Waters Sep-Pak DNPH-Silica Cartridges - Manual (Page 9)

[ Care and Use ManUal ]
Waters Sep-Pak DNPH-Silica Cartridge
9
5. Remeasure the concentration of DNPH derivatives by HPLC.
6. Calculate the difference in the concentration of each DNPH
derivative measured in steps 3 and 5 to yield the contribution
from the acetonitrile.
7. Calculate the percent hydrazone contributed by the acetonitrile
relative to the background level. The value for any hydrazone
should not exceed 25% of its value in the blank.
Example: Measuring Acetonitrile Purity
1. HPLC analysis of a fresh cartridge shows the sample contains:
2. Analysis of the concentrations of hydrazones after reacting with
acid yields:
3. The different between the concentrations of hydrazone from
steps 3 and 5 represent the amount of hydrazone contributed by the
acetonitrile.
4. The percent of the hydrazones contributed by the acetonitrile are:
Since the percent for formaldehyde and acetaldehyde arising from the
acetonitrile is less than 25% of the background level in the cartridge,
the acetonitrile is considered clean for these compounds. If the analysis
considers only these compounds, the acetonitrile is acceptable.
However, the amount of acetone arising from the acetonitrile is four
times the amount in the blank (much higher than the 25% suggested
maximum). Therefore, it is suggested that this lot of acetonitrile may
be unacceptable for the analyses.
Appendix B: Synthesizing DNPH Derivative Standards
DNPH-derivative standards are easily synthesized from DNPH supplied
by Aldrich Chemical Co. (70% DNPH and 30% water). To synthesize
98-99% pure hydrazones:
1. Prepare one liter of 2 M HCl solution: Add 172 mL concentrated
reagent-grade hydrochloric acid (HCl) to a 1 L volumetric flask.
Fill the flask to the mark with distilled deionized water.
2. Saturate the 2 M HCl solution with DNPH: Add 8 g DNPH and stir
for one hour at 20 to 25 °C. Filter through a 0.45 µm hydrophilic
membrane (HVLP) filter (Waters Part number: WAT200530).
3. Form hydrazone derivative: Add a two-fold molar excess of
reagent-grade aldehyde or ketone to the filtered 2 M HCl DNPH
solution. Stir for 30 minutes to one hour at 20 to 25 °C.
4. Filter the hydrazone slurry. Wash the hydrazone with 50 mL 2 M
HCl 3 times. Wash with 50 mL water 3 times. Dry the filter cake
in an oven at 50 to 60°C overnight.
5. Prepare standard solutions in acetonitrile at concentrations
between 1 to 10 mg hydrazone/L. The solutions are stable for
at least one month when stored in tightly-capped glass vials
at 4 °C.
Appendix C: Measuring Sample Breakthrough
Note: If several aldehydes and ketones are present in significant
concentration, estimate the maximum sample size from the total
concentration of all species.
The collection efficiency for Waters Sep-Pak DNPH-Silica cartridges is
greater than 95% for gaseous sampling rates of up to 2 L/min. The
sample may exhibit breakthrough if:
• The sampling flow rate is greater than 2 L/min
• The amount of sample collected is enough to react with more
than 50% of the DNPH (equivalent to 0.5 mg DNPH)
To measure DNPH-Silica cartridges for collection efficiency:
1. Connect two unused cartridges together by the Luer fittings and
mark each cartridge for identification.
2. Connect the cartridges to a pump with a short length of flexible
tubing.
3. Collect the sample.
4. Elute both cartridges and an unused third blank cartridge.