Waters Amino Acid Analysis Liquid Chromatography Column - Manual (Page 3)

[ 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.