Waters Pico-Tag Amino Acid Analysis Column User Manual

[ Care and Use ManUal ]

Pico•Tag amino acid analysis column

I. IntroductIon

Waters exclusive bonding and packing processes, developed for the Pico•Tag®

Column (3.9 mm x 15 cm), P/N 88131, produce a durable, high efficiency

stationary phase ideally suited for the reverse-phase separation of phenyl-

thiocarbamyl amino acids. Our stringent quality control procedures result

in a column which, when used in conjunction with Waters Pico•Tag reagents,

eluents, and HPLC system is guaranteed to provide the speed, resolution, and

sensitivity needed for amino acid analysis.

This information provides the means to ensure that you derive the full benefit

of our research, manufacturing, and quality control. For additional information

on the complete method, refer to Section 4.4 of the Amino Acid Analysis

Manual, P/N 07124, or the Pico•Tag Amino Acid Analysis System Operator’s

Manual, P/N 88140.

WARNING: THE AMPULES OF PHENYLISOTHIOCYANATE WHICH ARE

CONTAINED IN THE PICO•TAG CHEMICAL PACKAGE REACT ON CONTACT WITH

STRONG ACIDS, EMITTING HIGHLY TOXIC CYANIDE FUMES AND/OR OXIDES

OF SULPHUR. CARE MUST BE TAKEN TO ASSURE THAT THESE AMPULES AND

CONTENTS ARE DISPOSED OF PROPERLY AND THAT THEY DO NOT COME INTO

CONTACT WITH ACIDS.

contents

I. IntroductIon

II. PrePArAtIon For oPerAtIon

a. Column Installation

b. Solvent Requirements

c. Equilibration

III. cAre And use

a. Sample and Eluent Preparation and Filtration

b. Precautions

c. Storage Considerations

d. Troubleshooting

e. Service and Applications Information

IV. column eFFIcIency

V. test condItIons

VI. recommended sPAre PArts (steel columns)

Figure 1. Pico•Tag Method Amino Acid Analysis 250 pmol of

Pierce H Standard

Pico•Tag Amino Acid Analysis Column 1

VII. WArrAnty

[ Care and Use ManUal ]

II. PrePArAtIon For oPerAtIon

a. Column Installation

Remove the end plugs from your steel column with a 5/16” wrench and save

them for storage after the column has been removed from the system. The

column outlet is indicated by an arrow on the label (show-ing the direction

solvent should flow). Tighten the fittings 1/4-to-1/2 turn beyond finger tight.

DO NOT OVERTIGHTEN - THIS WILL DAMAGE THE FITTING SEAT. A properly

prepared and assembled com-pression fitting in good condition is all that

is required.

Follow the next four steps of this procedure if tubing cutting is required to

connect a new column or to improve the end connections on your existing

fittings.

1. Using a file with a cutting edge (such as the file included in the Startup

Tool Kit, P/N 96146, supplied with each Waters Liquid C hromatography

System) scribe the circumference of the tubing at the desired break.

2. Grasp the tubing on both sides of the scribe mark with cloth-covered

pliers (to prevent marring the tube surface) and gently work the tube

back and forth until it separates.

3. File the ends smooth and assemble as shown.

b. Solvent Requirements

Your Pico•Tag column is shipped containing an aqueous/organic mixture

compatible with the Pico•Tag eluents. It is recommended that only Waters

Pico•Tag eluents be used with this column.

Waters Pico•Tag Eluent A (P/N 88108)

Waters Pico•Tag Eluent B (P/N 88112)

Waters Pico•Tag Diluent (P/N 88119)

c. Equilibration

A necessary step to successful use of your column is the initial solvation

(or wetting) of the packing. Install the column in a column heater and bring

temperature to 38 °C. Purge the column at 1 ml/min with Eluent B, then

equilibrate with Eluent A for fifteen minutes. Equilibration between the mobile

phase and packing is established when a stable baseline can be produced. If

your result is un-satisfactory repeat the equilibration process.

III. cAre And use

Liquid chromatography columns have a finite life influenced by their care and

use, number of injections, sample and solvent cleanliness, frequency of sol-

vent changeover, and handling and storage procedures among other factors. If

a change is observed in the:

4. Slide the compression fitting, followed by the ferrule (large end of the

taper first) over the tube. Be certain to bottom the tube in the fitting

seat for which its use is intended to assure a leak-free connection.

Note: Attach a union in place of the column and flush the lines free of

microparticulates before attaching the column

Figure 5: Ferrule and Compression Assembly

.

Retention of a particular compound resolution between two compounds peak

shape.

Take immediate steps to determine the reason for the changes, and until the

determination is made, the results of any separations using the column must

not be relied upon. Follow generally accepted pro-cedures for quality control

and methods development when using these columns.

Important Note: Before running the first analysis on your new column perform

the Test Sample Separation given in the Test Conditions Section.

Pico•Tag Amino Acid Analysis Column 2