Waters Viridis SFC Columns - Manual (Page 2)

[ Care and Use ManUal ]
2 Viridis SFC Columns
II. GettInG stARteD
Each Viridis SFC column comes with a Certificate of Analysis and a
Performance Test Chromatogram. The Certificate of Analysis is specific
to each batch of packing material and includes the gel batch number,
analysis of unbonded particle and analysis of bonded particles. The
Performance Test Chromatogram is specific to each individual column
and contains the gel batch number, column serial number, USP plate
count, USP tailing factor, capacity factor, and chromatographic condi-
tions under normal-phase LC conditions. These data should be stored
for future reference.
a. Safety Considerations
An SFC column while being used is, by design, under pressure. The
major safety concern for even a small leak with just CO
2
is frostbite.
Pay attention to any frosting on the column or system connections.
This indicates a leak, usually with temperatures far below 0 °C. Any
small leak with solvent introduced could produce a situation where
the LEL (lower exposure limit) is reached.
b. Column Installation
Note: Scale the flow rate up or down accordingly based upon the i.d.,
length, particle size, and backpressure of the Viridis SFC column being
installed. See “Scaling Up/Down Isocratic Methods” for calculating
flow rates when changing column i.d. and/or length. See “Connecting
the Column to the SFC system” for a more detailed discussion on SFC
connections.
1. Make sure your co-solvent pump is primed and has an adequate
solvent/modifier supply before performing injections.
2. Connect both the inlet and outlet of the column to the SFC system.
3. If the column is still filled with a solvent, use a low flow rate and
back pressure setting (100 bar) to start pumping CO
2
and modifier
through the column.
4. If you see frosting on the column at the inlet or outlet, tighten the
compression screw on that side. If you see frosting on the column,
turn off the CO
2
and vent the system. Allow the column to depres-
surize fully before disconnecting the inlet or outlet to troubleshoot
the leaking issue.
c. Column Equilibration
Viridis SFC columns are shipped dry. Equilibrate the column with a mini-
mum of 10 column volumes of the mobile phase prior to use. (Refer to
Table 2 for a listing of empty column volumes.)
d. Initial Column Efficiency Determination
1. Perform an efficiency test on the column before using it. Waters
recommends using a suitable solute mixture to analyze the column
upon receipt.
2. Determine the number of theoretical plates (N) and use this value
as a benchmark for periodic comparisons.
3. Repeat the test at predetermined intervals to track column
performance over time. Slight variations may be obtained on
two different SFC systems due to the quality of the connections,
operating environment, system electronics, reagent quality and
column condition.
Table 2. Empty Column Volumes in mL (multiply by 10 for flush sol-
vent volumes)
Column
Dimension
Column Internal Diameter (mm)
4.6 mm 10 mm 19 mm 30 mm 50 mm
50 mm 0.8 4 14 35 98
100 mm 1.7 8 28 70 196
150 mm 2.5 12 42 106 294
250 mm 4 20 70 176 490
III. CoLUMn Use
To ensure the continued high performance of Viridis SFC columns,
follow these guidelines:
a. Sample Preparation
1. Sample impurities often contribute to column contamination.
Use Oasis ® solid-phase extraction cartridges/columns or Sep-
Pak ® cartridges of the appropriate chemistry to clean up the
sample before analysis. Link to www.waters.com/sampleprep.
2. Prepare the sample in the SFC mobile-phase modifier (typically,
methanol) for the best peak shape and sensitivity.