Waters Gen-Pak FAX Columns - Manual (Page 6)

[ Care and Use ManUal ]
Gen-Pak FAX Columns 6
Figure 4: Separation of 2 μg of Hae III Digest of ØX 174 RF DNA at
30 ˚C using a Shallow Salt Gradient
f. Temperature Optimization
Adjustments of gradient slope may not produce adequate
resolution of DNA fragments in every case. However, changes in the
temperature at which the separation is performed can alter the
relative retention of the fragments. As Figure 5 shows,
chromatography of the Hae III digest of ØX 174 at 60 °C yields
slightly less resolution of the 72 and 118 base-pair fragments.
Since it is not possible to predict the best separation temperature for
a particular sample, it is often useful to compare separations at two
temperatures, such as 30 °C and 60 °C. Although increased NaCl
concentration is required to elute DNA fragments from the column at
elevated temperatures, chromatography at such temperatures does
not shorten the column life nor affect the biological activity of the
collected samples.
Figure 5: Separation of 2 μg of Hae III Digest of ØX 174 RF DNA at
60 °C using a Shallow Gradient
VI. tRoUBLsHootInG
Liquid chromatography columns have a finite life that is directly
related to the care and use they recieve. Column life is influenced
by the number of injections, cleanliness of sample and solvent,
frequency of solvent changeover, and procedures for handling and
storage.
If you observe a change in the following areas, take immediate steps
to determine the reason for the changes.
• Retention of a particular compound
• Resolution between two compounds
• Peak shape
Until the cause of the change is determined, you should question the
validity of the results of any separation using the column
a. Pressure Buildup/Loss of Resolution
With continued use, it is possible that excessive pressure buildup
(more than 4,000 psi at 1.0 mL/min at 25°C) or loss of resolution
may be experienced. These may both be caused by the buildup of
particulates from samples or eluents on the inlet filter. Filtering
samples and eluents greatly extends inlet filter and column life.
b. Possible Corrective Actions
If pressure buildup or loss of resolution occurs, any of the following
corrective actions, listed in order preference, may help:
• Perform a 20 - 40% acetic acid wash as described in Column
Washing Section. The length of the wash can be extended to
10 to 15 minutes.
• Replace the inlet filter with a new one (Waters Part Number
WAT015715)
Absorbance at 260 nm
0.2
0
10
1 2
3 4
5-7
8
910
11
35
Column: Gen-Pak FAX (4.6 x 100 mm)
Buffer A: 25 mM Tris/Cl, 1 mM EDTA, pH 8.0
Buffer B: 25, mM Tris/Cl, 1mM EDTA, 1.0 M NaCl, pH 8.0
Gradient: Load at 30% B to 54% B in 0.01 min.
Then 54 to 67% in 30 min., linear.
Flow:
Temperature:
Minutes
0.75 mL/min
30˚C
Absorbance at 260 nm
1 2 3 4
5-7
8
9 10 11
0
0.2
Column: Gen-Pak ô FAX (4.6 x 100 mm)
Buffer A: 25 mM Tris/Cl, 1 mM EDTA, pH 8.0
Buffer B: 25 mM Tris/Cl, 1mM EDTA, 1.0 M NaCl, pH 8.0
Gradient: Load at 30% B to 54% B in 0.1 min.
Then 64 to 77% in 30 min., linear.
Flow: 0.75 mL/min
Temperature:
Minutes
60˚C
35 10