Appendix B. Sampling and Analysis Methods 

Table of Contents 

1.1 Time-Integrated Air Sample Collection and Analysis ................................................2 

1.2 BTEX by Solid Phase Microextraction (SPME).........................................................4 

1.2.1 Sampling ............................................................................................................4 

1.2.2 Analysis..............................................................................................................5 

1.3 Continuous Methods....................................................................................................8 

1.4 Breath Collection and Analysis.................................................................................10 

1
1.1 Time-Integrated Air Sample Collection and Analysis 

Time-integrated monitoring methods are used for quantification of the responses of 

continuous surrogate instruments. The methods include canister sampling for VOC (BTEX, 

1,3-butadiene, MTBE), solid adsorbent sampling (for ethanol) and DNPH-coated Sep Pak 

cartridges sampling for carbonyl compounds. The DRI Organic Analytical Laboratory (OAL) 

routinely uses these methods and DRI standard operating procedures (SOPs) for sampling 

and analysis are available upon request. Five to forty minute samples are collected 

depending on the ME sampled. 

Sampling. The DRI custom built sampler used for this study can sample 

simultaneously a canister, solid adsorbent cartridges (two in parallel), and a DNPHimpregnated Sep-Pac cartridge. The sampler is compact and can be set-up in a vehicle cabin 

and run from a battery. Prior to use, the sampler is checked for cleanliness by sampling zero 

air. If the concentration of any targeted compound exceeds 0.1 ppb, the sampler is 

thoroughly cleaned and re-tested. As noted earlier, a remote switch is installed to allow 

cabin on/off control of the sampler, no longer requiring access through the van tailgate. The 

protocol requires that the van engine be off during changes of sampler media. 

 The canister sampler uses a differential pressure flow controller to supply air to the 

sampler canister and a calibrated mass flow controller to check the flow rate. Since the actual 

flow rate is less important than that the flow rate remain constant, additional quality 

assurance checks on the flow controllers is not necessary. For the 5-minute canister samples 

an additional battery-operated sampler is used that allows flows up to 3 L/minute to 

pressurize the 3 L canister. 

 Both the solid adsorbent and DNPH samplers use a common vacuum pump controlled 

by mass flow controllers. These controllers are calibrated at the start of the field program by 

using a primary flow device (e.g. Gillibrator) and then periodically checked in the field to 

confirm that the flow rates are accurate. 

Canister samples. Prior to sampling, the canisters are cleaned by repeated evacuation 

and pressurization with humidified zero air, as described in the EPA document "Technical 

Assistance Document for Sampling and Analysis of Ozone Precursors" (October 1991, 

EPA/600-8-91/215). Six sequential cycles of evacuation to ~ 0.5 torr absolute pressure, 

followed by pressurization with ultra-high-purity (UHP) humid zero air to ~ 20 psig are used. 

The differences between the DRI procedure and the EPA recommended method are that the 

canisters are heated in the DRI method to 140°C during the vacuum cycle and that more 

cycles of pressure and vacuum are used. According to our experience and that of others 

(Rasmussen, 1992), heating is essential to achieve the desired canister cleanliness. Also, the 

canisters are kept longer under vacuum cycles, about one hour in the DRI method, as 

opposed to half an hour in the EPA method. At the end of the cleaning procedure, one 

canister out of 12 in a batch is filled with humidified UHP zero air and analyzed by the gas 

chromatograph/flame ionization detection (GC/FID) method. The canisters are considered 

clean if the total non-methane organic compound (NMOC) concentration is less than 20 

ppbC. The actual concentrations of blank-check canisters are typically below 10 ppbC. 

2
Canister samples are analyzed promptly upon receipt of samples from the field, using 

GC/FID according to guidance provided by the EPA Method TO-15. The GC/FID response 

is calibrated in ppbC, using NIST Standard Reference Materials (SRM) 1805 (254 ppb of 

benzene in nitrogen). Based on the carbon response of the FID to hydrocarbons, the response 

factors determined from these calibration standards are used to convert area counts into 

concentration units (ppbC) for every peak in the chromatogram. Identification of individual 

compounds in an air sample is based on the comparison of linear retention indices (RI) with 

those RI values of authentic standard compounds. A DB-1 column (60 m long 0.32 mm i.d., 

1 μm film thickness) is used for these analyses. Breath canisters are quantified for CO2, 

MTBE, and BTEX by the method of Pleil & Lindstrom using GC/MS. 

Blank checks are performed once daily, while performance standards are executed 

three times per week. Our analysis plan and data processing standards call for the replicate 

analysis of approximately 10% of the samples. For canisters the replicate analysis is 

conducted at least 24 hours after the initial analysis to allow for re-equilibration of the 

compounds within the canister. The replicate analyses are flagged in our database and the 

programs we have for data processing extract these replicates and determine a replicate 

precision. Replicate analysis is important because it provides us with a continuous check on 

all aspects of each analysis, and indicates problems with the analysis before they become 

significant. A portion (5%) of the canisters is also analyzed by a second independent 

laboratory (Battelle-Columbus). 

Solid adsorbent samples. Ethanol is quantified from solid adsorbent cartridges as 

well as canister methods. Although MTBE is stable in SUMMA canisters and can be 

quantified with high precision and accuracy, ethanol is relatively unstable and the replicate 

analyses of canister samples show a high degree of scatter (Goliff and Zielinska, 2001). 

Thus, the solid adsorbent samples are necessary for quantification of ethanol. For sample 

collection we use multibed adsorbent cartridges consisting of Tenax-TA, Carbotrap (or 

Carboxen) and Carbosieve (Shire et al., 1996; Tsai and Weisel. 2000; Vayghani et al., 1999). 

Prior to use the Tenax-TA solid adsorbent is cleaned by Soxhlet extraction with 

hexane/acetone (4/1 v/v) overnight and dried in a vacuum oven at ~ 80 °C. The dry Tenax is 

packed into Pyrex glass tubes together with Carbotrap and Carbosieve and thermally 

conditioned for four hours in an oven at 300 °C under nitrogen purge. Approximately 10% 

of the precleaned tubes are tested by GC/FID prior to sampling. After cleaning, the tubes are 

sealed with clean Swagelok caps (brass) with graphite/Vespel ferrules, placed in metal 

containers with activated charcoal on the bottom, and kept in a clean environment at room 

temperature until use. 

After sampling at monitored flows of 200-300 ml/minute, tube samples are analyzed by 

a thermal desorption-cryogenic preconcentration method, followed by high-resolution GC/MS. 

A Chrompack Thermal Desorption-Cold Trap Injection (TCT) unit is used for sample 

desorption and cryogenic preconcentration. The compounds of interest are quantified by MS, 

using the response factors of authentic standards, prepared at five different concentrations 

with a static dilution bulb. 

Carbonyl compounds. Formaldehyde and acetaldehyde are collected with Sep-Pak 

cartridges that have been impregnated with an acidified 2,4-dinitrophenylhydrazine (DNPH) 

3
reagent (Waters, Inc), according to the EPA Method TO-11A. When ambient air is drawn 

through the cartridge at nominal flow rates of 1 L/min, carbonyls are captured by reaction 

with DNPH to form hydrazones, which are separated and quantified in the laboratory using 

HPLC (Fung and Grosjean, 1981). The ambient measurement results are subject to various 

artifacts due to sorbent interactions with ozone so ozone is removed with a honeycomb 

denuder coated with sodium carbonate/sodium nitrite/glycerol mixture (Koutrakis, et al., 

1993). After sampling, the cartridges are eluted with acetonitrile. An aliquot of the eluent is 

transferred into a 1-ml septum vial and injected by autosampler into a high performance 

liquid chromatograph (Waters Alliance System) for separation and quantization of the 

hydrazones (Fung and Grosjean 1981). 

1.2 BTEX by Solid Phase Microextraction (SPME) 

1.2.1 Sampling 

 Carboxen/(poly)dimethylsiloxane (CAR/PDMS) coated (75 μm) quartz fibers are 

used for 10-minute BTEX sampling. SPME is a passive method, thus the rate of the fiber 

uptake is controlled by the diffusion rate of the analytes to the fiber. In a stationary 

environment without air movement, a concentration gradient is formed in the boundary layer 

between the fiber and the surrounding environment. This situation occurs during fiber 

calibration in a static system, such as a Tedlar bag. However, in an outdoor environment 

there is always some air movement and the sorption rate will be higher in this situation, since 

the thickness of the boundary layer will be smaller. This phenomenon explains some of the 

differences observed between canister and SPME samples collected in summer 2002. To 

improve the agreement between these methods the Summer 2003 SPME samples were 

obtained using the experimental set up in Figure 1. The outlet of the SPME sampling bulb 

was connected to the Tenax media sampling pump. Active sampling at low constant air 

flows was a suitable solution of outdoor field sampling variability on windy days. With these 

modifications, wind speed influence is avoided and a common air stream is being sampled by 

all instruments. The SPME was exposed to the air stream at a fixed flow rate of about 300 

ml/min. The sampling bulb temperature is not controlled. 

4
Figure 1. SPME Sampling System Set Up 

1.2.2 Analysis 

All SPME BTEX samples are analyzed in a mobile laboratory with a Model 8610C SRI 

Instruments GC equipped with a heated injection port suitable for SPME desorption, a CP-Sil 

5 (Varian, Inc.) capillary column (60m, 0.32mm i.d.), and a PID detector. The heated flash 

vaporization injector is maintained at 250°C and the PID at 150°C. The column temperature 

was programmed at 60°C for 2 min, 8°C/min to 165°C (held for one minute), and then 

45°C/min to 240°C. The helium carrier gas flow rate was 3 ml/min. 

Blanks and calibration checks are performed daily on the SRI GC in order to determine the 

performance of the instrument. Fibers are kept in the injector port throughout the sample run 

to guarantee that the entire sample was desorbed and to begin the conditioning of the fiber. 

Fibers are further cleaned in a fiber conditioner at 300°C for at least one hour. One fiber per 

batch of 5 is checked for cleanliness after conditioning. The fiber conditioner consisted of a 

70 cm x 16 cm stainless steel container with 5 ports and a Watlow heater/temperature 

controller capable of maintaining temperatures in the 200-300° C range. A helium flow of 

approximately 10 ml/min is maintained during fiber conditioning. Testing showed that the 

same fiber can be reused up to 40 times absent breakage of the fiber or failure of the fiber 

mounting mechanism. 

The SRI GC is calibrated with 1 μl injections of liquid standards prepared in pentane with 

BTEX at different concentrations (1, 5, 10, 20, 40, 50, and 100 ng/μl). Carboxen/PDMS 

fibers are calibrated by introducing the SPME fiber for a defined time into a flowing standard 

gas containing the calibration component under the same flow-through conditions used for 

field sampling). The fiber is then analyzed by GC/PID. SPME gas calibrations are done 

throughout the study as necessary. BTEX calibration is performed with certified gas 

standards at different concentrations (20, 80, 100 and/or 200 ppbv for each compound). 

5
The dependence of the SPME uptake rate on the sample flow rate is shown in Figure 2. The 

response changes logarithmically and reaches a plateau region after approximately 0.038 

cm/s when changing flow has a lesser effect on the amount of extracted mass. Optimal flow 

was determined to be 0.038 cm/s. The linear velocities studied were low (0.0044-0.1126 

cm/s) enough to characterize a “quasi static” environment. The flow profile showed two 

distinctive zones in Figure 2. In the first zone (0-0.0665 cm/s) mass transfer is controlled 

by diffusion through the well-developed fiber boundary layer; in the second zone (0.0665 

cm/s and higher) transfer is controlled by diffusion through the fiber pores. 

Sampling was performed within the plateau region where the SPME response is not greatly 

affected by changes in the flow. 

Figure 2. SPME Flow Profile 

Sampling temperature and humidity are also important parameters affecting the SPME data. 

Temperature correction factors are obtained in the laboratory. The sampling bulb is 

temperature controlled with a cooling/heating system. Temperature inside the glass bulb is 

measured with a thermocouple. For introducing humidity, a heated injection port is added 

before the glass bulb entrance in order to inject water with a syringe pump to produce the 

desired relative humidity (Tuduri, et al., 2001; Nelson, 1992; Martos, P. and J. Pawliszyn, 

1997, and Lodge 1989). 

6
Chai and Pawliszyn, 1995, performed several experiments to determine the influence of 

relative humidity (RH) on the 100 μm PDMS partition coefficient (Boy-Boland, 1998). RH 

usually decreases the response factor (Kfg) of each individual compound. The decrease of 

the response is dependent on temperature. At lower temperatures humidity has a larger effect 

on the response factor. The highest effect of humidity was observed for a 0°C-10°C 

temperature range for 0% to 25% RH. Changes in the area counts at 0°C-25°C for 25%-75% 

RH were almost negligible. It is understood that humidity affects the fiber response because 

water at high concentrations competitively adsorbs into the coating of the fiber. Some 

compounds are less affected by humidity than others. 

The 75 μm Carboxen/PDMS fiber showed no effect with RH at 100 ppbv of BTEX 

concentration. Experiments at 25°C and 12.5°C determined that RH does not affect the fiber 

uptake of BTEX compounds significantly. Figure 3 shows toluene response at 100 ppbv for 

different RH. All the other BTEX species behaved similarly. Data for BTEX showed a 

standard deviation between 5 to 13% for the range of humidity analyzed, 10% to 80% RH. 

Sensors are accurate as long as there is no condensation of water, which may happen above 

85% RH). These differences in the response of the fiber are within the precision of the 

method. Low RH effects on the fiber sensitivity may be due to the low concentration ranges 

used for calibration. At low BTEX concentrations, water is probably not an important 

competitive molecule. More research in this area is suggested. 

SPME Carboxen/PDMS coated (75 μm) fibers are used with portable or manual samplers. 

When manual samplers are used, the tip of the needle is closed with a septum or Teflon tape 

(Martos and Pawliszyn, 1997 and Chai and Pawliszyn, 1995). Prior to sampling, the precleaned fibers are kept at ambient temperature within an activated charcoal protector. This 

storage method has been tested in the laboratory and BTEX backgrounds are stable up to 48 

hours in storage following cleaning (the longest tested period). After sampling, fibers are 

kept in sealed Mylar bags inside a cooler with dry ice. Samples are analyzed 4-10 hours after 

sampling, on average, with a minimum of 1 hour and a maximum of 20 hours between 

sampling and analysis. 

7
0

5000

10000

15000

20000

25000

0 10 20 30 40 50 60 70 80 90

Relative Humidity (%)

Area Counts

Toluene 25C Toluene 12.5C Avg 25C Avg 12.5C

Figure 3. Humidity Effect in GC/PID response for a 100 ppbv BTEX standard at 25oC and 

12.5oC (with a standard deviation of 5% for the 12.5 °C and 8% for 25 °C from the respective 

mean) 

1.3 Continuous Methods 

The continuous Kore MS200 is used to monitor BTEX on a one-minute basis, sampling 

during the initial 10 second portion of each 60 second period. This instrument uses a time-offlight mass spectrometer to separate compounds of interest. Rather than using a gas 

chromatograph, this instrument uses a software solution and a library of 70 eV electron 

ionization fragmentation patterns to apportion the contribution of each parent species to the 

time-of-flight (TOF) mass spectrum recorded by the instrument. The software program does 

not distinguish between specific isomers so xylenes and ethylbenzene are reported together. 

The inlet uses a polydimethylsiloxane membrane preferentially to allow non-polar organic 

compounds into the analyzer while impeding polar species and air components to maintain 

the vacuum inside the analyzer chamber. The instrument performed well during the pilot 

studies and early field work, comparing favorably with the canister measurements for most 

samples, although it became increasingly unreliable in subsequent field studies. Given the 

corroborative role proposed for this device, we do not believe the information provided by 

this instrument, while desirable, is necessary to fulfill the goals of the screening study. 

Carbon monoxide is monitored continuously by the Langan T15 electrochemical 

monitor for CO and by a nondispersive infrared (NDIR) analyzer. The response time of 

Langan T15 instrument is slower than the response of the infrared-based instruments. Prior to 

each period of field measurement the instrument is calibrated using a zero-air generator and 

8
span gas to provide two reference points encompassing the expected range of concentrations 

anticipated during actual testing. The two-point calibration procedure is as follows: 

1. Allow instrument to stabilize for a minimum of 15 minutes. 

2. Record ambient concentration as determined by the instrument. 

3. Connect inlet lines from the instrument to a zero-air source (for a passive sampler 

use the flooder cap provided by manufacturer) and check for a flow rate of >1 

lpm with rotometer. 

4. Let instrument stabilize, record current baseline, then adjust zero. 

5. Connect inlet lines to a tank of span gas with an appropriate CO concentration for 

anticipated range and verify flow rate 

6. Let instrument stabilize, record current reading, and adjust span to correct value. 

7. Re-connect instrument to zero-air source, let stabilize and check baseline zero 

reading. 

8. Repeat steps 4-7 if necessary. 

9. Check a third concentration level with span gas if available. 

During field measurements the passive sampler is checked against the reference unit, which 

has automatic baseline stabilization and an internal zero-air source. Baseline readings will be 

recorded at the beginning and end of each sampling day. If significant deviations are 

observed a re-calibration will be performed. 

RAE Systems Model PGM-7240 (ppbRAE) portable PID monitors are used to continuously 

monitor ambient VOC levels in high end microenvironments. The monitor is sensitive to 

organic and inorganic gases that have an ionization potential of less than 10.6 eV, which 

includes most compounds of interest in this study. It does not respond to light hydrocarbons 

such as methane, ethane, propane, and acetyleneor to CO or formaldehyde. 

A Continuous Formaldehyde Monitor was purchased from Alpha-Omega Power Technology, 

Ltd. (Albuquerque, NM). The Alpha-Omega (AO) a wet instrument that utilizes the Hantzch 

reaction, absorbing formaldehyde in acidified water, reacting it with 2,4-pentanedione and 

ammonia to form a cyclized product, 3,5-diacetyl-1, and 4-dihydrolutidene, which is 

continuously detected by fluorescence. The method is sensitive and highly specific for 

formaldehyde. 

9
1.4 Breath Collection and Analysis 

Breath is collected from technicians who participate in a scripted exposure. Since this study 

requires the use of human subjects, the final protocol was reviewed by an Institutional 

Review Board that is certified with the National Institute of Health. 

Technicians were instructed to avoid exposure to materials that may compromise the 

exposure assessment prior to and during the scripted exposures. This includes avoiding 

alcohol ingestion (ethanol) and cigarette smoke for at least 3 days prior to the scripted 

exposures. Breath samples collected before the exposure were used to assess background 

levels in the technician. 

The test subject takes the three breath samples. One sample is taken before initiating ME 

sampling for background purposes; the second sample, ten seconds (timed with a stopwatch) 

immediately after completing an active refueling task or experiencing a measured peak 

exposure in another ME, and the third sample 1 minute immediately following the second 

sample. The 3rd backup sample is analyzed in case there is a problem with the second 

sample. The technician takes a 1-liter canister, places the tube in his mouth, and breathes 

smoothly and regularly through the mouth around the tube until a resting breath pattern is 

established. At the end of a normal exhalation, the technician closes the mouth, opens the 

canister valve, and continues to expel the expiratory reserve. The canister vacuum will 

collect 1 L of the expiratory reserve. When the flow stops, the technician closes the canister 

valve to capture the sample (Pleil and Lindstrom, 1995; 2002). Breath samples are collected 

in 1 L silico-steel steel canisters (Restek, Inc). . 

Gas Chromatography-Mass Spectrometry. The analysis of VOC in breath samples is 

conducted as described by Pleil and Lindstrom (1995, 2002). Prior to the commencement of 

sampling, the CO2 level in alveolar breath of all technician subjects is measured using 

GC/FID. Since the FID does not respond to CO2, this species is converted to methane by a 

methanator, positioned after the GC column, but ahead of the FID. Three breath samples are 

collected and analyzed, and the mean CO2 value is used for further comparison. After breath 

sample collection, the CO2 is measured using the same method. The target VOC (MTBE, 

1,3-butadiene, ethanol, BTEX) is measured using a GC/MS technique. The GC/MS system 

includes an Entech 7100 preconcentrator, a Varian 3800 gas chromatograph with FID and 

column switching valve, and a Varian Saturn 2000 ion trap mass spectrometer. 

10
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th

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