Waters Symmetry Columns - Manual (Page 4)

[ Care and Use ManUal ]
Symmetry Columns 4
d. Solvents
To maintain maximum column performance, use high quality
chromatography grade solvents. Filter all aqueous buffers prior to
use. Pall Gelman Laboratory Acrodisc
® filters are recommended.
Solvents containing suspended particulate materials will generally
clog the outside surface of the inlet distribution frit of the column.
This will result in higher operating pressure and poorer performance.
Degas all solvents thoroughly before use to prevent bubble formation
in the pump and detector. The use of an on-line degassing unit is
also recommended. This is especially important when running low
pressure gradients since bubble formation can occur as a result of
aqueous and organic solvent mixing during the gradient.
e. Pressure
Symmetry columns can tolerate pressures of up to 6,000 psi
(400 bar or 40 Mpa) although pressures greater than
4,000 – 5,000 psi should be avoided in order to maximize
column and system lifetimes.
f. Temperature
Temperatures between 20 ˚C – 45 ˚C are recommended for operating
Symmetry columns in order to enhance selectivity, lower solvent
viscosity and increase mass transfer rates. However, any temperature
above ambient will have a negative effect on lifetime which will vary
depending on the pH and buffer conditions used.
iV. 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
or
Injection volume
1
= Injection volume
2
(r
2
/r
1
) 2 (L
2
/L
1
)
Where: r = Radius of the column, in mm
F = Flow rate, in mL/min
L = Length of column, in mm
1 = Original, or reference column
2 = New column
V. troubleshootinG
Changes in retention time, resolution, or backpressure are often due
to column contamination. See “Column Cleaning, Regenerating and
Storage.” Information on column troubleshooting problems may be
found in the current Waters Chromatography Columns and Supplies
Catalog. You can also download a copy of the HPLC Troubleshooting
Guide at www.waters.com, click on “Literature Library”, then in the
Information Center Search Box, enter WA20769.
Vi. column cleaninG, reGeneratinG and storaGe
a. Cleaning and Regenerating
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 of HPLC-grade solvents (e.g., 80 mL total for
4.6 x 250 mm column). Increasing mobile phase temperature to
35-55 ˚C increases cleaning efficiency. If the column performance is
poor after regenerating and cleaning, call your local Waters office for
additional support.
Table 3: Column Sequence or Options
* Use low organic solvent content to avoid precipitating buffers.
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 isopro-
panol/water mixture*) Option 3: Flush column with
7M guanidine hydrochloride,
or 7M urea
6. Mobile Phase 6. Mobile Phase