Owner's manual

[ Care and Use ManUal ]
3
c. General Preparation Guide for One Liter of Eluent
• Dissolvesaltsinapproximately900mlofpurifiedwater.
• AdjustpHwithappropriateacidorbase.
• Adjusttofinalonelitervolumewithwater.
• FilterthroughtheSolventClarificationKitusingan0.45µmaqueous-
compatible filter.
• Transferbuffertoanamberglassbottleandpurgefor15minutes
with pure nitrogen.
• Transferbuffertoanalyzer.
• Bufferspreparedinthismannerwillbestableforatleastoneweek
at room temperature.
d. Use of Non-aqueous Solvents
• For additional resolution of the early-eluting aminoacids,small
amounts of methanol, ethanol, or propanol can be added to Buffer
A. This alcohol concentration should not exceed 10%
• Highconcentrationsofanon-aqueousmodifierwillcausetheresin
to swell resulting in high backpressure.
e. Backpressure
• With a new column flowing at0.4 ml/min in Buffer A at62°C.
typical system pressures will range from 800 psi - 1500 psi.
• As the column ages, pressure willslowlyincrease, althoughthis
increase is not usually associated with decreased efficiency.
• Themostcommoncausesofhighbackpressureare:particulatesin
the sample or buffer, proteinaceous material coating the resin packing,
mold formation in the buffers or in the column.
• Reducedtemperature,higherflowrateand highsolventviscosity
will all yield higher pressure.
• Topreventexcessivepressure,donotflowthroughthecolumnwith
the temperature control module off.
f. Sample Considerations
• Acidicproteinhydrolyzatesshouldbelyophilizedandreconstituted
in Buffer A.
• Convenient sample concentration range: 0.20 - 25 nmol amino
acid/5µl-50µlinjectionvolume.
• Samplevolumeandsensitivityrequirementsshouldbeoptimized
for individual needs.
• Samples that contain protein, lipid or heavy metals (e.g., tissue
extracts, food samples, feed samples, etc.) should not be introduced
directly on the column. Pre-purification on a strong exchange resin
or a Sep-Pak
®
C
18
cartridge is recommended.
g. Sep-Pak
®
C
18
Cartridge Cleanup for Amino Acid Samples
Prepare the following solutions:
Solution 1: 0.1 % trifluoroacetic acid (TFA) in water
Solution 2: 0.1 % TFA in H20:MeOH, 80:20
Solution 3: 0.1 % TFA in H2O:MeOH, 70:30
h. Preparation Scheme
1. Activate a new Sep-Pak
®
C
18
cartridge with two 10 ml volumes of
methanol.
2. Wash with two 10 ml volumes of Solution 1.
3. Wash with 10 ml of Solution 2.
4. Mix 1 ml sample with 2 ml Solution 3. Sample should be water or
acid solubilized and free of particulate matter.
5. Pass the sample through the Sep-Pak
®
C
18
cartridge.
6. Discard the first 1 ml of eluent and collect the next 2 ml. This fraction will
contain all amino acids. Lipids and high molecular weight pro¬teins
will be retained on the Sep-Pak
®
C
18
cartridge.