Waters XSelect HSS HPLC Columns User Manual

[ Care and Use ManUal ]

XseLeCt HIGH stRenGtH sILICa (Hss) HPLC CoLUMns

Contents

I. IntRoDUCtIon

II. ConeCtInG tHe CoLUMn to tHe HPLC sYsteM

a. Column Connection

b. Column Connectors and System Tubing Considerations

c. Band Spreading Minimization

d. Measuring System Band Spread Volume

e. Measuring Gradient Delay Volume

III. WateRs sMaLL PaRtICLe sIze (3.5 �m) CoLUMns

– Fast CHRoMatoGRaPHY

IV. CoLUMn eqUILIbRatIon

V. CoLUMn InstaLLatIon PRoCeDURe

VI. CoLUMn PeRFoRManCe VaLIDatIon

I. IntRoDUCtIon

Thank you for choosing an XSelect™ High Strength Silica (HSS) HPLC

column. The manufacture of XSelect HSS HPLC columns begins with

ultrapure reagents to control the chemical composition and purity of

the final product. XSelect HSS HPLC columns are manufactured in a

cGMP, ISO 9001:2000 certified plant with each step being conducted

within narrow tolerances. Every column is individually tested and

Certificates of Batch Analysis and a Performance Chromatogram are

provided with each column.

XSelect HSS HPLC columns are based on the same particle technol-

®

ogy and chemistries as ACQUITY UPLC

seamless transferability between HPLC and UPLC

HSS columns, thus enabling

®

separations.

VII. InItIaL CoLUMn eFFICIenCY DeteRMInatIon

VIII. CoLUMn UsaGe

a. Guard Columns

b. Sample Preparation

c. Recommended pH Range

d. Solvents

e. Pressure

f. Temperature

g. Scaling Up/Down Isocratic Methods

IX. CoLUMn CLeanInG, ReGeneRatInG anD stoRaGe

a. Cleaning and Regenerating

b. Storage

X. tRoUbLesHootInG

XSelect HSS HPLC Columns 1

[ Care and Use ManUal ]

II. ConneCtInG tHe CoLUMn 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-

quality 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

stainless steel tubing. When tightening or loosening the compression

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 associated with such exposure

including risks to health and electrical connections.

ferrule depth be reset for optimal performance prior to installing

the XSelect HSS HPLC column. In a proper tubing/column connection

(Figure 2), the tubing touches the bottom of the column endfitting,

with no void between them.

Figure 1: Waters and Parker Ferrule Types

Waters Ferrule Setting Parker Ferrule Setting

Figure 2: Proper Tubing/Column Connection

Tubing touches the bottom of the column endfitting, with no void

between them.

Attention: A void will occur if a Parker ferrule connection is used with

a Waters style endfitting (Figure 3). This will dramatically reduce the

efficiency of the column and cause peak shape distortion.

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

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

tubing and ferrule must be assembled.

It is important to realize that extra column peak broadening can

destroy a successful separation. The choice of appropriate column

connectors and system tubing is discussed in detail below.

b. Column Connectors and System Tubing Considerations

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

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

1). Each endfitting style varies in the required length of the tubing

protruding from the ferrule. XSelect HSS HPLC columns are 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

Figure 3: Parker Ferrule in a Waters Style Endfitting

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 4).

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

column with a Parker style endfitting.

XSelect HSS HPLC Columns 2

[ Care and Use ManUal ]

Figure 4: Waters Ferrule in a Parker 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-one PEEK

™

fitting (Waters Part Number PSL613315)

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

®

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 5).

Figure 5: Single and Double SLIPFREE Connectors

c. Band Spreading Minimization

Internal tubing diameter influences system band spreading and peak

shape. Larger tubing diameters cause excessive peak broadening and

lower sensitivity (Figure 6).

Figure 6: Effect of Connecting Tubing on System

0.005 inches

0.020 inches

0.040 inches

Diluted/Distorted Sample Band

d. Measuring System Band Spread Volume

This test should be performed on an HPLC system with a single wavelength

UV detector (not a Photodiode Array (PDA)).

1. Disconnect column from system and replace with a zero dead

volume union.

Table 1. Waters Part Numbers for SLIPFREE Connectors

SLIPFREE Type
and Tubing
Internal
Diameter

0.005” 0.010” 0.020”

Single 6 cm PSL 618000 PSL 618006 PSL 618012

Single 10 cm PSL 618002 PSL 618008 P SL 618014

Single 20 cm PSL 618004 PSL 618010 PSL 618016

Double 6 cm PSL 618001 P SL 618007 PSL 618013

Double 10 cm P SL 618 003 PS L 6180 09 PS L 618 015

Double 20 cm P SL 618005 PSL 618001 PSL 618017

Tubing Length

2. Set flow rate to 1 mL/min.

3. Dilute a test mix in mobile phase to give a detector sensitivity

0.5-1.0 AUFS (system start up test mix can be used which

contains uracil, ethyl and propyl parabens; Waters Part Number

WAT034544).

4. Inject 2 to 5 μL of this solution.

5. Using 5-Sigma Method measure the peak width at 4.4% of peak height:

Band Spreading (μL) = Peak Width (min) x Flow Rate (μL/min)

= 0.1 min x 1000 μL/min

= 100 μL

Figure 7: Determination of System Band Spread Volume Using

5-Sigma Method

XSelect HSS HPLC Columns 3