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3.6 Chromatography
Connections
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1.
Place the interconnected detector components next to the column outlet from the chroma-
tograph, or install your column inside the CC-5 (Figure 3.1). Several slots in the CC-
5 chassis along the sides and back provide tubing access, no matter what the orienta-
tion. On a BAS 480 system, the detector components may sit directly on top of the
PM-80 Pump. See Figures 3.2–3.4 and 3.7 for ideas.
MICROBORE INSTALLATION: A microbore column should be connected directly
between the injection valve and the flow cell. You must use a special UniJet injec-
tion valve (MF-4161) that uses nonconducting fittings. Connect the microbore column
to the correct output port of this valve with a high-pressure PEEK fingertight fitting (MF-
4165). Release the flow cell from the CC-5 cabinet by undoing the hex screw. Slide
the flow cell toward the column until the column bottoms in the entry port, and secure
with another fingertight fitting.
NOTE: It will be necessary to thoroughly degas the mobile phase prior to use. This is
particularly important when the cell preheater or column heater is set at 35 °C or
higher. The warmth reduces dissolved gas solubility to the point where bubbles may
appear in the tubing to and from the electrochemical cell.
If you are using a flow restrictor to provide back pressure, be sure not to exceed ap-
proximately 100 psi (6 atm).
2.
Flush the system with 200 mL of acetonitrile/water (40:60, v:v) and divert all column ef-
fluent to a waste receptacle. This flushing step cleans any chemicals remaining from
previous injections from the column, and also "wets" the hydrophobic stationary
phase for reproducible separations. For new columns see below.
NOTE: Always connect a BAS column so fluid flows in the direction you read the label
(because the inlet and outlet frits have different pore sizes).
Most new BAS columns must be washed before use.* Washing will not only remove
residual packing solvents, but will give sufficient time for elimination of bubbles from
the system. For C
and C
18
8
phase surface. To wash the column, pump at least 200 mL neat acetonitrile through it.
Then pump 50 mL 40:60 acetonitrile:water through it to remove the neat acetonitrile.
(It is important not to mix neat solvents and mobile phase in the system or column,
since buffer salts may precipitate.) Then pump mobile phase.
The column should be thoroughly equilibrated with mobile phase before connecting
the detector. If you are using an ION-PAIRING REAGENT in your mobile phase (e.g.
Sodium Octyl Sulfate), you should allow up to 8 hours for equilibration at a flow rate
of 1 mL/min.
* Exceptions include enzyme columns, acetylcholine columns, and preloaded catecho-
lamine columns, which must never be washed with solvents. SepStik microbore col-
umns should be washed with acetonitrile for 15 minutes at microbore flow rates.
stationary phases, this step also "wets" the stationary
Section 3. Installation
15
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