Waters Delta-Pak High Pressure Insert HPLC Column User Manual

[ Care and Use ManUal ]

delta-Pak HIGH-Pressure Inert HPlc coluMn

I. IntroductIon

a. Overview

Waters High Pressure Inert (HPI) HPLC columns are a metal free alternative
for both bio-chromatographic and ion-chromatographic applications. In this

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advanced design, the sample contacts only polyetherether ketone (PEEK
and ultrahigh molecular weight polyethylene (UHMWPE), two materials
which are widely accepted for their biocompatibility and inertness. The
chromatographic performance of these columns is essentially identical
to their metal counterparts with the added advantage of greater chemical
inertness.

b. Delta-Pak Columns

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The Delta-Pak
phase. It is synthesized from 5 µm spherical silica particles having an aver­age pore diameter of 100 Å or 300 Å. Delta-Pak
analysis and purification of peptides and proteins.

column packing material is an endcapped C18 or C4 bonded

columns are ideal for the

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contents

I. IntroductIon

a. Overview

b. Delta-Pak Columns

II. InstallatIon

a. Installing the Column

b. Equilibration

III. MoBIle PHase and saMPle GuIdelInes

a. Solvent Compatibility

b. Mobile Phase Requirements

c. Sample Preparation and Filtration

IV. oPeratIon

a. Chromatography Guidelines

b. Efficiency Testing
c. Typical Column Backpressure and Eluent Viscosity
d. Column Temperature Limits

V. care and MaIntenance

a. Troubleshooting

b. Cleaning the Column
c. Storing the Column

VI. orderInG InforMatIon

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[ Care and Use ManUal ]

II. InstallatIon

a. Installing the column

Use only plastic tubing and plastic finger-tight endfittings with the High­Pressure Inert column. The Waters endfitting design is shown in Figure 1.
Over-tightening the endfittings can cause irreversible damage to the filter
housing.

Figure 1: Finger-tight Endtting

To install a new fitting or replace a worn fitting:

1) Using a single-edge razor, make a straight and square cut on the
plastic tubing. Make this cut in front of the compression screw on the
worn endfitting.

2) Slide a compression screw fitting and a washer over the end of the cut
tubing.

3) Place the gripper ring in the top depression of the tool (see Figure 2).
Insert the end of the tubing into the gripper ring tool as far as it will
go. This seats the gripper ring at the proper length of the tubing.

4) Remove the tubing from the tool and slide the other washer and ferrule
over the end of the tubing. Insert a union over the endfitting assembly
and tighten. This seats the assembly.

Make sure that any mobile phase used for startup is miscible with the ship­ping solvent. Before placing the column in the flow path:

1) Attach a union between the column inlet and outlet lines.

2) Flush the lines to remove any microparticulates and old solvents. Flush
the injector loop if applicable.

3) Remove the union.

Remove the finger-tight end plugs from our column and save them to store
the column when it is removed from the system. Attach the column so that
flow follows the direction of the arrow on the column label. To install the
column, read the inlet and outlet fittings into the column until finger-tight.

b. Equilibration

The column is delivered in the mobile phase indicated in Table 1. Prior to
use, the column should be equilibrated with the mobile phase that will be
used in a analysis. Check mobile phase/shipping solvent miscibility.

Delta-Pak material is highly hydrophobic. If mobile phases with high concen­trations of water are to be used, it is necessary to thoroughly solvate the
packing with the non-aqueous component before starting. To do so, pure the
column with 5 to 10 column volumes of the non-aqueous solvent to be used
in the mobile phase prior to equilibrating the column with the mobile phase.

Should the mobile phase contain a buffer salt, flush the column with 30
mL of water prior to equilibrating the column with the buffer. With PIC
ion- pairing reagents, whose concentration in the mobile phase is very
low (typically 5 mmol/l), 100 to 300 mL of mobile phase is required for
equilibration.

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Figure 2: Gripper Ring Installation

III. MoBIle PHase and saMPle GuIdelInes

Liquid chromatography columns have a finite lifetime directly related to the
care and use they receive. Column life is reduced by:

• contamination from the mobile phase or sample

• improper storage and handling

• frequent solvent switching

• exposure to high or low pH eluents (for example, less than

2.0, greater than 8.0)

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[ Care and Use ManUal ]

a. Solvent Compatibility

The HPI column is constructed from non-metallic components which
have been chosen on the basis of their chemical inertness. While the
materials of construction are resistant to a wide spectrum of organic
and inorganic chemicals, the following list of chemicals attack the
surface and cause irreparable damage.

Caution: Use of any of these solvents with the column will result in a
void of the warranty:

• Chlorinated hydrocarbons

• Concentrated sulfuric acid

• Tetrahydrofuran (THF)

• Concentrated nitric acid

b. Mobile Phase Requirements

The following precautions are recommended for the preparation
of eluents:

• Use LC-grade solvents which have been filtered to remove

microparticulate matter above 0.45 µm. Ultrapure water
(18 megohm) is recommended.

• Use Gelman Sciences Aerodisc

to filter samples and prevent the high backpressure that results
from a blocked column inlet filer.

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syringe filters from Waters

IV. oPeratIon

Simple procedures such as those outlined in this and the previous

chapter can significantly extend the column lifetime.

Should a change in peak shape, retention of a particular compound,
or resolution between two compounds be observed, take immediate
steps to determine a reason for the changes (see Section V. a.,
Troubleshooting). Until the cause of the change is determined, do not
rely on the results of the analyses.

Note: Before running the first analyses on your new column, perform
the test sample separation given in Efficiency Testing, Section IV, b.

a. Chromatography Guidelines

The following operating guidelines will help you obtain the best
performance from your Waters analytical HPIC column.

• Do not exceed an operating pressure of 40 Mpa (400 atm or

6,000 psi).

• Use vacuum filtration and/or sonification to remove dissolved

gases which could affect your pump. Care should be exercised

when vacuum filtering or sparging mixtures of solvents, because

the composition of the mixture could change. The best way to

degas the mobile phase is to place it into an ultrasonic bath and
apply vacuum and ultrasonic power simultaneously for about
30 seconds.

• Use ultrapure water as an intermediate solvent when changing

from aqueous salt solutions to organic solvents. Perform this
changeover gradually. Use care when adding organic solvents to
aqueous buffer solutions, as salt precipitation may occur.

c. Sample Preparation and Filtration

If the sample contains dissolved contaminants or particulates that
may bind irreversibly to the column, the following procedure is
recommended:

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• Use Sep-Pak

sample that may be adsorbed onto the packing material surface
causing changes in performance and reduced column lifetime.

cartridges to remove contaminants from the

• Avoid using concentrated acids and bases. T he use of mobile

phases whose pH is below 2.0 or above 8.0 results in signifi­cantly reduced column lifetime due to the hydrolysis of the
bonded phase.

• Filter all aqueous buffers through a 0.45 µm filter prior to use.

Never use turbid or cloudy mobile phases.

• Protect the column from vibration, mechanical shock, and rapid

changes in pressure which can result from rapidly changing the
composition of the eluent.

• Use ultrapure water (18 megohn). Deionized water is not accept

able because it contains organic compounds that may alter
column selectivity.

b. Efficiency Testing

Waters’ columns are tested for compliance with our specifications. It
is possible that columns may be damaged during shipment. Test the
column before using it. The results of the efficiency test may be used
as a benchmark or future references.

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