Waters Pico-Tag Columns for Free Amino Acids User Manual

[ Care and Use ManUal ]

Pico•Tag column for free amino acids

I. IntroductIon

Waters Pico•Tag® Column for Free Amino Acids uses the exclusive bonding

and pack-ing processes developed by Waters for the original Pico•Tag column.

This has resulted in a durable, high efficiency, 3.9 mm x 30 cm column ideally

suited for the reversed-phase separation of phenylthiocarbamyl amino acids.

Our stringent quality control procedures assure that this column, when used

in conjunction with Waters Pico•Tag reagents, eluents, and HPLC system,

will provide the speed, resolution, and sensitivity needed for amino acid

analysis.

Use these procedures to ensure that you obtain quality results from your

Pico•Tag column and take full advantage of the features it offers.

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. InstallatIon

a. Solvent Requirements

b. Sample and Solvent Preparation

c. Column and Cartridge Installation

d. Equilibration

III. operatIng tIps

IV. column effIcIency

a. Column Testing

b. Troubleshooting

V. column storage

VI. warranty/serVIce InformatIon

VII. orderIng InformatIon

The Pico•Tag Column for Free Amino Acids 1

[ Care and Use ManUal ]

Figure 1: Separations Method Using Pico•Tag Column For Free

Amino Acids

Abbreviation Component Name Retention Time

PSER Phosphoserine 3.05

ASP Aspartic Acid 3.50

GLU* Glutamic Acid 4.01

AAD α-Aminoadipic Acid 5.62

HYP RO Hydroxyproline 7.12

PEA Phosphoethanolamine 7.53

SER* Serine 9.20

ASN* Asparagine 9.50

GLY* Glycine 10.30

_-ALA β Aline 12.62

TAU Taurine 14.82

HIS* Histidine 17.00

GABA

CIT Citrulline 18.62

NH2 Ammonia 19.62

THR Threonine 20.35

ALA Alanine 21.52

BAIB B-Aminoisobutyric Acid 23.65

CARN Carnosine 24.72

ARG* Arginine 26.02

METSO2 Methionine Sulfone 27.00

PRO Proline 30.15

1MH 1-Methyl Histidine 31.32

3MH 3-Methyl Histidine 32.25

AAB A-Aminobutyric Acid 37.99

TYR* Tyrosine 44.89

VA L* Valine 47.62

MAT* Methionine 49.22

CYS T Cystathionine 51.35

RG1 Reagent 1 52.90

CYS2* Cystine 52.99

RG2 Reagent 2 54.40

ILE Isoleucine 54.95

LEU* Leucine 55.65

HYLYS 1 Hydroxylsine 1 58.15

HYLYS 2 Hydroxylsine 2 58.90

PHE* Phenylanlanine 60.02

TRP* Tyrptophan 60.32

ORN Orninthine 60.77

LYS* Lysine 65.52

γ-Aminobutyric Acid

18.00

II. InstallatIon

a. Solvent Requirements

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

patible with the Pico•Tag eluent. For best results use Pico•Tag Eluent 1 (P/N

10960) and Waters Pico•Tag Eluent 2 (P/N 10965) with this column.

b. Sample and Solvent Preparation and Filtration

• Use LC grade solvents, filtered to remove microparticulate matter above

0.45 µm. Pico•Tag prefiltered eluents eliminate the need for this step

by providing a prepared, ready-to-use solvent to reduce the problem of

plugged filters and preserve column life.

• Vacuum filtration or sonification may be used to remove dissolved

gasses which could affect your solvent delivery system.

• Waters Sample Clarification Kit, (P/N 26865) is recommended to filter

prepared samples and prevent excessive pressure buildup.

• Use of a Waters In-line Precolumn Filter (P/N 84560),is recommended.

• Pico•Tag Diluent, (P/N 88119), is recommended for reconstitution of

derivatized samples.

c. Column and Cartridge Installation

Remove the end plugs from your column with a 5/16” wrench. (Be sure

to replace the end plugs when the column is removed from the system for

storage.) The column outlet is indicated by an arrow on the label show-

ing the direction solvent should flow. Tighten the fittings to turn. DO NOT

OVERTIGHTEN - THIS WILL DAMAGE THE FITTING SEAT. A properly prepared and

assembled compression fitting in good condition is all that is required.

Follow the next three steps of this procedure if you must cut tubing to con-

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

fittings.

1. Using a three-cornered file with a cutting edge 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.

The Pico•Tag Column for Free Amino Acids 2