Waters Delta-Pak High Pressure Insert HPLC Column User Manual

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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

®

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

TM

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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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.

®

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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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.

®

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:

®

• 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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INJECT

N = 25

2

(

)

To perform an efficiency test, prepare the test sample as follows:

1) Prepare an acetone/acenaphthene sample by dissolving 0.05 g
acenaphthene and 600 µl acetone in 100 mLs of mobile phase.
While acenaphthene is the recommended sample, you can
substitute the following simple aromatic hydrocarbons:

Figure 3: 5 Sigma Test Method

• naphthalene

• p-cymene

• xylene

• dibutyl-, dipropyl-, or diethylphthalate

• propylbenzene

• toluene

• ethylbenzene

Toluene and diethylphthalate are the least desirable substitutes

since they have the lowest k.

2) Equilibrate the column employing the conditions summarized
below. Note: Incomplete equilibration results in tailing, fronting
or split peaks.

Table 1: Column Efciency Test Conditions

Column Mobile Phase Flow Rate Test Samples

Delta-Pak C
Delta-Pak C

18

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and

50/50
acetonitrile/

0.7 ml/min Acenaphthene

water

Do not substitute methanol for acetonitrile in reversed-phase

column tests due to the much higher viscosity of methanol/water

mixtures.

If problems occur during normal operation of the column, repeat th

efficiency test and compare the results. This may help locate the
source of the problem.

c. Typical Column Backpressure and Eluent Viscosity

The column hardware has been designed to withstand operating
pressures as high as 50 Pa (500 atm or 7,000 psi). However, normal

operating backpressure should not exceed 40 Mpa (400 atm or

6,000 psi).
The normal operating backpressure of a column varies considerably
and is affected by column length, flow rate, mobile phase viscosity,
temperature, and particle size. The backpressure may rise signifi­cantly during the course of a gradient.
The following equation can be used to calculate the backpressure of
your column:

Pressure (atm) at 1 L/min in H

0 = 210 x l • v

2

dp2 x d2

Where:

L = column length, mm

dp = particle diameter, µm

d = column diameter, mm

v = mobile phase viscosity, centipose (see Table 2)

3) Set a UV detector at 254 nm with an attenuation of 0.05 AUFS.

The resulting detector deflection should be approximately 90

percent of full scale. The minimum deflection is 60%.

4) Inject about 2 µL of sample. Large amount may overload the

column.

5) Measure and record plate count, asymmetry, backpressure, and

instrument settings. Waters uses the 5 sigma method, shown
in Figure 3, to measure column efficiency. Unlike the tangent
method, this more stringent method takes peak asymmetry into

account

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Table 2: Viscosity

Mobile Phase Viscosity (20 °C),

Centipoise

n-Pentane 0.24

n-Hexane 0.33

wn-Heptane 0.42

Isoctane 0.50

Acetone 0.32

Dioxane 1.54

Nitromethane 0.65

Acetonitrile 0.37

n-Propanol 2.3

Ethanol 1.2

Methanol 0.6

Water 1.0

60/40 Methanol/Water 2.0

The typical operating backpressure for Waters® Delta-PakTM columns
is summarized below.

Table 3: Typical Operating Backpressure at 1 mL/min

Column Dimension Mpa Methanol

Delta­Pak

Delta­Pak

2.1 x 150

TM

mm

3.9 x 150

TM

mm

(Water)

17 (28) 170 (285) 2500 (4200)

6.0 (10.0) 60 (100) 900 (1500)

Atm Metha­nol (Water)

Psi Methanol
(Water)

A variation of plus or minus 10-15% is acceptable.

d. Column Temperature Limits

The column hardware has been designed to operate between 4 °C and
50 °C.

Note: Column failure due to operation outside of these temperature
limits voids the warranty.

V. care and MaIntenance

a. Troubleshooting

Table 4, on the following page, provides the corrective action for
specific problems that may occur with the Waters HPLC High Pressure
Inert columns.

b. Cleaning the Column

Flush reversed-phase columns with methanol or acetonitrile. It is
useful to monitor the UV absorbance during this procedure to see if
contaminants are being removed from the column.

If flushing does not resolve the problem, wash the column with sol­vents of decreasing polarity, always making sure the solvent in the
column and a washing solvent are miscible. Return the column to the
standard reversed-phase conditions by reversing the wash sequence.

If you suspect a particular contaminant is building up on the column,
use solvents or chemicals that are known to dissolve a material.
Be sure to remain within the limits of pH 2.0 to pH 8.0. Also,

ensure that no precipitate forms as a result of mixing the washing

solution with the solvent in the column. Proteinaceous material can
sometimes be removed by making repeated injections of 200 µL of

dimethylsulfoxide (DMSO).

c. Storing the Column

Leaving the column unused for less than 72 hours does not generally
require specialized storage procedures. However, never let columns
dry out. For longer storage:

• DO NOT store the column in water alone, as this may result

in bacterial growth in the column. Storing the column in a 10%
aqueous solution of isopropanol or methanol will hinder
bacterial growth.

• DO NOT store the column in buffered solution or solutions

containing salts, as these may precipitate.

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

for storage. Allowing the column to dry out may result in poor
chromatographic performance.

• DO NOT leave a column at elevated temperatures with no mobile

phase flow.

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Table 4: Troubleshooting

Symptom Cause Corrective Action Prevention

Buildup in system operating
pressure.

Loss of resolution, low plate
count.

Inlet filter plugged with
particulates from dirty sample
or mobile phase. Injector seal or
pump seal shredding.

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

Clogged tubing Replace the tubing. Identify and eliminate the source

Sample solvent is incompatible
with or stronger than the mobile
phase.

Failing injector (measure system
band spreading periodically).

Contaminated column. Wash with strong solvent. Isolate the source of the con-

Insufficient equilibration. Continue equilibration.

Replace the filter. Install an in-line filter between

the pump and the injector. Filter
the sample and/or mobile phase.

Wash the column using a solvent
that will dissolve the sample.

Dissolve sample in another
solvent.

Repair the injector. Filter mobile phases and use an

Use a mobile phase in which the
sample is soluble.

of the material clogging the
tubing.

Dissolve sample in another
mobile phase. If not possible,
change mode of separation.

in-line filter.

tamination and use a Sep-Pak
cartridge to clean up the sample.
If this is not possible, use a
Guard-Pak™ holder and insert
to protect the column from
contaminants. Use HPLC grade
solvents.

®

Incorrect connecting tubing. Replace with 0.0009” internal

diameter tubing.

All tubing from injector to the

detector should be 0.0009”

internal diameter.

VI orderInG InforMatIon

Delta-Pak HPI (High Pressure Inert Analytical Columns (PEEK)

Column (non metallic) Particle Size Pore Size Dimensions Part No.

DeltaPak HPI C18 5 µm 100 Å 2.1 mm x 150 mm WAT052750

DeltaPak HPI C18 5 µm 300 Å 2.1 mm x 150 mm WAT052765

DeltaPak HPI C4 5µm 100 Å 2.1 mm x 150 mm WAT052760
DeltaPak HPI C4 5 µm 300 Å 2.1 mm x 150 mm WAT052755

DeltaPak HPI C18 5 µm 300 Å 3.9 mm x 150 mm WAT035571

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©2007 Waters Corporation, Waters, The Science of W hat’s Pos­sible, Delta-Pak, Guard-Pak, PIC and Sep-Pak are trademarks of
Waters Corporation. Aerodisc is a trademark of Gelman Sciences.
PEEK is a trademark of Victrex plc.

October 2007 WAT035904 Rev 4 V W-PDF

Waters Corporation

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

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