Waters Sep-Pak DNPH-Silica Cartridges - Manual (Page 4)

[ Care and Use ManUal ]
Waters Sep-Pak DNPH-Silica Cartridge
4
d. Eluting the Derivatives
There are two recommended methods for elution of the derivatives
from the cartridge:
Volumetric Method:
1. Elute the DNPH derivatives from the cartridge directly into a
5 mL volumetric flask. Use 3 mL HPLC-grade acetonitrile at a
flow rate of less than 3 mL/min. Higher flow rates (>3 mL/min)
can result in reduced recovery.
2. Dilute to volume with HPLC-grade acetonitrile.
Gravimetric Method:
1. Elute the DNPH derivatives from the cartridge directly into a
tared vessel. Weigh the eluate and divide by the density of the
acetonitrile to obtain the sample volume. Acetonitrile has a
density of 0.785 g/mL at 20 °C.
III. AnALYZInG tHe dnPH derIVAtIVes
a. Operating Guidelines
To ensure success in your HPLC analysis:
• Use a pre-column filter between the injector and column.
• Use HPLC-grade unstabilized tetrahydrofuran for making mobile
phases.
• Use HPLC-grade water and HPLC-grade acetonitrile.
• Degas the mobile phases by simultaneously applying vacuum
and ultrasound to the mobile phases for 30 seconds. If you are
using a low-pressure mixing gradient system, sparging with
helium may be necessary.
• Waters Nova-Pak ® C
18
columns are shipped containing
water/acetonitrile. Equilibrate the column at 1.5 mL/min for
10 minutes in mobile phase before the first analysis.
b. Performing HPLC Analysis
To analyze the sample:
1. Prepare the standard solution of the DNPH derivatives that you
need to quantify. The concentrations of the standards should be
in the same range as the expected concentrations in the sample.
To synthesize DNPH derivatives, see Appendix B.
2. Prepare a cartridge blank from the same sample lot as the
cartridge used to collect the sample, using the sample
procedure and same bottled solvent.
3. Analyze the standard solution to determine the response
factor for each derivative. Due to the high linearity of the
detector response, a single point calibration is sufficient for
Waters detectors.
Note: Use an injection volume appropriate for your column. Inject
≤20 µL for a 3.9 x 150 mm Nova-Pak C
18
column, and ≤10 µL for a
3.0 x 75 mm Nova-Pak C
18
column.
4. Analyze the cartridge blank to determine background levels.
Ensure that the blank values are in the normal range
(see Table 2).
5. Analyze the samples.
6. Subtract the blank values from the sample values. Run standards
at regular intervals between samples.
c. Analyzing a Cartridge Blank
Analyze a blank to determine background levels. Figure 5 shows a
typical result from a blank cartridge extraction. Since background
levels may change during storage (see Section V), always compare
samples to blank cartridges from the same lot stored under the same
conditions.
Note: When preparing a blank sample, ensure that you use the exact
bottled reagents that were used for the preparation of the sample.