Download [ Care and Use ManUal ] Pico•Tag column for free amino

[ Care and Use Manual ]
Pico•Tag Column for Free Amino Acids
I. Introduction
Contents
Waters Pico•Tag® Column for Free Amino Acids uses the exclusive bonding
I.introduction
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
II.installation
suited for the reversed-phase separation of phenylthiocarbamyl amino acids.
a. Solvent Requirements
Our stringent quality control procedures assure that this column, when used
b. Sample and Solvent Preparation
in conjunction with Waters Pico•Tag reagents, eluents, and HPLC system,
c. Column and Cartridge Installation
will provide the speed, resolution, and sensitivity needed for amino acid
d. Equilibration
analysis.
III.Operating tips
Use these procedures to ensure that you obtain quality results from your
Pico•Tag column and take full advantage of the features it offers.
IV. column efficiency
WARNING: The ampules of phenylisothiocyanate which are contained in the
a. Column Testing
b. Troubleshooting
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
V. column storage
assure that these ampules and contents are disposed of properly and that
they do not come into contact with acids.
vi. warranty/service information
vii.ordering information
The Pico•Tag Column for Free Amino Acids
1
[ Care and Use Manual ]
II.installation
Figure 1: Separations Method Using Pico•Tag Column For Free
Amino Acids
a. Solvent Requirements
Abbreviation
Component Name
Retention Time
Your Pico•Tag column is shipped containing an aqueous/organic mixture com-
PSER
Phosphoserine
3.05
patible with the Pico•Tag eluent. For best results use Pico•Tag Eluent 1 (P/N
ASP
Aspartic Acid
3.50
10960) and Waters Pico•Tag Eluent 2 (P/N 10965) with this column.
GLU*
Glutamic Acid
4.01
AAD
α-Aminoadipic Acid
5.62
HYPRO
Hydroxyproline
7.12
PEA
Phosphoethanolamine
7.53
0.45 µm. Pico•Tag prefiltered eluents eliminate the need for this step
SER*
Serine
9.20
by providing a prepared, ready-to-use solvent to reduce the problem of
ASN*
Asparagine
9.50
plugged filters and preserve column life.
GLY*
Glycine
10.30
_-ALA
β Aline
12.62
TAU
Taurine
14.82
HIS*
Histidine
17.00
GABA
γ-Aminobutyric Acid
18.00
CIT
Citrulline
18.62
NH2
Ammonia
19.62
THR
Threonine
20.35
ALA
Alanine
21.52
b. Sample and Solvent Preparation and Filtration
•
•
Use LC grade solvents, filtered to remove microparticulate matter above
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.
BAIB
B-Aminoisobutyric Acid
23.65
c. Column and Cartridge Installation
CARN
Carnosine
24.72
Remove the end plugs from your column with a 5/16” wrench. (Be sure
ARG*
Arginine
26.02
to replace the end plugs when the column is removed from the system for
METSO2
Methionine Sulfone
27.00
storage.) The column outlet is indicated by an arrow on the label show-
PRO
Proline
30.15
1MH
1-Methyl Histidine
31.32
ing the direction solvent should flow. Tighten the fittings to turn. DO NOT
3MH
3-Methyl Histidine
32.25
AAB
A-Aminobutyric Acid
37.99
TYR*
Tyrosine
44.89
Follow the next three steps of this procedure if you must cut tubing to con-
VAL*
Valine
47.62
nect a new steel column or to improve the end connections on your existing
MAT*
Methionine
49.22
fittings.
CYST
Cystathionine
51.35
RG1
Reagent 1
52.90
CYS2*
Cystine
52.99
RG2
Reagent 2
54.40
2. Grasp the tubing on both sides of the scribe mark with cloth-covered
ILE
Isoleucine
54.95
pliers (to prevent marring the tube surface) and gently work the tube
LEU*
Leucine
55.65
back and forth until it separates.
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
The Pico•Tag Column for Free Amino Acids
OVERTIGHTEN - THIS WILL DAMAGE THE FITTING SEAT. A properly prepared and
assembled compression fitting in good condition is all that is required.
1.
Using a three-cornered file with a cutting edge scribe the circumference
of the tubing at the desired break.
2
[ Care and Use Manual ]
IV. column efficiency
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
Liquid chromatography columns have a finite life which is directly related
seat to assure a leak-free connection.
to the care and use they receive. Column life is influenced by the number of
Note: Attach a union in place of the column and flush the lines free of previous
injections, sample and solvent cleanliness, frequency of solvent changeover,
solvents before attaching the column.
and handling and storage procedures.
Figure 2: Ferrule and Compression Assembly
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
d. Equilibration
must not be relied upon.
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
Follow generally accepted procedures for quality control and methods devel-
the temperature to 46 °C. Purge the column with 30 mls of Eluent 2, then
opment when using these columns.
equilibrate with Eluent 1. Equilibration between the mobile phase and pack-
Note: Before running the first analysis on your new column perform the test
ing is established when a stable baseline can be produced. If your result is
sample separation given in the test conditions section.
unsatisfactory repeat the equilibration process.
a. Column Testing
The w5 sigma method shown in Figure 3 is used to measure column efficiency.
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
Unlike the tangent method used to determine system efficiency, this stringent
method considers naturally occurring peak asymmetry.
Figure 3. 5 Sigma Test Method
(avoid using concentrated acids or bases). Maximum column life (pH)
3.5 - 6.5.
•
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.
•
Columns are thoroughly tested in our quality control laboratories for adher-
Protect column from vibration, mechanical shock and rapid changes in
ence to our specifications. Since slight variations in your results will occur
pressure. Column packings are based on a highly porous and delicate
depending on the equipment used, test sample makeup and equipment set-
silica gel alignment. Any thermal, physical or chemical shock (such as
tings and conditions, perform the test sample run given here for your new
changing solvents rapidly or at high flow rates) can cause the particles
column and record the results (retention time and the settings used) before
to shift and may result in a loss of efficiency.
•
attempting the first analysis. Use these results for comparison throughout the
When using water, distill or treat with a Milli-Q or equivalent system.
life of your column.
De-ionized water is not acceptable because it contains organic com-
Note: Be sure to record results and instrument settings (and configurations)
pounds which alter column selectivity.
•
to allow exact reproduction and comparison in the future.
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
[ Care and Use Manual ]
Test Sample:
Acenaphthene (0.05% in Acetonitrile)
vi. warranty/service information
Solvent:
Pico•Tag Eluent B (P/N 88112)*
a. Service and Applications Assistance
Flow Rate:
1.0 ml/min
Chart Speed:
10 cm/min
Injection Volume:
8 µl
Waters’ staff of experienced service specialists provide maintenance assistance 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
Detection:254 nm @ 0.2 AUFS
be contacted at 1-800-252-HPLC in Milford.
*or equivalent made by mixing 600 ml of HPLC grade acetonitrile with 400
b. Warranty
ml Milli-Q° water.
Warrants its high performance liquid chromatography columns in accordance
b. Troubleshooting
with the following terms and conditions:
Table 1: Typical Column Problems and Solutions
Problem
Excess pressure
buildup
Cause
• Filters plugged
with Particulates
• Sample precipitates on column
(sample not
soluble in mobile
phase)
Loss of resolution, • Filters partially
broad peaks, low
plugged
plate counts
• Contaminated
colum, insufficent
equilibration
• Column collapse
and void formation
Waters will repack or replace (at our discretion) without cost any steel col-
Solution
• 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.
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 incompatible solvents or buffers, or at an incorrect pH.
•
rect temperatures or pressures.
•
or sample filtration practices.
Leaving the column unused for less than 72 hours does not generally require
vii.ordering information
storage procedures. Follow the procedures below to store column for periods
of more than 72 hours.
•
Item
Part Number
DO NOT store the column in water alone; this practice will promote
Phenylisothiocyanate (PITC)
88120
microbial contamination. Store the column in Pico•Tag Eluent 2.
Triethylamine (TEA)
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
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.
•
Particulate buildup or precipitation in the column or end fittings causing
high internal pressure which has occurred because of improper solvent
V. column storage
•
Physical damage to the packing material because of operation at incor-
Return the column to its box with the end plugs firmly in place for storage. Allowing steel columns to dry out can result in poor chromatography performance.
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 +7 495 3367000
Czech Republic 42 02 617 11384
Denmark 45 46 59 8080
Finland +358 9 5659 6288
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 +31 (0)76-50 87 200
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 What’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