Waters Ultrahydrogel Columns - Manual (Page 4)

[ Care and Use ManUal ]
Ultrahydrogel Columns 4
NOTE: ATTACH A UNION IN PLACE OF THE COLUMN AND FLUSH
THE LINES FREE OF PREVIOUS SOLVENTS BEFORE ATTACHING THE
COLUMN.
Figure 1. Ferrule and Compression Screw Assembly
If you wish to use Ultrahydrogel columns in series:
1. Connect the columns in order of decreasing pore size (e.g. Ultra
500 before Ultra 250).
2. To minimize dead volume insert interconnecting tubing into the
compression fittings before tightening the fittings.
3. Connect the outlet end of the last column to the detector.
II. operatIng tIps
• Use the column only in the direction indicated by the arrow.
Flow in the reverse direction may cause degradation in
column performance.
• Be careful not to introduce air into the column during
installation or removal. Air bubbles will cause voids in the
column packing.
• Slowly increase flow rates and change solvents.
• Continue to pump solvent through the column after separation is
complete. Air may be sucked into the column by contraction of
the solvent if the pump is turned off while the column is still hot.
• Maintain a slow flow rate (below 0.2 ml/min) through the
column to prevent precipitation of buffer salts during daily
operation. If halides are being used, replace the buffer with
distilled water for overnight storage.
• Ultrahydrogel columns maybe operated at a temperature range
of 10 °C to 80 °C. High temperature analysis reduces viscosity,
increases theoretical plate count and resolution and reduces
adsorptivity.
III. COLUMN EFFICIENCY
Liquid chromatography columns have a finite life that is directly
related to the care and use they receive. Column life is influenced by
the number of injections, sample and solvent cleanliness, frequency
of solvent changeover, and handling and storage procedures.
To perform a plate count to check for column efficiency proceed
as follows:
Flow Rate: 0.8 ml/min
Detector: Refractive Index
Eluent: Water
Temperature: Ambient
Injection Volume: 20 µl
Marker: Ethylene Glycol
Test Standard Conc: 0.0025 ml/ml
Calculate the plates (MN) at the half height using the calculations
shown in Figure 2.
Half Height Method Test Calculations
Figure 2. Half Height Method Test Calculations
a. System Efficiency
Column degeneration may not always be the cause of unacceptable
sample resolution values. Troubleshooting the entire system may
be necessary to determine the exact cause of low performance.
In order to isolate the cause of poor system efficiency, always
measure bandspreading before installing a column. If loss of
resolution is observed after column use, repeat the test for system
bandspreading and compare with the initial bandspreading results.
If bandspreading has increased, focus troubleshooting efforts on the
system hardware (such as the injector or an in-line filter). Isolating
or eliminating the column as the source of reduced efficiency in an
COMPRESSION SCREW OR NUT
TUBE
FERRULE
END MUST BE STRAIGHT
AND SMOOTH TO ACHIEVE
MAXIMUM COLUMN EFFICIENCY
CRTITICAL DISTANCE TO BE DETERMINED BY
EACH APPLICATION (UNION, COLUMN FITTING ETC.)
IN
JE
C
T
W½ ½h
h
= Column Efficiency (plates)
= Volume to peak apex (µL)
= Volume of peak at ½ peak height (µL) N
VR
W ½
N = 5.54 x
2