Waters Atlantis T3, DC18 and HILIC Silica Columns - Manual (Page 5)

[ Care and Use ManUal ]
Atlantis Columns 5
Figure 10: Chromatograms Obtained Using Non-Optimized vs.
Optimized LC/MS/MS System
7.00 7.50
Non-optimized LC/MS/MS System Optimized System
8.00 7.00 7.50 8.00
III. systeM ModIfIcAtIon recoMMendAtIons
1. Use a microbore detector flowcell with ≤ 2.1 mm i.d. columns.
Attention: 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) i.d. tubing between pump and
injector.
4. Use 0.009 inch (0.25 mm) i.d. tubing for rest of connections with
> 3.0 mm i.d. columns. Use 0.005 inch (0.12 mm) i.d. tubing for
narrow-bore (≤ 2.1 mm i.d.) systems.
5. Use perfect (pre-cut) connections (with a variable depth inlet if
using columns from different suppliers).
6. Detector time constants should be shortened to less than 0.2 seconds
and the sampling rate adjusted to obtain at least 20 data points
across peaks of interest.
IV. WAters sMALL PArtIcLe sIze (3 µM) coLuMns
– fAst cHroMAtoGrAPHy
Waters columns with 3 µm particles provide faster and more efficient
separations without sacrificing column lifetime. This section describes
five parameters to consider when performing separations with columns
containing 3 µm particles.
Note: Columns that contain 3 µm particles have smaller pore-size outlet
frits to retain packing material than are used at the inlet. These columns
should not be backflushed.
1. Flow Rate — Compared to columns with 5 µm particles, columns
with 3 µm particles have higher optimum flow rates and are used
when high efficiency and short analysis times are required. These
higher flow rates, however, lead to increased backpressure.
Note: Use a flow rate that is practical for your system.
2. Backpressure — Backpressures for columns with 3 µm particles
are higher than for 5 µm columns with the same dimensions.
Waters suggests using a shorter column to compensate for
increased back pressure and to obtain a shorter analysis time.
3. Temperature — Use a higher temperature to reduce backpressure
caused by smaller particle sizes. The recommended temperature
range for Atlantis columns is 20 °C to 45 °C. See “Column Usage”
for a discussion of elevated temperature use with Atlantis columns.
4. Sampling Rate — Use a sampling rate of about 10 points per
second.
5. Detector Time Constant — Use a time constant of 0.1 seconds for
fast analyses.
V. coLuMn equILIbrAtIon
Atlantis columns are packed and shipped in 100% acetonitrile. It is
important to ensure solvent compatibility before changing to a new
solvent.
Atlantis T3 and dC
18
- Equilibrate with a minimum of 10 column vol-
umes of the mobile phase to be used (refer to Table 2 for a listing of
standard column volumes).
Atlantis HILIC Silica – Upon receipt, equilibrate with 50 column volumes
of 50:50 acetonitrile:water with 10 mM final buffer concentration (refer
to Table 2 for a listing of standard column volumes). Prior to the first
injection, equilibrate with 20 column volumes of initial mobile phase
conditions. See “HILIC Getting Started” for additional information.
Table 2. Standard Column Volumes in mL (Multiply by 10 for
Equilibration Mobile Phase Volumes)
Column Volume (mL)
Column Column internal diameter (mm)
Length 1.0 2.1 3.0 3.9 4.6 7.8 10 19 30 50
15 mm – 0.1 – – – – – – – –
20 mm – 0.1 0.1 – 0.3 – – – – –
30 mm – 0.1 0.2 – 0.5 – 2.4 8 – –
50 mm 0.1 0.2 0.3 – 0.8 2.4 4 14 35 98
100 mm 0.1 0.4 0.7 1.2 1.7 5 8 28 70 –
150 mm 0.1 0.5 1.0 1.8 2.5 7 12 42 106 294
250 mm – 0.9 1.8 – 4 – 20 70 176 490
300 mm – – – – – 14 24 85 212 589