Waters XSelect CSH130 C18 3.5 μm and 5 μm Columns - Manual (Page 5)

[ CARE AND USE MANUAL ]
XSelect CSH130 C
18
Columns 5
III. SCALING UP/DOWN ISOCRATIC METHODS
The following formulas will allow scale up or scale down, while maintaining
the same linear velocity, and provide new sample loading values:
If column i.d. and length are altered:
F
2
= F
1
(r
2
/r
1
)
2
Load
2
= Load
1
(r
2
/r
1
)
2
(L
2
/L
1
)
Injection volume
2
= Injection volume
1
(r
2
/r
1
)
2
(L
2
/L
1
)
Where: r = Radius of the column
F = Flow rate
L = Length of column
1 = Original, or reference column
2 = New column
IV. TROUBLESHOOTING
Changes in retention time, resolution, or backpressure are often due to
column contamination. See the Column Cleaning, Regeneration and Storage
section of this Care and Use Manual. Information on column troubleshooting
problems may be found in HPLC Columns Theory, Technology and Practice,
U.D. Neue, (Wiley-VCH, 1997), the Waters HPLC Troubleshooting Guide
(Literature code # 720000181EN) or visit the Waters Corporation website
for information on seminars (www.waters.com).
VI. COLUMN CLEANING, REGENERATION AND STORAGE
a. Cleaning and Regeneration
Changes in peak shape, peak splitting, shoulders on the peak, shifts in
retention, change in resolution or increasing backpressure may indicate
contamination of the column. Flushing with a neat organic solvent,
taking care not to precipitate buffers, is usually sufficient to remove the
contaminant. If the flushing procedure does not solve the problem, purge
the column using the following cleaning and regeneration procedures.
Use the cleaning routine that matches the properties of the samples and/or
what you believe is contaminating the column (see Table 3 ). Flush columns
with 20 column volumes each of HPLC-grade solvents (e.g., 80 mL total for
4.6 x 250 mm column) listed in Table 3. Increasing mobile phase temperature
to 35-55 ˚C increases cleaning efficiency. If the column performance is
poor after cleaning and regeneration, call your local Waters office for
additional support.
Table 3: Cleaning and Regeneration Sequence or Options
Polar Samples Proteinaceous Samples
1. water Option 1: Inject repeated 100 µL aliquots of dimethylsulfoxide
(DMSO) using a reduced flow rate delivering 50% Eluent A
and 50% Eluent B
2. methanol Option 2: gradient of 10% to 90% B where:
A = 0.1% trifluoroacetic acid (TFA) in water
B = 0.1% trifluoroacetic acid (TFA) in acetonitrile (CH
3
CN)
3. isopropanol
Option 3: Flush column with 7M guanidine hydrochloride, or
7M urea
Note: To avoid potentially damaging precipitation within your column
(e.g., if your separation eluent contains phosphate buffer), be certain to
flush column with 5 to 10 column volumes of water BEFORE using suggested
organic eluent column wash procedures.
b. Storage
For periods longer than four days at room temperature, store the column
in 100% acetonitrile. Immediately after use with elevated temperatures
and/or at pH extremes, store in 100% acetonitrile for the best column
lifetime. Do not store columns in highly aqueous (<20% organic) mobile
phases, as this may promote bacterial growth. If the mobile phase con-
tained a buffer salt, flush the column with 10 column volumes of HPLC
grade water (see Table 1 for common column volumes) and replace with
100% acetonitrile for storage. Failure to perform this intermediate step
could result in precipitation of the buffer salt in the column or system
when 100% acetonitrile is introduced. Completely seal column to avoid
evaporation and drying out of the bed.
Note: If a column has been run with a mobile phase that contains formate
(e.g., ammonium formate, formic acid, etc.) and is then flushed with 100%
acetonitrile, slightly longer equilibration times may be necessary when the
column is re-installed and run again with a formate-containing mobile phase.