Waters Delta-Pak High Pressure Insert HPLC Column - Manual (Page 4)

[ Care and Use ManUal ]
4
To perform an efficiency test, prepare the test sample as follows:
1) Prepare an acetone/acenaphthene sample by dissolving 0.05 g
acenaphthene and 600 µl acetone in 100 mLs of mobile phase.
While acenaphthene is the recommended sample, you can
substitute the following simple aromatic hydrocarbons:
• naphthalene
• p-cymene
• xylene
• dibutyl-, dipropyl-, or diethylphthalate
• propylbenzene
• toluene
• ethylbenzene
Toluene and diethylphthalate are the least desirable substitutes
since they have the lowest k.
2) Equilibrate the column employing the conditions summarized
below. Note: Incomplete equilibration results in tailing, fronting
or split peaks.
Table 1: Column Efficiency Test Conditions
Do not substitute methanol for acetonitrile in reversed-phase
column tests due to the much higher viscosity of methanol/water
mixtures.
3) Set a UV detector at 254 nm with an attenuation of 0.05 AUFS.
The resulting detector deflection should be approximately 90
percent of full scale. The minimum deflection is 60%.
4) Inject about 2 µL of sample. Large amount may overload the
column.
5) Measure and record plate count, asymmetry, backpressure, and
instrument settings. Waters uses the 5 sigma method, shown
in Figure 3, to measure column efficiency. Unlike the tangent
method, this more stringent method takes peak asymmetry into
account
Figure 3: 5 Sigma Test Method
If problems occur during normal operation of the column, repeat th
efficiency test and compare the results. This may help locate the
source of the problem.
c. Typical Column Backpressure and Eluent Viscosity
The column hardware has been designed to withstand operating
pressures as high as 50 Pa (500 atm or 7,000 psi). However, normal
operating backpressure should not exceed 40 Mpa (400 atm or
6,000 psi).
The normal operating backpressure of a column varies considerably
and is affected by column length, flow rate, mobile phase viscosity,
temperature, and particle size. The backpressure may rise signifi-
cantly during the course of a gradient.
The following equation can be used to calculate the backpressure of
your column:
Pressure (atm) at 1 L/min in H
2
0 = 210 x l • v
dp2 x d2
Where:
L = column length, mm
dp = particle diameter, µm
d = column diameter, mm
v = mobile phase viscosity, centipose (see Table 2)
Column Mobile Phase Flow Rate Test Samples
Delta-Pak C
18
and
Delta-Pak C
4
50/50
acetonitrile/
water 0.7 ml/min Acenaphthene
INJEC
T
N = 25
2
( )