Enhancing Haloacetic Acid Detection: The Role of Nitrogen Evaporation in Alternative Sample Preparation Methods

HAAs are chemical compounds consisting of an acetic acid backbone with one or more halogen atoms (chlorine, bromine, or iodine) attached to the methyl carbon. The U.S. Environmental Protection Agency (EPA) currently regulates five haloacetic acids, collectively known as HAA5: monochloroacetic acid (MCAA), dichloroacetic acid (DCAA), trichloroacetic acid (TCAA), monobromoacetic acid (MBAA), and dibromoacetic acid (DBAA). The maximum contaminant level (MCL) for total HAA5 is set at 0.06 mg/L (60 μg/L) in the United States, European Union, and Scotland.

Four additional haloacetic acids—bromochloroacetic acid (BCA), bromodichloroacetic acid (BDCA), dibromochloroacetic acid (DBCA), and tribromoacetic acid (TBA)—have been monitored under the Unregulated Contaminant Monitoring Rule, bringing the total to nine compounds often referred to as HAA9. There is increasing regulatory interest in monitoring all nine compounds due to health concerns, particularly regarding the more toxic brominated species which show enhanced toxicity relative to their chlorinated analogues. The World Health Organization recommends monitoring of all nine haloacetic acids, and this is likely to become more relevant as pressure on water resources results in the need to use lower quality source waters or to increase the use of desalinated water.

Long-term exposure to HAAs above the MCL has been associated with increased cancer risk, with several epidemiological studies of exposure to chlorinated water finding an association with increased risk of urinary bladder cancer. The National Toxicology Program (NTP) concluded that six HAAs are reasonably anticipated to be human carcinogens: bromochloroacetic acid (BCA), bromodichloroacetic acid (BDCA), dibromochloroacetic acid (DBCA), dibromoacetic acid (DBA), dichloroacetic acid (DCA), and tribromoacetic acid (TBA).

Animal studies have demonstrated that HAAs can cause liver and lung tumors in mice of both sexes and male rats, mesothelioma in rats, mammary gland tumors in female rats, and various other tumor types depending on the specific HAA compound. Beyond cancer, animal studies have also shown reproductive issues including birth defects and spontaneous abortions, and harmful effects on fetal growth and development.

BCA, BDCA, DBA, and DCA are listed as reasonably anticipated human carcinogens based on sufficient evidence of carcinogenicity from studies in experimental animals, while DBCA and TBA are listed based on convincing information indicating they would likely cause cancer in humans, including studies showing these HAAs are metabolized in the body to known rodent carcinogens. The EPA and other health agencies have established individual maximum contaminant level goals at 0.0 mg/L for dichloroacetic acid and 0.30 mg/L for trichloroacetic acid.

HAA formation depends on several critical water quality parameters and disinfection conditions. Water temperature significantly influences HAA production, with higher concentrations forming during warmer months as reaction kinetics increase. The concentration and character of natural organic matter in the source water—expressed through parameters including color, chemical oxygen demand (COD), total organic carbon (TOC), and UV254 absorbance—directly correlates with HAA formation potential. Phenolic compounds and aromatic structures within natural organic matter are particularly reactive precursors, with chlorinated phenols showing high HAA formation potentials exceeding 400 μg/mgC.

The type of disinfectant also plays a crucial role: chlorine produces the highest amount of HAAs, while chlorine dioxide alone generates the smallest amounts, with mixtures of chlorine and chlorine dioxide giving intermediate results. Bromide concentration in source water is perhaps the most critical factor affecting HAA speciation—even low bromide concentrations of 0.1 mg/L can result in mixed bromochlorinated HAAs constituting at least 10% of total HAA concentration. As bromide levels increase, brominated and mixed halogenated HAA species become dominant and contribute a high percentage of total organic halogen formation.

EPA Method 552.3: The GC-ECD Approach

EPA Method 552.3 represents the traditional approach to HAA analysis, utilizing gas chromatography with electron capture detection (GC-ECD) following liquid-liquid microextraction and derivatization. This method targets nine HAAs and dalapon but requires extensive sample preparation that can exceed four hours.

The method involves multiple labor-intensive steps performed on a 40-mL water sample: pH adjustment to 0.5 or less with concentrated sulfuric acid, addition of approximately 18 grams of pesticide-grade anhydrous sodium sulfate, extraction with 4 mL of methyl tert-butyl ether (MTBE) or tert-amyl methyl ether (TAME) containing internal standard, derivatization by adding 3 mL of 10% sulfuric acid in methanol followed by heating for 2 hours at 50°C (for MTBE) or 60°C (for TAME), phase separation by adding 7 mL of sodium sulfate solution, and neutralization with saturated sodium bicarbonate solution before GC analysis. The derivatization converts HAAs into their methyl ester forms to enable volatilization and gas chromatographic separation.

While Method 552.3 has been widely used for decades and established the foundation for HAA monitoring, it presents several challenges including lengthy preparation time exceeding four hours per batch, multiple extraction and derivatization steps increasing potential for analytical errors, difficulty achieving acceptable recoveries for all analytes particularly the brominated tri-halogenated species, and sensitivity to interferents such as phthalates from plastic materials and low-level contaminants in sodium sulfate.

EPA Method 557: Direct Injection IC-MS/MS

EPA Method 557 represents a modern alternative that uses ion chromatography coupled with tandem mass spectrometry (IC-MS/MS) for HAA analysis. This method employs direct injection of water samples with negative-ion electrospray ionization (ESI), eliminating sample preparation entirely.

The direct injection approach offers significant advantages over Method 552.3: no derivatization or extraction required, resulting in dramatically reduced sample preparation time; excellent sensitivity with detection limits ranging from 0.02 to 0.11 μg/L depending on the specific HAA compound; improved recoveries consistently exceeding 90% across all nine HAAs; ability to handle high ionic strength samples containing up to 320 mg/L chloride, 250 mg/L sulfate, 150 mg/L bicarbonate, and 20 mg/L nitrate without sample pretreatment; and simultaneous analysis of nine HAAs plus bromate and dalapon in a single 7.5- to 10-minute chromatographic run.

For laboratories processing more than 24 batches of 25 samples per month, Method 557 can provide substantial cost savings compared to Method 552.3 due to reduced labor, faster turnaround time, and elimination of consumables associated with liquid-liquid extraction and derivatization. Real-world comparisons of the two methods using actual drinking water samples have demonstrated that the LC-MS/MS approach is significantly quicker and easier while showing improved performance in terms of accuracy and precision, particularly for the more toxic brominated compounds.

Alternative Sample Preparation: Solid Phase Extraction with Nitrogen Evaporation

While EPA Method 557's direct injection approach eliminates sample preparation for most drinking water applications, certain analytical scenarios benefit from sample concentration and cleanup using solid phase extraction (SPE) coupled with nitrogen evaporation. These scenarios include samples with very low HAA concentrations requiring improved detection limits below the direct injection capability; complex matrices with high levels of interfering compounds that exceed Method 557's capacity; non-potable water samples from environmental monitoring or industrial processes; research applications exploring HAA formation mechanisms and kinetics; and method development for emerging HAA species not covered by standard EPA methods.

SPE Methodology for HAA Extraction

Solid phase extraction provides targeted isolation and concentration of HAAs from water samples, enabling detection at ultra-trace levels. Recent research has demonstrated successful SPE methods for HAA analysis using polymeric sorbents based on styrene-divinylbenzene copolymers that offer broad analyte retention, good recoveries across all nine HAAs, and compatibility with LC-MS analysis.

A validated SPE approach for HAAs utilizes polymeric cartridges (such as HR-P or similar non-polar polymeric phases) with the following optimized protocol: cartridge conditioning with 5 mL methanol followed by 3 mL acidified water adjusted to pH 2.5 with sulfuric acid, delivered at 1 mL/min flow rate; sample loading of 50-100 mL acidified water sample at 5 mL/min without allowing the cartridge to dry, which would cause irreversible loss of retention; brief washing with 1 mL water at pH 2.5 to remove residual matrix components; and elution with 2-4 mL of organic solvent optimized for HAA recovery.

Alternative elution approaches have been systematically evaluated for HAA extraction. One comprehensive study found that pure acetonitrile as an elution solvent achieved good recoveries for all nine HAAs with better chromatographic peak shapes than mobile phase mixtures or methanol-based eluents. The optimal elution volume was determined to be 2.0 mL acetonitrile, providing a balance between complete analyte recovery (>85% for all HAAs) and a concentrated extract suitable for subsequent nitrogen evaporation and analysis. Other researchers have successfully employed methanol-acetone mixtures (1:1, v/v) totaling 4 mL as the elution solvent, with slightly higher total volume compensated by higher individual HAA concentrations in the eluate.

The SPE approach achieves remarkable preconcentration factors. Starting with a 50 mL water sample and eluting into 2 mL of organic solvent provides a 25-fold concentration before considering any subsequent evaporation steps. This dramatic improvement in sensitivity reduces limits of quantification from 10-500 μg/L achievable by direct aqueous injection to 0.08-2.0 μg/L after SPE extraction. For research applications or monitoring programs targeting ultra-trace HAA levels, this sensitivity enhancement makes SPE an invaluable tool despite the additional sample preparation time compared to direct injection methods.

The Critical Role of Nitrogen Evaporation

Following SPE elution, the organic solvent extract must be concentrated or exchanged into a more suitable solvent for the analytical instrument. This is where nitrogen evaporation becomes an essential technique in alternative HAA sample preparation workflows, bridging the gap between SPE extraction and final instrumental analysis.

Nitrogen blowdown evaporation removes excess solvent by applying a steady stream of high-purity nitrogen gas just above the sample surface, lowering vapor pressure above the liquid and allowing rapid yet controlled evaporation while preventing thermal degradation and oxidation of analytes. The process works by continuously pushing away vapor-saturated air immediately above the sample, preventing solvent molecules from returning to the liquid phase and thereby accelerating evaporation rates by factors of 10 or more compared to passive evaporation. Gentle heating in a temperature-controlled water bath further enhances evaporation kinetics while maintaining conditions well below solvent boiling points to ensure analyte stability.

For HAA analysis following SPE, the typical nitrogen evaporation protocol involves several carefully controlled steps: transfer of the 2-4 mL SPE eluate (acetonitrile, methanol-acetone, or other organic solvent) to clean evaporation tubes made of borosilicate glass; application of gentle nitrogen flow at 10-15 psi to create visible swirling in the tubes without causing splashing or cross-contamination between adjacent samples; use of a heated water bath set to 35-50°C, a few degrees below the solvent boiling point to optimize evaporation kinetics while protecting thermally labile compounds; evaporation until only a small volume remains (for mixed water-organic eluates) or to near-dryness for pure organic solvents; and immediate reconstitution in 100-500 μL of LC-MS compatible mobile phase, aqueous buffer, or formate solution optimized for the specific analytical method.

The nitrogen evaporation step is critical for multiple reasons that directly impact analytical performance and data quality. First, it concentrates analytes from the relatively large SPE elution volume (2-4 mL) into a much smaller volume suitable for microvolume LC-MS injection (typically 100-500 μL), achieving additional concentration factors of 4 to 40-fold beyond the SPE preconcentration alone. This additive concentration effect means that combining 25-fold SPE preconcentration with 10-fold nitrogen evaporation provides an overall 250-fold concentration enhancement, pushing detection capabilities well into the sub-μg/L range.

Second, nitrogen evaporation enables essential solvent exchange from SPE elution solvents (methanol, acetone, acetonitrile) to LC-MS compatible solvents such as aqueous buffers, formate solutions, or mobile phase mixtures that provide optimal chromatographic retention and peak shape for the highly polar HAA analytes. This solvent exchange is particularly critical for reversed-phase LC-MS methods where injection of pure organic solvents would cause severe peak distortion and loss of retention for early-eluting polar compounds like monochloroacetic acid.

Third, controlled evaporation under a gentle stream of high-purity nitrogen (≥99.999%) prevents oxidation and degradation of HAAs, which are reactive compounds containing labile halogen-carbon bonds. The inert nitrogen atmosphere displaces ambient oxygen and moisture that could promote hydrolysis, oxidation, or other degradation pathways, ensuring that the analytes measured in the final extract accurately represent those present in the original sample.

Fourth, modern nitrogen evaporation systems handle multiple samples simultaneously in configurations ranging from 6 to 96 positions, maintaining laboratory efficiency and enabling batch processing that matches the throughput of automated LC-MS systems. This parallel processing capability is essential for environmental monitoring programs or research studies involving large sample sets where individual sequential evaporation would create unacceptable bottlenecks.

Integration with LC-MS Analysis

After nitrogen evaporation and reconstitution in LC-MS compatible solvent, the concentrated HAA extract is ready for liquid chromatography-mass spectrometry analysis. Modern reversed-phase LC-MS/MS methods using C18 or other hydrophobic stationary phases with gradient elution can baseline-separate all nine HAAs in 7.5-10 minutes with detection limits generally between 0.003 and 0.99 μg/L depending on the specific compound and matrix, far exceeding regulatory requirements and enabling ultra-trace monitoring.

Hydrophilic interaction liquid chromatography (HILIC) coupled with MS/MS represents an alternative separation mode that has been successfully employed for HAA analysis, offering excellent retention of these highly polar analytes without the need for ion-pairing reagents that can suppress ionization efficiency. HILIC methods typically use acetonitrile-rich mobile phases with small amounts of aqueous buffer, making them particularly well-suited for samples reconstituted after nitrogen evaporation in acetonitrile or other organic solvents.

The combination of SPE preconcentration (25-50-fold), nitrogen evaporation for additional solvent reduction and exchange (4-40-fold), and sensitive LC-MS/MS detection creates a powerful analytical workflow capable of detecting HAAs at 0.01-0.1 μg/L levels in complex matrices—sensitivity levels 100 to 1000-fold better than direct injection approaches. This three-component workflow (SPE + nitrogen evaporation + LC-MS/MS) is particularly valuable for research investigating HAA formation mechanisms in bench-scale chlorination studies, evaluating effectiveness of alternative water treatment methods for DBP mitigation, analyzing non-potable water samples from surface waters or industrial processes with complex matrices, conducting epidemiological studies requiring accurate exposure assessment at low concentrations, and validating new analytical methodologies for emerging halogenated DBPs beyond the regulated HAA9.

While EPA Method 557's direct injection IC-MS/MS approach has revolutionized routine HAA monitoring in drinking water by eliminating sample preparation entirely and providing excellent sensitivity and throughput, solid phase extraction coupled with nitrogen evaporation remains a powerful and essential alternative sample preparation strategy for specialized applications that extend beyond routine compliance monitoring.

This combination of techniques enables ultra-sensitive detection reaching sub-μg/L and even ng/L concentration ranges; effective cleanup of complex matrices containing interferents, high dissolved solids, or unusual chemical backgrounds; flexibility in method development for emerging analytical challenges including novel DBPs and alternative water matrices; research applications investigating HAA formation mechanisms, treatment effectiveness, and exposure assessment; and validation of new analytical methodologies that will become tomorrow's standard methods.

Nitrogen evaporation serves as the critical bridge between SPE extraction and instrumental analysis, providing controlled concentration and solvent exchange that maximizes sensitivity while protecting analyte integrity through inert atmosphere processing. Modern nitrogen blowdown systems offer the automation, reproducibility, temperature control precision, and multi-sample processing efficiency needed to generate high-quality analytical results that meet or exceed the most stringent data quality objectives while maintaining acceptable sample throughput for research and monitoring programs.

For laboratories conducting HAA research, developing new analytical methods, analyzing challenging water matrices, pursuing ultra-trace detection, or preparing for future regulatory requirements that may expand monitoring to additional DBP species, the integration of SPE with advanced nitrogen evaporation technology represents a proven and robust approach that combines traditional sample preparation principles with modern analytical capabilities and green chemistry principles.

As our understanding of disinfection byproducts continues to grow through toxicological research and environmental monitoring, as regulatory requirements become more stringent with expanded compound lists and lower action levels, and as water quality challenges intensify due to source water degradation and increased water reuse, these flexible and powerful sample preparation techniques will remain essential tools in the analytical chemist's arsenal for protecting public health through accurate and reliable water quality assessment.