Waters Sep-Pak DNPH-Silica Cartridges - Manual (Page 5)

[ Care and Use ManUal ]
Waters Sep-Pak DNPH-Silica Cartridge
5
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.
Figure 5: Typical DNPH-Silica Sep-Pak Cartridge.
d. Selecting Separation Conditions
Isocratic Separation of Derivatives
Use the conditions in Table 3 to separate DNPH and DNPH
derivatives of C
1
through C
3
aldehydes and ketones in less than
10 minutes. This separation requires a relatively weak mobile phase
(high water content) to separate the formaldehyde-DNPH derivative
from trace impurities. Figure 6 shows a representative chromatogram
using the isocratic conditions listed in Table 3.
Table 3: Isocratic Separation of C
1
-C
3
Aldehyde and Ketone
Derivatives
Figure 6: Isocratic Separation of C
1
-C
3
Aldehyde and Ketone
Derivatives
Gradient Separation of Derivatives
Use the gradient conditions listed in Table 4 to separate DNPH and
DNPH derivatives from a complex mixture of aldehydes and ketones
in 15 minutes. Figure 7 shows an example separation.
Table 4: Gradient Separation of C
1
-C
9
Aldehyde and Ketone
Derivatives
Figure 7: Gradient Separation of C
1
-C
9
Aldehyde and Ketone
Derivatives
Column Nova-Pak C
18
3.9 x 75 mm
Mobile Phase Water/Acetonitrile/Tetrahydrofuran 65/30/5 v/v
Flow Rate 1.5 mL/min
Injection Volume 10 µL
Detection Adsorbance at 360 nm
1. DNPH
2. Formaldehyde-DNPH
3. Acetaldehyde-DNPH
4. Acetone-DNPH
5. Acrolein-DNPH
6. Propionaldehyde-DNPH
Time (minutes)
Column Nova-Pak C
18
3.9 x 150 mm
Mobile Phase A: Water/Acetonitrile/Tetrahydrofuran 60/30/10 v/v
B: Water/Acetonitrile 40/60 v/v
Gradient 100% A for 1 min (adjust as necessary to give gradient delay >
3mL) then a linear gradient from 100% A to 100% B in 10 min
Flow Rate 1.5 mL/min
Injection Volume 20 µL
Detection Adsorbance at 360 nm
Time (minutes)
1. DNPH
2. Formaldehyde-DNPH
3. Acetaldehyde-DNPH
4. Acetone-DNPH
5. Acrolein-DNPH
6. Propionaldehyde-DNPH
7. Crotonaldehyde-DNPH
8. Butanone-DNPH
9. Butyraldehyde-DNPH
10. Benzaldehyde-DNPH
11. Isovaleraldehyde-DNPH
12. Valeraldehyde-DNPH
13. o-Tolualdehyde-DNPH
14. m-Tolualdehyde-DNPH
15. p-Tolualdehyde-DNPH
16. Hexaldehyde-DNPH
17. 2,5-Dimethylbenzaldehyde-DNPH
1. DNPH
2. Formaldehyde-DNPH
3. Acetaldehyde-DNPH
4. Acetone-DNPH
Time (minutes)
1
1. DNPH
2. Formaldehyde-DNPH
3. Acetaldehyde-DNPH
4. Acetone-DNPH
1
Cartridge Blank
2 3 4
0 2 6 8 10 12 14