Waters Atlantis T3, DC18 and HILIC Silica Columns - Manual (Page 8)

[ Care and Use ManUal ]
Atlantis Columns 8
f. Temperature
Temperatures between 20 ˚C - 45 ˚C are recommended for operating
Atlantis 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 depend-
ing on the pH and buffer conditions used. The combination 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 main-
taining 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
XII. HILIc GettInG stArted
a. Equilibration of Atlantis HILIC Silica Columns
1. Upon receipt, equilibrate in 50% acetonitrile/50% aqueous buffer
(10 mM final buffer concentration) for 50 column volumes.
2. Prior to first injection, equilibrate with 20 column volumes of
initial mobile phase conditions.
3. When running gradients, equilibrate with 10 column volumes
between injections.
Failure to appropriately equilibrate the column could result in drifting
retention times.
b. HILIC Mobile Phase Considerations
1. Always maintain at least 5% polar solvent in the mobile phase
or gradient (e.g., 5% water, 5% methanol or 3% methanol/2%
aqueous buffer, etc.). This ensures that the Atlantis HILIC Silica
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 OK).
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) 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.
c. Injection Solvents for HILIC
1. If possible, injection solvents should be 100% organic solvent.
Water must be eliminated or minimized. Choose weak HILIC
solvents such as acetonitrile, isopropanol, methanol, etc.
2. A generic injection solvent is 75:25 acetonitrile methanol. This is
a good compromise between analyte solubility and peak shape.
3. Avoid water and dimethylsulfoxide (DMSO) as injection solvents.
These solvents will produce very poor peak shapes.
4. Exchange water or DMSO with acetonitrile by using reversed-
phase solid-phase extraction. If this is not possible, dilute the
water or DMSO with organic solvent.
d. Additional HILIC Recommendations
1. For initial scouting conditions, run a gradient from 95% acetoni-
trile to 50% acetonitrile. If no retention occurs, run isocratically
with 95:3:2 acetonitrile:methanol:aqueous buffer.
2. Alternate polar solvents such as methanol, acetone or isopro-
panol can also be used in place of water in the mobile phase to
increase retention.
3. Be sure that your needle wash solvent/purge solvent contains the
same high organic solvent concentration as your mobile phase,
else peak shapes will suffer.