Waters XBridge Peptide BEH C18 130A and 300A Columns - Manual (Page 5)

c. 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 poor
performance.
d. Pressure
XBridge Peptide BEH C
18
, 130Å and 300Å 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.
e. Temperature
Temperatures between 20 ˚C – 60 ˚C are recommended for
operating XBridge Peptide BEH C
18
, 130Å and 300Å 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.
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 volume1(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).
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
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.
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 7 M guanidine
hydrochloride, or 7 M urea
5
[ CARE AND USE MANUAL ]
XBridge Peptide BEH C
18
, 130Å and 300Å Columns