Waters PAH Columns User Manual
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Waters Pah Column
I. IntroduCtIon
We are sure you will find that Waters built-in quality helps solve many of
your challenging separation problems. We strive to provide products with the
highest degree of lot-to-lot and column-to-column reproducibility to mini-
mize variations in your chromatographic results. Waters PAH columns are
manufactured and packed under highly controlled conditions. Each must pass
a series of stringent tests before being accepted for shipment. Included with
each column is the final Certificate of Analysis.
Polynuclear Aromatic Hydrocarbons (PAHs) are among the most frequently
monitored environmental contaminants. Standard and official methods for the
analysis of PAHs are found in compendia for air, drinking water, wastewater,
solid waste, and food analysis1.
Many of these methods specify HPLC, usually with UV and fluorescence detec-
tion, as the recommended analytical procedure.
Waters PAH columns are optimized for the HPLC analysis of PAHs. Figure 1
shows a chromatogram of 16 PAH compounds, listed as target pollutants by
the U.S. EPA. The Waters PAH columns achieve baseline resolution and excel-
lent peak symmetry for all 16 target analytes in less than 25 minutes, while
employing a simple water; acetonitrile binary gradient. The resolving power
of the PAH Columns provides superior peak identification and quantitation
for PAHs.
Florida Administrative Code 17.700 includes 2 additional compounds
(1-methyl naphthalene and 2-methyl naphthalene ) in addition to the 16 com-
pound EPA 610 mix that we currently use to show the proficiency of Waters
instrumentation to analyze PAH compounds. The new Waters PAH columns
resolve these two compounds along with the other 16, (see Figure 2).
Contents
I. IntroduCtIon
II. ConneCt Ing the Column to the hP lC Inst rument
a. Column Connectors and System Tubing Considerations
b. SLIPFREE Connectors
c. Minimization of Band Spreading
d. Measuring System Bandspread Volume
e. Measuring Gradient Delay Volume
f. Use of Narrow-Bore Columns (3.0 mm i.d.)
g. Impact of bandspreading on Column performance
(2.1 mm i.d. column)
h. System Modification Guidelines
III. Column equIlIbratIon
IV. Column usage
V. Column CleanIng, regeneratIng and storage
a. Cleaning and Regeneration
b. Storage
VI. t roubleshootIng
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Minutes
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Minutes
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Column: Waters PAH Column 5 μm 4.6 x 250 mm @ 27 °C Peaks:
System: Waters Alliance System with 2996 Photodiode Array Detector
Eluent A: Water
Eluent B: Acetonitrile
Gradient: 60% B to 100% B using curve 9 in 12 minutes,
hold 11 minutes, back to initial conditions
Flow Rate: 1.2 mL/min
Injection: 20 μL
Sample: EPA-610 mixture
UV @ 254 nm
Figure 1: PAH Analysis using Waters PAH Columns
Column: Waters PAH Column 5 μm 4.6 x 250 mm @ 27 °C Peaks:
Eluent A: Water
Eluent B: Acetonitrile
Gradient: 60% B to 100% B using curve 9 in 12 minutes
hold 11 minutes, back to initial conditions
Flow Rate: 1.2 mL/min
Injection: 20 μL
Sample: EPA-610 mixture plus two compounds*
1. Naphthalene - 20 ppm
2. Acenaphthylene - 40 ppm
3. Acenaphthene - 20 ppm
4. Fluorene - 4 ppm
5. Phenanthrene - 2 ppm
6. Anthracene - 2 ppm
7. Fluoranthene - 4 ppm
8. Pyrene - 2 ppm
1. Naphthalene - 20 ppm
2. Acenaphthylene - 40 ppm
3*. 1-methyl naphthalene - 25 ppm
4*. 2-methyl naphthalene - 25 ppm
5. Acenaphthene - 20 ppm
6. Fluorene - 4 ppm
7. Phenanthrene - 2 ppm
8. Anthracene - 2 ppm
9. Fluoranthene - 4 ppm
9. Benzo(a)anthracene - 2 ppm
10. Chrysene - 2 ppm
11. Benzo(b)fluoranthene - 4 ppm
12. Benzo(k)fluoranthene - 2 ppm
13. Benzo(a)pyrene - 2 ppm
14. Dibenzo(a,h)anthracene - 4 ppm
15. Benzo(g,h,I)perylene - 4 ppm
16. Indeno(1,2,3-cd)pyrene - 2 ppm
10. Pyrene - 2 ppm
11. Benzo(a)anthracene - 2 ppm
12. Chrysene - 4 ppm
13. Benzo(b)fluoranthene - 4 ppm
14. Benzo(k)fluoranthene - 2 ppm
15. Benzo(a)pyrene - 2 ppm
16. Dibenzo(a,h)anthracene - 4 ppm
17. Benzo(g,h,I)perylene - 4 ppm
18. Indeno(1,2,3-cd)pyrene - 2 ppm
UV @ 254 nm
Figure 2: PAH Analysis According to Florida Administrative Code 17,700
II. CONNECTING THE COLUMN TO THE HPLC INSTRUMENT
Handle the column with care. Do not drop or hit column on a hard surface
as it 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 chromato-
graphic results.
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2. 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
the 5/16 inch wrench on the compression screw and the other 3/8 inch
wrench on the hex head of the column endfitting. Note: If one of the
wrenches is placed on the column flat during this process, the endfitting
will be loosened and leak.
[ Care and Use ManUal ]
0.090 inches
0.130 inch
e
Gap
Void
3. 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.
4. An arrow on the column identification label indicates correct direction
of solvent flow.
It is important to realize that extra column peak broadening can destroy suc-
cessful separation. The choice of appropriate column connectors and system
tubing is discussed in detail below.
a. Column Connectors and System Tubing Considerations
Due to the absence of an industry standard, various column manufacturers
have employed different styles of chromatographic column connectors. The
chromatographic performance of your separation can be negatively affected if
the style of your column endfittings do not match the existing instrumenta-
tion tubing ferrule setting. This page explains the difference between Waters
style and Parker style endfittings, which vary in the required length of the
tubing protruding from the ferrule. The PAH column is equipped with Waters
style endfittings which require a 0.130 inch ferrule depth (see next section
for setting ferrule depth). If you are presently using a non-Waters style
column, it is critical that you reset the ferrule depth for optimal performance.
A void appears if a tube with Parker ferrule setting is connected to a Waters
style column.
The presence of a void in the flow stream downgrades the column perfor-
mance. There is only one way to fix the problem: Cut the end of the tubing
with the ferrule, put a new ferrule on the tubing and make the connection.
Before tightening the screw, make sure that the tubing bottoms out in the
endfitting of the column.
If tubing with a Waters style ferrule setting is connected to a column with
Parker style endfitting, the end of the tubing will bottom out before the fer-
rule reaches its proper sealing position. This will leave a gap creating a leak.
There are two ways to fix the problem:
1. Just tighten the screw a little bit more. The ferrule moves forward, and
reaches the sealing surface. Do not overtighten because this may end in
breaking the screw.
The Proper Tubing/Column Connection
Tubing touches the bottom of the column endfitting, with no void
between them.
2. Cut the tubing, put a new ferrule on it and make the connection.
An alternative is to replace the conventional compression screw fitting with
an all-in-one PEEKTM fitting.
(Waters part number PSL613315) that allows you to reset the ferrule depth.
Another approach is to use a SLIPFREE® fitting to always ensure the correct
fit. The finger-tight SLIPFREE connectors automatically adjust to fit all
compression screw type fittings without the use of tools.
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