Waters Sep-Pak XPoSure Aldehyde Sampler - Manual (Page 5)

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b. Performing the 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 Waters Symmetry C
18
column,
and ≤ 10 µL for a 3.0 x 75 mm Waters Symmetry C
18
column.
4. Analyze the cartridge blank to determine background levels.
Ensure that the blank values are in the normal range (see Table
3). Figures 4 and 5 show a representative separation and blank
sample, respectively.
5. Analyze the samples.
6. Subtract the blank values from the sample values. Run
standards at regular intervals between samples.
Table 4: HPLC Separation Conditions
Column Waters Symmetry C
18
, 3.9 x 150 mm
Mobile Phase 45:55 Acetonitrile/Water
Flow Rate 1.3 mL/min
Injection Volume 20 µL
Detection Wavelength
Absorbance at 360 nm
(<50 mV of baseline noise)
Limit of Detection for
Formaldehyde Based on a signal to noise ration of 3
(55 picograms in 20 µL injection)
Limit of Quantification for
Formaldehyde Based on a signal to noise ratio of 10
(183 picograms in 20 µL injection)
Figure 4. Isocratic Separation of C
1
-C
3
Hydrazone Derivatives.
Figure 5: Typical Sampler Blank.
c. Analyzing a Cartridge Blank
Analyze a blank to determine background levels. Since
background levels may change during storage, 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.
To prepare a cartridge blank:
1. Elute a fresh DNPH-Silica Sep-Pak cartridge from the same lot
as the cartridges used to collect your sample
2. Analyze the solution by HPLC using the same conditions as
those used for the sample.
3. Multiply the concentration of each DNPH derivative by the
volume of the eluate to determine the amount of background
DNPH derivative.
1. DNPH
2. Formaldehyde-DNPH
3. Acetaldehyde-DNPH
4. Acetone-DNPH
5. Acrolein-DNPH
6. Propionaldehyde-DNPH
Time (minutes)
0 2 4 6 8 10 12 14
0
.0005
.0010
.0015
.0020
.0025
1. DNPH
2. DNPH-Formaldehyde
3. DNPH-Acetaldehyde
4. DNPH-Acetone
.0030
1
2
3
4
5
[ CARE AND USE MANUAL ]
Sep-Pak XPoSure Aldehyde Sampler