Waters Amino Acid Analysis Liquid Chromatography Column - Manual (Page 5)

[ Care and Use ManUal ]
5
f. regeneration of heavy metal contaminated column
• Often metals can be removed by chelation under basic and acidic
conditions.
• Reverse direction of buffer flow through column. Elevate column
heater temperature to 90°-950C and pump 0.2 N NaOH solution
containing 0.25 g EDTA/liter through the column for minimum of 4
hours.
• Switch buffer tube to second container and pump 2 N HNO, solution
containing 0.25 g EDTA/liter through the column for another 2-4
hours.
• Restore normal flow direction and temperature. Reequilibrate column
with Buffer A and test column with amino acid standard solution; If
problem still exists, repeat the above basic/acidic EDTA treatment.
CAUTION: Never attempt to clean the column using hydrochloric acid
(HCI) or sulphuric acid (H2SO,) solutions. Such acids are harmful to
stainless steel.
g. available service and applications Information
The Waters Corporation staff of trained and e¬perienced service
specialists provides maintenance for Waters instruments on
preventive and/or corrective levels. Contact Waters Corporation at
508-478-2000 or your local Waters representative for further
details. For answers to specific chromatography questions in areas
such as methods development, applications and quality control,
contact your local Waters representative or Waters’ home office
applications laboratory.
h. chromatographic troubleshooting
When a new column is installed, it is recommended that a separation
of standard hydrolyzate amino acids be performed using Elution
System 1. A typical separation is shown on the following page
(Figures 3 & 4).
The more advanced Waters HPLC System allows for the generation of
a continuous gradient using only two buffers. This is equivalent to an
“infinite step gradient”. Superior resolution is achieved without baseline
disturbances. The constant ionic strength, pH gradient (0.2N Na
+ ,
pH 3.00 to pH 9.60) allows for rapid reequilibration without NaOH
regeneration.
i. typical separation
Figure 2: Amino Acid Analysis – Continuous Gradient
As well as the standard hydrolyzate amino acids, unusual amino acids
can be separated by the two-buffer, gradient system. Note cysteic acid,
hydroxyproline, methionine sulfoxide, hydroxylysine, and tryptophan.
Slight modifications of the pH and/or constituency of the A or B buffers
can quickly be used to optimize new separations.