9
CORTECS and CORTECS Premier 2.7
µ
m Columns
[ CARE AND USE MANUAL ]
highest quality solvents possible. HPLC-grade water
is not UPLC-grade water.
■
Consider sample preparation (e.g., solid-phase
extraction, filtration, etc.).
5. Avoid (where possible):
■
100% aqueous mobile phases (if possible)
■
HPLC-grade bottled water
■
“Topping off” or adding “new” mobile phase
to “old” mobile phase
■
Old aqueous mobile phases. Remember to rinse bottles
thoroughly and prepare fresh every 24 to 48 hours
■
Using phosphate salt buffer in combination with high
ACN concentrations (e.g., >70%) due to precipitation.
6. Don’t assume a “bad” column is the culprit when high
backpressure or split peaks are observed. Investigate
cause of column failure by checking:
■
Backpressure
■
Mobile phase(s), bacteria, precipitation,
and/or samples
■
Peak splitting
■
Sample quality
■
Injection solvent strength
7. To reduce the chances of mobile-phase contamination or
degradation, only prepare what you need for analysis or
store excess bulk quantities in a refrigerated environment.
8. Mobile phase-related questions to ask:
■
Am I using 100% aqueous mobile phases?
Am I able to add a small amount of organic
modifier to my mobile phase A?
■
Do I filter my aqueous mobile phases through
0.2 µm filters?
■
How old is my mobile phase? Do I label the bottle
with the preparation date?
■
Do I “top off” or do I prepare fresh mobile phases
every 24–48 hours?
■
What is the quality of my water? Has the quality
recently changed? How is my water purification
system working? When was it last serviced?
■
Am I working with pH 7 phosphate buffer (which
is VERY susceptible to bacterial growth)?
9. Sample-related questions to ask:
■
If I inject neat standards prepared in mobile
phase do I observe these problems?
■
If I prepare my standards in water and prepare them
like samples (e.g., SPE, filtration, etc.) do I still observe
these problems?
■
Has the quality of my samples changed over time?
b. Getting Started with CORTECS HILIC Columns
1. Because CORTECS HILIC columns do not possess a
bonded phase, the pH operating range is 1–5, and they can
be operated at temperatures up to 45 °C.
2. As with any LC column, operating at the extremes of pH,
pressures, and temperatures will result in decreased
column lifetime.
Column Equilibration
1. When the column is first received, flush in 50% acetonitrile:
50% water with 10 mM final buffer concentration for
50 column volumes.
2. Equilibrate with 20-column volumes of initial mobile-phase
conditions before making first injection.
3. If gradient conditions are used, equilibrate with
8–10 column volumes between injections.
4. Failure to appropriately equilibrate the column could
result in drifting retention times.
Mobile-phase Considerations
1. Always maintain at least 3% polar solvent in the mobile
phase or gradient (e.g., 3% aqueous/3% methanol or
2% aqueous/1% methanol, etc.). This ensures that the
CORTECS particle is always hydrated.
2. Maintain at least 40% organic solvent (e.g., acetonitrile)
in your mobile phase or gradient.
3. Avoid phosphate salt buffers to avoid precipitation in
HILIC mobile phases. Phosphoric acid is okay.
4. Buffers such as ammonium formate or ammonium acetate
will produce more reproducible results than additives such
as formic acid or acetic acid. If an additive (e.g., formic
acid, etc.) must be used instead of a buffer, use 0.2% (v:v)
instead of 0.1%.
5. For best peak shape, maintain a buffer concentration
of 10 mM in your mobile phase/gradient at all times.