Waters High Strength Silica Columns - Manual (Page 4)

[ Care and Use ManUal ]
HSS HPLC Columns 4
e. 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
Time
Inflection
point time
1.0
0.8
0.6
0.4
Au
0.2
0.0
The dwell volume should be less than 1 mL.
III. WaTERS SMaLL PaRTICLE SI zE (3.5 �M) COLUMNS
– FaST CHROMaTOGRaPHY
Waters columns with 3.5 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 3.5 μm particles.
Note: Columns that contain 3.5 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 3.5 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 3.5 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 HSS HPLC columns is 20 °C to 45 °C. See “Column
Usage” for a discussion of elevated temperature use with HSS
HPLC 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.
IV. COLUMN EqUILIBRaTION
HSS HPLC columns are packed and shipped in 100% acetonitrile. It
is important to ensure solvent compatibility before changing to a
new solvent. Equilibrate with a minimum of 10 column volumes of
the mobile phase to be used (refer to Table 2 for a listing of standard
column volumes).
Table 2. Standard Column Volumes in mL (Multiply by 10 for
Equilibration Mobile Phase Volumes)
Column Volume (mL)
Column
Column internal diameter (mm)
Length
3.0 4.6 10
30 mm 0.2 0.5 –
50 mm 0.3 0.8 4
100 mm 0.7 1.7 8
150 mm 1.0 2.5 12
250 mm – 4 –
300 mm – – –