Waters High Strength Silica Columns - Manual (Page 6)

[ Care and Use ManUal ]
HSS HPLC Columns 6
b. Sample Preparation
1. Sample impurities often contribute to column contamination. Use
Waters Oasis ® or Sep-Pak ® solid-phase extraction cartridges/
columns of the appropriate chemistry to cleanup the sample
before analysis.
2. It is preferable to prepare the sample in mobile phase or a solvent
that is weaker (less organic modifier) than the mobile phase.
3. If the sample is not dissolved in the mobile phase, ensure that
the sample and diluent are miscible in the mobile phase(s) in
order to avoid sample and/or diluent precipitation.
4. Filter sample through a 0.2 µm membrane to remove particulates.
If the sample is dissolved in a solvent that contains an organic
modifier (e.g., acetonitrile, methanol, etc.) ensure that the
membrane material does not dissolve in the solvent. Contact the
membrane manufacturer with solvent compatibility questions.
Alternatively, centrifugation for 20 minutes at 8000 rpm,
followed by the careful transfer of the supernatant liquid to an
appropriate vial, could be considered.
c. Recommended pH Range
HSS T3 and C
18
SB : 2-8 HSS C
18
: 1-8
Column lifetime will vary depending upon the temperature, type
and concentration of buffer used. A listing of recommended and
non-recommended buffers is given in Table 3. Please use this as a
guideline when developing methods.
Attention: Operating at the upper or lower end of the pH range in
combination with elevated temperatures will lead to shorter column
lifetime and/or may result in the column generating high backpressure.
Table 3: Buffer recommendations for using HSS HPLC columns
from pH 1 to 7
Additive or Buffer pKa
Buffer range
(±1 pH unit)
Volatility
Used
for Mass
Spec?
Comments
TFA 0.3 – Volatile Yes
Ion pair additive, can suppress MS signal. Used
in the 0.01-0.1% range.
Formic Acid 3.75 – Volatile Yes
Maximum buffering obtained when used
with Ammonium Formate salt. Used in
0.1-1.0% range.
Acetic Acid 4.76 – Volatile Yes
Maximum buffering obtained when used
with Ammonium Acetate salt. Used in
0.1-1.0% range.
Formate
(NH
4
COOH)
3.75 2.75 – 4.75 Volatile Yes
Used in the 1-10mM range. Note: sodium or
potassium salts are not volatile.
Acetate
(NH
4
CH
2
COOH)
4.76 3.76 – 5.76 Volatile Yes
Used in the 1-10mM range. Note: sodium or
potassium salts are not volatile.
Phosphate 1 2.15 1.15 – 3.15 Non-volatile No
Traditional low pH buffer, good UV
transparency.
Phosphate 2 7.2 6.20 – 8.20 Non-volatile No
Much shorter colum lifetimes will be realized
using phosphate at pH 7.
d. Solvents
To maintain maximum column performance, use high quality chrom-
atography grade solvents. Filter all aqueous buffers prior to use. The
addition of at least 5% organic to buffers is recommended to discour-
age bacterial growth. Pall Gelman Laboratory Acrodisc
® filters are
recommended. Solvents containing suspended particulate materials
will generally clog the outside surface of the inlet 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
HSS HPLC 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, and
the risk of system shutdowns and leaking.
f. Temperature
Temperatures between 20 ˚C - 45 ˚C are recommended for operating
HSS HPLC columns in order to enhance selectivity, lower solvent
viscosity and increase mass transfer rates. However, any temperature
rise above ambient will have a negative effect on lifetime which will
vary depending on the pH and buffer conditions used. The combina-
tion of operating at elevated temperatures and at pH extremes should
be avoided.
g. Scaling Up/Down Isocratic Methods
The following formulas will allow scale up or scale down, while
maintaining the same linear velocity (retention time), and provide
new sample loading values:
If column i.d. and length are altered: F
2
= F
1
(r
2
/r
1
) 2
or: Load
2
= Load
1
(r
2
/r
1
) 2 (L
2
/L
1
)
or: Inj vol
1
= Inj vol
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