Waters Atlantis T3, DC18 and HILIC Silica Columns - Manual (Page 7)

[ Care and Use ManUal ]
Atlantis Columns 7
To ensure the continued high performance of Atlantis columns and
cartridges, follow these guidelines:
a. Guard Columns
Use a Waters Sentry guard cartridge of matching i.d., chemistry and
particle size between the injector and main column. For best results, the
guard column should be replaced prior to the observation of a substantial
loss in resolution or increase in system backpressure. It is important to use
a high-performance matching guard column to protect the main column
while not compromising or changing analytical resolution.
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. For reversed-phase separations (Atlantis T3 and dC
18
) prepare the
sample in mobile phase or a solvent that is weaker (less organic
modifier) than the mobile phase. For Hydrophilic Interaction
Chromatography (HILIC) separations (Atlantis
HILIC Silica), the
samples must be prepared in 100% organic solvents (e.g., aceto-
nitrile). See “HILIC Getting Started” for additional information.
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.
c. Recommended pH Range
Atlantis HILIC Silica: 1-5 Atlantis T3: 2-8 Atlantis dC
18:
: 3-7
Column lifetime will vary depending upon the temperature, type and
concentration of buffer used. A listing of recommended and non-rec-
ommended 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 com-
bination 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 Atlantis columns
from pH 1 to 7
Additive or Buffer range Used for
Buffer pKa (±1 pH unit) Volatility 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 3.75 2.75 – 4.75 Volatile Yes Used in the 1-10mM range. Note: sodium or
(NH
4
COOH) potassium salts are not volatile.
Acetate 4.76 3.76 – 5.76 Volatile Yes Used in the 1-10mM range. Note: sodium or
(NH
4
CH
2
COOH) 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
* Phosphate salt buffers are not recommended for HILIC (phosphoric acid is OK) due to phosphate buffer
salt insolubility at high acetonitrile concentrations.
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 neutral pH buffers is recommended
to prevent bacterial growth. Pall Corporation 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
Atlantis 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.