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

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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

[ Care and Use ManUal ]

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

taper first) over the tube. Be cer-tain to bottom the tube in the fitting

seat to assure a leak-free connection.

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

solvents before attaching the column.

Figure 2: Ferrule and Compression Assembly

d. Equilibration

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

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

the temperature to 46 °C. Purge the column with 30 mls of Eluent 2, then

equilibrate with Eluent 1. Equilibration between the mobile phase and pack-

ing is established when a stable baseline can be produced. If your result is

unsatisfactory repeat the equilibration process.

III. operatIng tIps

• Normal recommended pressure should not exceed 3500 psi.

• For all silica based packing materials, stay within a pH range of 2-8

(avoid using concentrated acids or bases). Maximum column life (pH)

3.5 - 6.5.

IV. column effIcIency

Liquid chromatography columns have a finite life which is directly related

to the care and use they receive. Column life is influenced by the number of

injections, sample and solvent cleanliness, frequency of solvent changeover,

and handling and storage procedures.

If a change is observed in the:

• Retention of a particular compound

• Resolution between two compounds

• Peak shape

Take immediate steps to determine the reason for the changes. Until the cause

of the change is determined, the results of any separation using the column

must not be relied upon.

Follow generally accepted procedures for quality control and methods devel-

opment when using these columns.

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

sample separation given in the test conditions section.

a. Column Testing

The w5 sigma method shown in Figure 3 is used to measure column efficiency.

Unlike the tangent method used to determine system efficiency, this stringent

method considers naturally occurring peak asymmetry.

Figure 3. 5 Sigma Test Method

• Filter all aqueous buffers (Pico•Tag eluents are prefiltered). Avoid using

turbid or cloudy buffers. Be sure that any solutions containing buffers,

salts, etc. are compatible with the wetted surfaces of the column and

equipment.

• Protect column from vibration, mechanical shock and rapid changes in

pressure. Column packings are based on a highly porous and delicate

silica gel alignment. Any thermal, physical or chemical shock (such as

changing solvents rapidly or at high flow rates) can cause the particles

to shift and may result in a loss of efficiency.

• When using water, distill or treat with a Milli-Q or equivalent system.

De-ionized water is not acceptable because it contains organic com-

pounds which alter column selectivity.

• Protect the column from rapid changes in solvent composition. DO NOT

change the flow rate faster than 0.5 ml/min increments.

The Pico•Tag Column for Free Amino Acids 3

Columns are thoroughly tested in our quality control laboratories for adher-

ence to our specifications. Since slight variations in your results will occur

depending on the equipment used, test sample makeup and equipment set-

tings and conditions, perform the test sample run given here for your new

column and record the results (retention time and the settings used) before

attempting the first analysis. Use these results for comparison throughout the

life of your column.

Note: Be sure to record results and instrument settings (and configurations)

to allow exact reproduction and comparison in the future.

[ Care and Use ManUal ]

Test Sample: Acenaphthene (0.05% in Acetonitrile)

Solvent: Pico•Tag Eluent B (P/N 88112)*

Flow Rate: 1.0 ml/min

Chart Speed: 10 cm/min

Injection Volume: 8 µl

Detection: 254 nm @ 0.2 AUFS

*or equivalent made by mixing 600 ml of HPLC grade acetonitrile with 400

ml Milli-Q° water.

b. Troubleshooting

Table 1: Typical Column Problems and Solutions

Problem Cause Solution

Excess pressure
buildup

Loss of resolution,
broad peaks, low
plate counts

• Filters plugged
with Particulates

• Sample precipi­tates on column
(sample not
soluble in mobile
phase)

• Filters partially
plugged

• Contaminated
colum, insufficent
equilibration

• Column collapse
and void formation

• Clean in an ultrasonic bath
or replace.

• Always alter mobile phases
and samples.

• Slowly purge with a strong
mobile phase that is both
appropriate to dissolve the
contaminate and compat­ible with the column.

• Replace or clean inlet
and outlet filters in an
ultrasonic bath.

V. column storage

Leaving the column unused for less than 72 hours does not generally require

storage procedures. Follow the procedures below to store column for periods

of more than 72 hours.

• DO NOT store the column in water alone; this practice will promote

microbial contamination. Store the column in Pico•Tag Eluent 2.

• DO NOT allow buffers or other potentially harmful materials to remain

in the system when not being used. Flush and replace with Pico•Tag

Eluent 2 or 10% organic in water mixture.

• Return the column to its box with the end plugs firmly in place for stor-

age. Allowing steel columns to dry out can result in poor chromatogra-

phy performance.

VI. warranty/serVIce InformatIon

a. Service and Applications Assistance

Waters’ staff of experienced service specialists provide maintenance assis-

tance on both preventative and/or corrective levels. For complete information

and assistance, please call Waters Service Department at 1-800-252-HPLC.

For solutions to particular applications questions Waters team of technical

support personnel are available to help you with specialized support. They may

be contacted at 1-800-252-HPLC in Milford.

b. Warranty

Warrants its high performance liquid chromatography columns in accordance

with the following terms and conditions:

Waters will repack or replace (at our discretion) without cost any steel col-

umn that fails to perform satisfactorily if notified within 90 days from your

receipt. Any column that is returned must have a Return Authorization Number

granted by the Waters Customer Ser-vice Department. Approval is subject to

the following exclusions:

• Physical damage to the column because of misuse or abuse.

• Chemical damage to the packing material because of use with incompat-

ible solvents or buffers, or at an incorrect pH.

• Physical damage to the packing material because of operation at incor-

rect temperatures or pressures.

• Particulate buildup or precipitation in the column or end fittings causing

high internal pressure which has occurred because of improper solvent

or sample filtration practices.

VII. orderIng InformatIon

Item Part Number

Phenylisothiocyanate (PITC) 88120

Triethylamine (T EA) 88121

Physiological Standard A/N 10948

Physiological Standard B 10949

Pico•Tag Eluent 1 10960

Pico•Tag Eluent 2 10965

Sample Clarification Kit 26865

In-Line Precolumn Filter Kit 84560

Pico•Tag Diluent 88119

The Pico•Tag Column for Free Amino Acids 4

[ Care and Use ManUal ]

Sales Offices:

Austria and European Export

(Central South Eastern Europe,

CIS and Middle East) 431 877 18 07

Australia 2 9933 1777

Belgium 32 2 726 1000

Brazil 55 11 5094 3788

Canada 800 252 4752

China 8621 6495 6999

CIS/Russia [phone]

Czech Republic 42 02 617 11384

Denmark 45 46 59 8080

Finland [phone]

France (33) 1 30 48 72 00

Germany 49 6196 400600

Hong Kong 852 29 64 1800

Hungary 36 1 350 5086

India and India Subcontinent

91 80 2 837 1900

Ireland 353 1 448 1500

Italy 39 02 274 211

Japan (81) 3 3471 7191

Korea (82) 2 820 2700

Mexico 5255 5200 1860

The Netherlands [phone]

Norway 47 63 84 60 50

Poland (48) 22 833 4400

Puerto Rico 787 747 8445

Singapore 65 6273 1221

Spain 34 93 600 93 00

Sweden 46 8 555 11500

Switzerland 41 62 889 2030

Taiwan 886 2 2543 1898

United Kingdom 44 208 238 6100

©2007 Waters Corporation. Waters, The Science of W hat’s Pos­sible, and PicoTag are trademarks of Waters Corporation. MilliQ
is a trademark of Millipore Corporation.

November 2007 WAT010954 Rev 4 VW-PDF

The Pico•Tag Column for Free Amino Acids 5

Waters Corporation

34 Maple Street
Milford, MA 01757 U.S.A.
T: 1 508 478 2000
F: 1 508 872 1990
www.waters.com