Waters Atlantis T3, DC18 and HILIC Silica Columns User Manual

[ Care and Use ManUal ]

AtLAntIs t3, dc18 And HILIc sILIcA coLuMns

I. IntroductIon

Thank you for choosing an Atlantis® Column. The manufacture of Atlantis

columns begins with ultrapure reagents to control the chemical composition

and p urity of the final product. Atlantis columns are manufactured in a cGMP,

ISO 9001:2000 certified plant with each step being conducted within nar-

row tolerances. Every column is individually tested and Certificates of Batch

Analysis and a Performance Chromatogram are provided with each column.

™

Waters recommends the use of Sentry

lifetime and protect the column from contaminants.

guard columns to maximize column

contents

I. IntroductIon

II. connectInG tHe coLuMn or cArtrIdGe to tHe

III. systeM ModIfIcAtIon recoMMendAtIons

HPLc systeM

IV. WAters sMALL PArtIcLe sIze (3 µM) coLuMns

– fAst cHroMAtoGrAPHy

V. coLuMn equILIbrAtIon

VI. coLuMn InstALLAtIon Procedure

VII. coLuMn PerforMAnce VALIdAtIon

VIII. InItIAL coLuMn effIcIency deterMInAtIon

IX. coLuMn usAGe

XII. HILIc GettInG stArted

Atlantis Columns

XIII. coLuMn cLeAnInG, reGenerAtInG And storAGe

XIV. troubLesHootInG

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[ Care and Use ManUal ]

II. CONNECTING THE COLUMN OR CARTRIDGE TO THE HPLC
SYSTEM

a. Column Connection

Handle the column with care. Do not drop or hit the column on a hard

surface as this may disturb the bed and affect its performance.

1. Correct connection of 1/16 inch outer diameter stainless steel

tubing leading to and from the column is essential for high-qual-

ity chromatographic results.

2. An arrow on the column identification label indicates correct

direction of solvent flow.

3. When using standard stainless steel compression screw fittings,

it is important to ensure proper fit of the 1/16 inch outer diameter

stai nless s teel t ubing. W hen t igh teni ng or l oose ning the c omp res -

sion screw, place a 5/16 inch wrench on the compression screw

and a 3/8 inch wrench on the hex head of the column endfitting.

Caution: If one of the wrenches is placed on the column flat during this

process, the endfitting will be loosened and leak. Under-tightening

compression screws or using worn ferrules can lead to solvent

leaking. Care should be taken to check all column connections

for leaks to avoid exposure to solvents and the hazards associ-

ated with such exposure including risks to health and electrical

connections.

4. If a leak occurs between the stainless steel compression screw fit-

ting and the column endfitting, a new compression screw fitting,

tubing and ferrule must be assembled.

b. Cartridge Connection

Handle the cartridge with care. Do not drop or hit the cartridge on a

hard surface as it may disturb the bed and affect its performance. Refer

to Figure 1 for an exploded view of an Atlantis cartridge column with

a Sentry guard column.

1. Unscrew end connectors from the old cartridge. Leave them con-

nected to the inlet and outlet lines of the instrument.

2. Attach new cartridge column between connectors so that the

direction of the flow arrow on the label is pointing in the direc-

tion of mobile phase flow (toward detector).

3. Fingertighten all fittings.

Caution: Under-tightening the connectors can lead to solvent leak-

ing. Care should be taken to check all column connections for leaks

to avoid exposure to solvents and the hazards associated with such

exposure including risks to health and electrical connections.

4. Check for leaks once flow has been initiated. If a leak occurs

between the connector and the column endfitting, the column

may be misaligned in the connector or the Kalrez O-ring must be

replaced in the connector.

It is important to realize that extra column peak broadening can destroy

a successful separation. T he choice of appropriate column connectors

and system tubing is discussed in detail below.

c. Column Connectors and System Tubing Considerations

Due to the absence of an industry standard, various column man

facturers have employed different types of chromatographic column

connectors. The chromatographic performance of the separation can

be negatively affected if the style of the column endfittings does

not match the existing tubing ferrule setting. This section explains

the differences between Waters style and Parker style ferrules and

endfittings (Figure 2). Each endfitting style varies in the required

length of the tubing protruding from the ferrule. The Atlantis column

is equipped with Waters style endfittings, which require a 0.130 inch

ferrule. If a non-Waters style column is presently being used, it is

critical that ferrule depth be reset for optimal performance prior to

installing an Atlantis column. In a proper tubing/column connection

(Figure 3), the tubing touches the bottom of the column endfitting,

with no void between them.

Figure 1: Installation of Atlantis Cartridge Column with Sentry Guard Column

Spacer

Sentry™ Guard

0.25 mm (0.009 inch) Tubing Atlantis® Cartridge Column

Atlantis Columns

Column

C-Clip

2

End Connector

[ Care and Use ManUal ]

Diluted/Distorted Sample Band

0.005 inches

0.020 inches

0.040 inches

Figure 2: Waters and Parker Ferrule Types

Waters Ferrule Setting Parker Ferrule Setting

Figure 3: Proper Tubing/Column Connection

Tubing touches the bottom of the column endfitting, with no void between them.

Attention: A void will occur if tubing with a Parker ferrule is connected

to a Waters style endfitting (Figure 4). This will dramatically reduce the

efficiency of the column and cause peak shape distortion.

Figure 4: Parker Ferrule in a Waters Style Endfitting

There are two ways to fix the problem:

1. Tighten the screw a bit more. The ferrule moves forward, and

reaches the sealing surface. Do not overtighten since this may

break the screw.

2. Cut the tubing, replace the ferrule and make a new connection.

Alternatively, replace the conventional compression screw fitting with

™

an all-in-on e PE EK

fitting (Waters Part Number PSL613315) that allows

resetting of the ferrule depth. Another approach is to use a Thermo

™

Hypersil

Keystone, Inc. SLIPFREE® connector to ensure the correct

fit. The fingertight SLIPFREE connectors automatically adjust to fit all

compression screw type fittings without the use of tools (Figure 6).

Figure 6: Single and Double SLIPFREE Connectors

To fix this problem: Cut the end of the tubing with the ferrule, place a

new ferrule on the tubing and make a new connection. Before tighten-

ing the screw, make sure that the tubing bottoms out in the endfitting

of the column.

Conversely, if tubing with a Waters ferrule is connected to a column

with Parker style endfitting, the end of the tubing will bottom out

before the ferrule reaches its proper sealing position. This will leave a

gap and create a leak (Figure 5).

Caution: The connection will leak if a Waters ferrule is connected to a

column with a Parker style endfitting.

Figure 5: Waters Ferrule in a Parker Style Endfitting

Table 1. Waters Part Numbers for SLIPFREE Connectors

SLIPFREE Type and Tubing Internal Diameter
Tubing Length 0.005” 0.010” 0.020”

Single 6 cm PSL 618000 PSL 618006 PSL 618012
Single 10 cm PSL 618002 PSL 618008 PSL 618014
Single 20 cm PSL 618004 PSL 618010 PSL 618016
Double 6 cm PSL 618001 PSL 618007 PSL 618013
Double 10 cm PSL 618003 PSL 618009 PSL 618015
Double 20 cm PSL 618005 PSL 618001 PSL 618017

d. Band Spreading Minimization

Internal tubing diameter influences system band spreading and peak

shape. Larger tubing diameters cause excessive peak broadening and

lower sensitivity (Figure 7).

Figure 7: Effect of Connecting Tubing on System

Atlantis Columns

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