Waters Protein-Pak SEC Columns - Manual (Page 4)

[ CARE AND USE MANUAL ]
Protein-Pak SEC Columns 4
– Avoid precipitation. DO NOT inject sample directly into the mobile
phase whenever possible. Dissolve (or dilute) samples in an appropri-
ate volume of the mobile phase first. If other solvents must be used,
be sure that no precipitation occurs when they are injected into the
mobile phase.
– Protect the column from rapid changes in solvent composition. DO NOT
change the flow rate faster than 0.5 mL/min. increments.
C. STORAGE CONSIDERATONS
– DO NOT store column in water alone for more than three days, this practive
will promote microbial contaimination. Store this series of columns in a
0.05% sodium azide in water solution.
– DO NOT allow buffers or other potentially harmful materials to remain in
the system overnight when not being used. Flush and replace with 0.05%
sodium azide in water solution.
– Return the column to its box with the end caps firmly in place for
storage. Allowing steel columns to dry out can result in poor chro-
matographic performance.
IV. T ROUBLESHOOTING
a. Service and Applications Information
The Waters staff of trained and experienced Service Specialists
provides maintenance for Waters instruments on preventative and/or
corrective levels. Contact Waters Office at 1-800-252 HPLC or your
local Waters Representative for answers to specific chromatography
questions in methods development, applications, quality control, and
service related matters.
b. Column Efficency
Waters measures the efficiency of the Protein-Pak 60 and 125
columns by using the 5 sigma method. Plate count as an expression of
effieciency, is determined by measuring the peak width at 4.4% of the
peak height method.
Figure 2: Half Height Method
Problem Cause Solution
Excess pressure
buildup • Filters plugge
with Particulates
• Sample precipitates
on column (sample
not soluble in mobile
phase)
• Clean in an ultrasonic
bath or replace.
• Always alter mobile
phases and samples.
• Slowly purge with a
strong mobile phase that
is both appropriate to
dis-solve the contaminate
and compatible with the
column.
Loss of
resolution, broad
peaks, low plate
counts • Filters partially
plugged
• Contaminated
colum, insufficent
equilibration
• Column collapse
and void formation
• Replace or clean inlet
and outlet filters in an
ultrasonic bath.
INJEC
T
W½
½h
h
Ve
N= Column Efficiency
V
e
= Elution time (min)
W 1/2= Half width value of
sample peak (min) taken at
half height (1/2 h)
N = 5.54 x
2 e