps for WPS vials, 25 pcs. 6820.0028
96 Well Microplate, PP, V-Bottom 6820.4113
LCi Solutions Library CD 6830.0400
Operating Instructions for automated off-line 2D-LC of peptides and proteins 164208
2.11.3 Installation tips
• Follow the General Recommendations for Applications (→ page 6).
• The design of the nano trap column provides the easiest connections, but must only be
used in forward flush operation. Please check the indicated flow direction when
installing a nano trap column.
• If the loss of hydrophilic peptides is observed, the concentration of acetonitrile in the
loading solvent can be decreased down to 99/1 water/ACN + 0.05% TFA.
• Optimal trapping in the second dimension is achieved by adding a strong, acidic ion-
pairing agent to the fractions and a combination of evaporating and diluting the ACN
from the first dimension.
• When two VWD-3400RS detectors are available, they should be placed on top of each
other.
• Depending on the location of the MS outlet and the application of UV detection (1
st
or
2
nd
Dimension), the WPS can be placed on the left side of the system.
2.11.4 Testing the application
The automated off-line application allows, as any off-line application, optimization of the
individual separation dimensions. Thermo Fisher Scientific recommends testing and
optimizing the performance of each separation dimension (RP basic and RP acidic)
individually in a one-dimensional application.
This means that the test criteria for a nano RP second dimension are identical as described in
the part of Pre-concentration onto a Nano Column on page 21.
Property Setting
NCS NC_Pump A
100% water, 1% (72 mM) triethylamine (TEA) titrated to pH = 9.6 with acetic
acid
NCS NC_Pump B
19%/80% (v/v) water/ACN, 1% (72 mM) triethylamine (TEA), titrated to pH =
9.6 with
acetic acid
Loading pump A 98%/2% (v/v) water/ACN + 0.05% TFA
NCP NC_Pump A 100% water + 0.05% TFA
NCP NC_Pump B 20%/80% (v/v) water/ACN + 0.04% TFA
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