Waters XSelect CSH HPLC Columns - Manual (Page 5)

[ CARE AND USE MANUAL ]
5 XSelect CSH HPLC Columns
III. SCALING UP/DOWN ISOC RATIC MET HODS
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. T ROUBLESHOOTING
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).
V. 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 4). 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 4. 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 4: Cleaning and Regeneration Sequence or Options
Polar Samples Non-polar Samples Proteinaceous Samples
1. Water
1. Isopropanol
(or an appropriate
isopropanol/water mixture*) Option 1: Inject repeated
aliquots of dimethyl sulfoxide
(DMSO)
2. Methanol 2. Tetrahydrofuran (THF) 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. Tetrahydrofuran
(THF)
3. Dichloromethane
4. Methanol 4. Hexane
5. Water
5. Isopropanol
(followed by an appropriate
isopropanol/water mixture*) Option 3: Flush column with 7M
guanidine hydrochloride, or
7M urea
6. Mobile phase 6. Mobile phase
*Use low organic solvent content to avoid precipitating buffers.
b. Storage
For periods longer than four days at room temperature, store
XSelect CSH columns 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 contained 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
packed bed.