Environmental quality issues are extremely demanding, heterogeneous and ever expanding. Regulatory agencies around the world are constantly increasing the amount of environmental testing requirement to ensure public health and safety.Carbonyl compounds may be formed in water during ozonization and chlorination of natural organic matter. These, hazardous pollutants released from diverse sources including motor vehicles and industrial emissions, have been shown to have adverse effects on human health. EPA method 556 addresses this issue of carbonyl compounds in detail. This method applies to 15 carbonyl compounds. The compounds are derivatised using pentafluoro benzyl hydroxylamine and determined on Gas chromatograph equipped with an Electron Capture detector. This GC-ECD method enables the separation, detection and quantitation of parts per billion (ppb) concentrations of low molecular weight carbonyls in water samples, safeguarding human health and ensuring compliance with industry regulations. This method is a gas chromatographic method optimized to determine the carbonyl compounds in drinking water and raw source water. The analytes are derivatised to their corresponding penta fluorobenzyl oximes, which are extracted from water with hexane. The hexane extracts are then analyzed by GC-ECD. A PerkinElmer Elite -5 (30 meter, 0.53 mm i.d., 0.5 µm df) was used for in the method at a flow rate of 3.5 ml/min helium at constant flow mode. The oven temperature was programmed to separate the aldehyde oximes. The method is simple, fast and reproducible. The micro extraction procedure is simple and uses very small quantity of solvents which greatly reduces waste management steps and prevents pollution.

1. Environmental contaminants in
finished drinking water and raw
source water : carbonyl
compounds by EPA method 556
Dr. Padmaja Prabhu Application Specialist-
Chromatography, India.
Andrew Tipler, Chromatography R&D Manager,
Shelton, CT.
March 14, 2012
Jan 30, 2010
© 2009 PerkinElmer

2. Pure drinking water is a major CONCERN in many countries
'Super sand' to help clean up dirty drinking water, By Katia Moskvitch Technology
reporter, BBC News
 In many countries around the world, access to clean drinking water and sanitation facilities is still limited
Shale gas drilling 'contaminates drinking water‘, By Mark Kinver Science and
environment reporter, BBC News
 Shale gas drilling operations increase the risk of nearby drinking water becoming
contaminated with methane, a study has suggested.
Water rates to rise by 10% in Alderney
 Water rates in Alderney will rise by 10% to help pay for cleaner drinking water, the island's
States has agreed.
2

3. The concern………..
Emerging Substances of Concern
 Global Organic Contaminants
 Pharmaceuticals and Personal Care Products
 Endocrine Modulating Chemicals
 Nanoparticles
 Industrial Chemicals (new and recently recognized)
 Biological Metabolites and Toxins
3

4. Choice of Samples
Carbonyl compounds may be formed in water during ozonization and chlorination of natural
organic matter
Hazardous pollutants released from diverse sources including motor vehicles and industrial
emissions, have been shown to have adverse effects on human health.
EPA method 556 addresses this issue of carbonyl compounds in detail and suggests
methodologies for its determination
We chose to examine:
• Bottled drinking water
• Well water
4 Take Away Here

5. Method 556
A gas chromatographic method – ECD detector
Carbonyl compounds are derivatized to pentafluorobenzyloximes using
(pentafluorobenzylhydroxylamine) PFBHA
Oxime derivatives were extracted in hexane
 Only 4 ml of hexane was used for the extraction, Micro extraction helps in reducing pollution
and ensures easy waste management
Analysed on capillary GC with ECD
5

6. Challenge with the method
Getting low background levels of formaldehyde and acetaldehyde
 Usually present in laboratory atmosphere, water , glassware and other reagents
 Aldehyde free water can be achieved by exposure to UV or distillation from permanganate
 Avoid exposure of reagents used to laboratory atmosphere as much as possible
 Glassware can be cleaned by detergent, hot water, tap water and distilled water rinses
A good skill level in liquid-liquid extractions and derivatization procedure is needed
6

7. Carbonyl compounds in water - Analytical Method Summary…
Gas Chromatograph PerkinElmer Clarus 580
Analytical Column PerkinElmer Elite-5 (30 meter, 0.53 mm i.d., 0.5 µm df)
GC column Flow 3.5 mL/min helium in constant flow mode
GC inlet temperature 220 ºC
Split ratio 5:1
Oven temperature 50ºC hold for 1.0 min, 4 ºC/min to 220 ºC and hold for 1.0 min,
programme 20 ºC/min to 250 ºC and hold for 5.0 min, runtime is 56 minutes
Detector ECD at 375 º C
Make-up Flow 30 mL/min
Extraction procedure The analytes are derivatized at 35 ºC for 2 hours with O-
(2,3,4,5,6-pentafluorobenzyl)-hydroxylamine (PFBHA) reagent.
The derivatives are extracted in water with hexane. The extract
is finally processed through an acidic wash step.
Injection volume 1.0 µL
7

8. PerkinElmer Clarus 580
Sensitive and
selective
Collector Responds to halogen-
containing compounds
63Nickel foil (Cl, F, Br, I), as well as
other electron
capturing substances
Ideal choice for
Make-up gas in
pesticide residue
analysis
Column connection
Trace analysis
(example: PCBs)
8

9. Carbonyl compounds for EPA method 556 in drinking water and raw source water
# Analyte # Analyte
1. Formaldehyde 9. Nonanal
2. Acetaldehyde 10. Decanal
3. Propanal 11. Cyclohexanone
4. Butanal 12. Crotonaldehyde
5. Pentanal 13. Benzaldehdye
6. Hexanal 14. Glycoxal (ethanedial)
7. Heptanal 15. Methyl Glyoxal
8. Octanal
9 Take Away Here

10. Calibration
Stock Solution: All the aldehydes standards were procured from Alfa aeasar. Ten mg of each of the aldehydes were weighed in 10
ml flask and diluted to volume with acetonitrile, to make 1000 μg/mL of each of the aldehydes.
Primary dilution standard (PDS) : 2.5 mL of each of Stock solution was then mixed in a 50 mL flask and made up to volume with
acetonitrile (50 μg/mL)
Calibration spiking solution (CSS): Four mL of PDS was diluted to 10 mL with acetonitrile, to make a mixture 20 μg/mL of each of
the analyte. This CSS solution was further used to prepare calibration solutions of the analytes.
Internal standard solution : 40 mg of 1,2 dibromopropane was diluted to 100 mL with hexane. Further 0.1 mL of this was again
diluted to 100 mL with hexane to make a solution containing 0.4 μg/mL of IS. This solution was used for the extraction of the aldehyde
oximes from water.
Surrogate Standard solution : 50 mg of 2,4,5 trifluoroacetophenone was diluted to 100 mL with acetonitrile. Further 0.4 mL of this
was again diluted to 100 mL with acetonitrile to make a solution containing 2.0 μg/mL of SS. 0.2 μL of this solution was spiked in each
of the calibration and recovery levels.
PFBHA solution : Freshly prepared 15 mg/mL solution in reagent water was used for derivatizing the analytes in samples and
standard.
Cal level no. Conc.of each analyte (ppb) CSS added (µL) Final volume (mL)
1 2 2 20
2 5 5 20
3 10 10 20
4 20 20 20
5 30 30 20
6 40 40 20
10

11. Summary of calibration experiment
# Analyte Linearity r2 LOQ level LOD level
1. Formaldehyde 2-40 ng/mL 0.997 2 ng/mL 0.5 ng/mL
2. Acetaldehyde 2-40 ng/mL 0.993 2 ng/mL 0.5 ng/mL
3. Propanal 2-40 ng/mL 0.992 2 ng/mL 1.0 ng/mL
4. Butanal 2-40 ng/mL 0.993 2 ng/mL 0.5 ng/mL
5. Pentanal 2-40 ng/mL 0.993 2 ng/mL 0.5 ng/mL
6. Hexanal 2-40 ng/mL 0.996 2 ng/mL 0.5 ng/mL
7. Heptanal 2-40 ng/mL 0.993 2 ng/mL 0.5 ng/mL
8. Octanal 2-40 ng/mL 0.992 2 ng/mL 1.0 ng/mL
9. Nonanal 5-40 ng/mL 0.988 5 ng/mL 1.0 ng/mL
10. Decanal 5-40 ng/mL 0.990 5 ng/mL 0.5 ng/mL
11. Cyclohexanone 2-40 ng/mL 0.998 2 ng/mL 0.5 ng/mL
12. Benzaldehdye 2-40 ng/mL 0.998 2 ng/mL 0.5 ng/mL
13. Glyoxal (ethanedial) 5-40 ng/mL 0.994 5 ng/mL 0.5 ng/mL
14. Methyl Glyoxal 2-40 ng/mL 1.000 2 ng/mL 0.5 ng/mL
11

12. Typical calibration curve for some aldehydes……………….
formaldehyde Linearity Acetaldehyde Linearity Butanal Linearity
4000000 800000 120000024,628.723x + 12,997.276
y=
R² = 0.993
3500000 y = 80,487.570x + 137,144.662 700000y = 17,131.675x + 19,860.468 1000000
R² = 0.997 R² = 0.993
3000000 600000
800000
2500000 500000
2000000 400000 600000
1500000 300000 400000
1000000 200000
Series2
200000
500000 Series2 100000 Series2 Linear (Series2)
0 Linear (Series2) Linear (Series2) 0
0
0 10 20 30 40 50 0 10 20 30 40 50 0 10 20 30 40 50
Benzaldehyde Linearity
Pentanal Linearity Octanal Linearity
900000
800000 20,675.236x - 13,665.289
y= 700000 1400000 28,980.950x - 9,799.143
y=
R² = 0.993 y = 16,542.117x - 11,962.838 R² = 0.998
700000 600000 1200000
R² = 0.992
600000 500000 1000000
500000 400000 800000
400000 300000 600000
300000
200000 400000
200000 Series2 Series2
100000 Linear (Series2) 200000 Linear (Series2)
100000 Series2
0 Linear (Series2) 0 0
0 10 20 30 40 50 0 10 20 30 40 50 0 10 20 30 40 50
12

13. Typical chromatogram for aldehyde mixture
Elution order
1. Formaldehyde 3. Propanal 6. Hexanal 9. Nonanal 12. Benzaldehyde
2. Acetaldehyde 4. Butanal 7. Heptanal 10. Decanal 13. Glycoxal
5. Pentanal 8. Octanal 11. Cyclohexanone 14. Methyl Glyoxal
13

14. Sample preparation
Samples were collected from a local Mumbai market. Bottled water samples were
collected at random from the market. Raw water samples were collected from some
wells in the vicinity of our laboratory. These samples were extracted as per the
following extraction procedure.
20 mL of sample 200 mg Potassium One mL of freshly
in a Test tube Hydrogen Phthalate prepared PFBHA solution
Keep the tubes in water bath
at 35ºC for 2 hours, remove
and cool at RT
3 mL hexane layer + 4 mL of hexane 0.05 mL of concentrated
3 mL of 0.2N sulfuric containing IS sulphuric acid
acid shake shaken for 3 min.
1 µL injected to
GC/ECD
14

15. Results of sample analysis
Sample raw source
# Name of aldehyde Sample 1 Sample 2 Sample 3 Sample 4 Sample 5
water
Concentration in ng/mL
1. Formaldehyde 22.46 21.00 23.50 18.47 10.74 8.77
2. Acetaldehyde 30.55 42.58 86.81 52.30 1.04 4.67
3. Propanal ND ND ND ND ND ND
4. Butanal 0.80 0.81 0.64 0.72 ND 1.42
5. Pentanal 1.03 1.25 1.14 ND ND 0.98
6. Hexanal 2.17 2.08 2.40 1.65 ND 1.50
7. Cyclohexane 2.45 2.32 1.44 2.07 ND 0.69
8. Heptanal 0.66 1.08 1.16 0.54 0.17 0.90
9. Octanal 1.31 1.55 1.77 ND ND ND
10. Benzaldehyde 2.30 1.07 1.28 ND ND ND
11. Nonanal 5.58 4.96 5.51 4.39 ND 3.32
12. Decanal 12.31 9.72 8.62 3.77 0.79 4.91
13. Glyoxal 10.78 5.93 15.14 2.02 2.93 3.90
14.
Methyl Glyoxal ND 0.56 0.73 ND ND 1.19
15

16. Precision and Accuracy Results
Fortified conc. Mean Conc. found
# Name of aldehyde RSD % Mean Recovery (%)
(µg/mL) (µg/mL)
1. Formaldehyde 20.0 18.7 4.61 94
2. Acetaldehyde 20.0 19.4 9.66 97
3. Propanal 20.0 19.8 3.74 99
4. Butanal 20.0 19.7 4.55 98
5. Pentanal 20.0 19.5 4.43 98
6. Hexanal 20.0 19.8 3.29 99
7. Cyclohexane 20.0 20.5 1.46 102
8. Heptanal 20.0 19.0 9.37 95
9. Octanal 20.0 19.1 6.94 95
10. Benzaldehyde 20.0 20.1 2.70 100
11. Nonanal 20.0 19.3 3.30 97
12. Decanal 20.0 21.5 7.69 108
13. Glyoxal 20.0 20.6 3.85 103
14. Methyl Glyoxal 20.0 20.7 0.94 103
16

17. Results and Conclusion
There is a very strong need to control the aldehyde contaminants in water.
Sulfuric acid added during solvents extraction prevents extraction of reagent, which
may cause chromatographic interferences.
The method uses a micro-extraction procedure which requires very small quantities of
organic solvents, reducing the need for waste management.
Formaldehyde and acetaldehyde were seen in all the drinking water samples at varying
levels
17 Take Away Here

18. References
1. EPA method 556. Determination of carbonyl compounds in drinking water by
pentafluorobenzylhydroxylamine derivatisation and capillary gas chromatography with
electron capture detection.
18 Take Away Here

19. Thank you
Padmaja.prabhu@perkinelmer.com
19 Andrew.tipler@perkinelmer.com