[ CARE AND USE MANUAL ]
Atlantis T3, dC18 and HILIC Silica Columns
I. Int roduction
Thank you for choosing an Atlantis® Column. The manufacture of Atlantis columns begins with ultrapure reagents to control the chemical composition and purity of the final product. Atlantis 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.
Waters recommends the use of Sentry™ guard columns to maximize column lifetime and protect the column from contaminants.
Cont ents
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
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 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.
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.
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 connected 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 direction of mobile phase flow (toward detector).
3.Fingertighten all fittings.
Caution: Under-tightening the connectors 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.
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. The 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 |
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C-Clip |
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Sentry™ Guard |
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0.25 mm (0.009 inch) Tubing |
Column |
Atlantis® Cartridge Column |
End Connector |
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[ CARE AND USE MANUAL ]
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.
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 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
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
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 tightening 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 |
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Tubing Internal Diameter |
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Tubing Length |
0.005” |
0.010” |
0.020” |
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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 |
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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
0.005 inches
0.020 inches
0.040 inches
Diluted/Distorted Sample Band
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