ANALYSES OF FAT, OIL AND GREASE IN WASTEWATER OR RECEIVING WATER USING FOURIER TRANSFORM INFRA-RED SPECTROSCOPY (FTIR) Philip

A. Russell Littleton/Englewood Wastewater Treatment Plant 2900 S. Platte River Drive Englewood, Colorado 80110 ABSTRACT The insoluble component of wastewater consisting of a mixture of various natural and petroleum organic oils and fats, typically referred to as Fats, Oils and Grease (FOG), interferes with facility processes and becomes a regulatory issue when it enters a receiving stream. Typically, the concentration of FOG in influent wastewater and effluent is estimated gravimetrically by extracting 1 liter of water sample with an organic solvent followed by the subsequent evaporation of all of the solvent. The mass of the remaining residue is measured gravimetrically and corrections are applied to produce a concentration estimate; no other information about the nature of the FOG is provided by this method. Gravimetric protocols also result in (1) the release of significant amounts of organic solvent to the atmosphere and (2) a relatively inaccurate mass estimate because of the error introduced by measuring a small mass change in a relatively massive container. A method was developed by our laboratory that utilizes Fourier Transform Infra Red (FTIR) spectroscopy to analyze FOG in water/wastewater samples. Advantages of this procedure include: 1. The ability to quantify a wide range and low levels of FOG (400 to <1.0 mg/l) 2. Reducing analytical sample size to <5 ml organic solvent 3. The ability to characterize the chemical nature of the sample extracted from wastewater and water samples. For quantifying FOG in influent and effluent samples, FTIR provides a method that is more sensitive and/or precise than gravimetric methods. FTIR also permits the identification of FOG components, including determining whether an observed ambient slick is produced by FOG from a wastewater facility or another source. KEYWORDS Fat, oil, grease, FOG, FTIR, IR, infrared spectroscopy, analyses

INTRODUCTION Infrared (IR) Spectroscopy is not an analytical method commonly associated with water analyses. At least, in part, that is because water strongly absorbs IR radiation and effectively masks IR absorption bands. IR spectroscopy is, however, a powerful tool for quantifying organic chemicals and identifying the chemical structure of the molecules being investigated. By using an appropriate solvent it is possible to extract organic chemicals from water and take advantage of the analytical advantages of IR spectroscopy. We used a combination of organic chemical solvent extraction and FTIR spectroscopy to quantify and reveal the chemical nature of the class of organic chemical broadly referred to as Fats, Oils and Grease (FOG) present in municipal wastewater. FTIR Fourier Transform Infrared Spectroscopy (FTIR) is an optical interference method for collecting a complete infrared spectrum in real time. The method can be used in both transmitted and reflective modes. IR energy or frequency is usually expressed as a wavenumber, W (cm-1), which is the reciprocal of the wavelength (1/). The region of interest for IR organic analyses is between ~4000 and 400 cm-1 (commonly referred to as the Mid Infrared range). The reason that infrared is such a good tool to gain information on organic chemicals is that their chemical bonds really rock and roll when stimulated by IR radiation; specifically the atomic bonds vibrate when stimulated by IR and predictably resonate at frequencies related to each bonds energy. Different bond groups resonate at specific frequencies. The frequency spectra of IR radiation will exhibit absorption of radiation at specific wavelengths depending upon the chemical structure of the chemical. Figure 1a illustrates a typical absorption spectra showing wavelength versus percent transmittance; the relatively strong regions of absorption are produced by the resonance of functional groups within the sample. Figure 1b is the same spectra displayed using the more commonly accepted wavenumber versus absorption convention. A good reference on IR principles and analytical theory can be found in Smith 1996. The spectra in the ~2900 wavenumber region are created by C-H stretching vibrations and are routinely used for quantitative analyses. The spectra in the ~1600 to 500 wave number fingerprint region are created by other chemical bonds including C-H bending vibrations, branched alkanes, double carbon bonds (alkenes), triple carbon bonds (alkynes), aromatic structures, C-O bonds (alcohols and phenols), C-O-C bonds (ethers), C=O bonds (carbonyls, ketones, aldehydes, carboxylic acids, esters), nitrogen compounds (amides, imides, nitriles), organic compounds containing sulfur, silicon or halogens, and some inorganic compounds. A good reference on IR spectral interpretation can be found in Smith 1999.

Figure 1a. An example of a raw IR spectra (Octane) showing a spectra using wavelength (cm-1) versus percent transmittance.

Figure 1b. An example of an IR spectra (Octane) showing IR absorption using wavenumber versus absorption.

FOG Fats, Oils and Grease (FOG) is a method defined protocol. FOG is typically that component in wastewater (or environmental waters) which can be extracted using a hydrophobic solvent. They are a group of chemicals that because of their hydrophobic nature and ability to collect debris, can cause severe problems in sewer systems and wastewater treatment plants. FOG may be composed of natural oils and fats, and petroleum based products. Typically the major component observed in wastewaters will be natural oils and fats which are of animal or plant origin. These lipids are based on the triglyceride (triacylglycerol) chemical structure which consists of a glycerol molecule where each hydroxyl group has been esterfied with a fatty acid. The resulting structure looks like figure 2 where each R represents a fatty acid. It is the nature of the fatty acid components that cause the characteristics of FOG; in general, the more carbon atoms in the tail of the fatty acid and the more prone to polymerization, the more likely the resultant molecule is classified as a fat or grease versus an oil. Fatty acids typically found in natural food oils will have the following structures: CH3 -(CH2)14-COOH CH3 -(CH2)16-COOH CH3 -(CH2)7-CH=CH-(CH2)7-COOH CH3 -(CH2)3-(CH2-CH=CH)2-(CH2)7-COOH Palmitic acid Stearic acid Oleic acid Linoleic acid

It should be recognized that a FOG sample collected in the field will rarely be a pure chemical; even a purified natural oil will be a mix of glycerols of similar but different atomic masses, electrical charges and saturation (number of double bonds). Compared to most petroleum products, triglycerides are polar compounds and therefore capable of interaction with ions in solution in water. Figure 2. Chemical structure of a triglyceride.

METHODOLOGY Instrumentation The instrument used to quantify and qualify samples for this study is a Nicolet Impact 410 FTIR spectrometer equipped to handle both samples appropriate for transmitted and reflected IR radiation. The instruments spectral range is ~5000 to 400 W. The instrument is equipped to examine a sample by direct transmittance using a 1 cm path length quartz cell or IR transparent thin film and by reflection using Horizontal Attenuated Total Reflection (HATR). A schematic view of a transmission setup is shown if figure 3a; the actual instrument configuration is shown in figure 3b. The HATR sample holder permits the analysis of small samples of less than 50ul of extract solution; multiple evaporations can be used to concentrate a dilute sample. Because the HATR sample holder uses a zinc-selenium crystal, the lowest W that can be observed is ~650. A schematic view of the transmission setup is shown if figure 4a, the actual instrument configuration is shown in figure 4b. The program used to collect, display and assist in interpretation of IR spectra is Nicolets Omnic software. Quantitative data is exported to Statsoft Statistica for analytical comparative analyses and display. Sample preparation All samples for FTIR analyses, except qualitative and quantitative standards, were prepared using EPAs Oil and Grease method 1664 (USEPA 1999) sample extraction procedure. Water samples were prepared from three extractions from 1000 ml of water using a total of ~100ml Freon 113 instead of n-hexane. Extracted samples were centrifuged to improve separation of the hydrophobic component from the water phase. Freon was recycled by distillation reducing analytical cost and air pollution, and produced a solvent of higher quality than the original product. The amount of solvent required for quantitative analyses was less than 5 ml and could be recycled; the amount of solvent required for HATR consisted of from 40 200ul. Extracted samples were stored in 125ml glass containers with Teflon caps; repeat temporal analyses demonstrated that samples could be stored at room temperatures for over a year without notable changes in quantity or chemical composition of the extracted samples.

Figure 3a. A schematic representation of a transmission IR setup.

Figure 3b. The instrument configuration for transmission IR spectra collection.

Figure 4a. A schematic representation of a HATR setup.

Figure 4b. The instrument configuration for a HATR IR spectra collection; the yellow strip in the center of the sample holder is the ZnSe crystal.

Quantitative analysis Samples were analyzed using USEPA method 413.2 extraction procedure and USEPA 1664s standard solution of 50%stearic acid and 50% hexadecane for calibration purposes. A series of dilutions were used to create a calibration curve using the C-H2 asymmetric stretching vibration absorption peak at 2930 W; an example is shown in figure 5. Standard solutions made from natural oils and petroleum products were also prepared and compared with the 1664 standard. Sample concentrations were calculated as follows C = (ax (K) + b) * (S * 0.1) where C K S 0.1 Concentration of FOG in sample Correction for O&G standard (1.0 for USEPA 1664 standard) Correction for actual sample size in ml Correction for extraction magnification (1000ml HOH 100ml solvent)

For quantitative analysis, only a portion of the total spectra from 3500 to 2500 W was collected and stored for subsequent mass estimate using standard table values. Figure 5. A set of transmittance spectra showing spectral results from EPA method 1664 standard calibration.

Qualitative analyses For qualitative analysis of the IR fingerprint, guidance was used from Smith 1996, Smith 1993 and the manufacturers HATR user manual (Nicolet __). The extract sample used for quantitative analyses is suitable for HATR analysis and requires only ~40 200 ul of extract solution. Standard samples were typical applied from a diluted solution. The use of Freon 113 facilitated very rapid sample preparation because of rapid, complete evaporation as verified by time series IR analyses. For fingerprint comparison, the total spectra from 3500 to 600 W was collected and stored for reference and display. The FTIR spectrometer was set to collect and average 32 complete spectra at a resolution of 4 W and ATR correction; total acquisition time per sample was 31 seconds. Typical fingerprint spectra are shown in figures 6, 7, 8 and 9. A sample of a simple linear organic chemical, hexadecane, is presented in figure 6; the absorption peaks are produced by C-H3 and C-H2 bond vibrations. A sample of a fatty acid, stearic acid, is presented in figure 7; the absorption peaks are produced by C-H3, C-H2 and C=O bond vibrations. A sample of a glycerol is presented in figure 8; the absorption peaks are produced by C-H3, C-H2 and strong, broad O-H bond vibrations. A sample of a natural oil, canola, is presented in figure 9; the absorption peaks are produced by C-H3, C-H2 and carbonyl/ester bond vibrations. Figure 6. IR spectra a sample of hexadecane.

Figure 7. IR spectra a sample of stearic acid.

Figure 8. IR spectra a sample of glycerol.

Figure 9. IR spectra a sample of olive oil.

RESULTS An intensive study of FOG was conducted at the two influents and combined effluent at the Littleton/Englewood Wastewater Treatment Plant (LEWWTP), Englewood, Colorado, from 1996 through 2000. FOG was analyzed again in 2003 with a possible indication of a positive Pretreatment Program impact on one influent wastewater stream. Quantitative analysis of effluent and influent samples The results of analyses are presented graphically in figure 10; trends have been shown using a least square regression curve fit. Note that there are two scales in the graph to differentiate effluent and influent results. Table 1 summarizes data from all FOG quantitative measurements. Effluent samples were not collected in 2003.

Figure 10. Influent and effluent FOG values from 1996 through 2000 expressed in EPA 1664 equivalent values.

Table 1. Summary of LEWWTP FOG data derived from FTIR analyses form 1996 through 2000, and 2003. Sample Location Englewood Influent Littleton Influent Effluent
1 2

FOG (mg/L)1 1996-2000 33.6 9.9 27.6 7.5 0.62 0.51

FOG (mg/L) 1 2003 25.2 4.8 33.9 18.8 28.8 2.82 NA

EPA 1664 standard equivalents Without outlier value of 86.6

Qualitative analyses of effluent and influent samples The results of selected influent and effluent sample fingerprint IR HATR spectra are presented graphically in figures 11 and 12. The spectra show a basic consistency; when compared with the spectra of natural oils, it is obvious that the composition of the collected FOG samples closely match those of natural triglycerides as shown in Figures 13 and 14. Some differences are possibly present but because of the nature of triglycerides, problems with mixtures of different animal and vegetable oils, and possible chemical reactions in the collection or treatment systems it would be very difficult to tease out subtle chemical information. The results also indicate that most, if not all, the FOG components entering the facility are natural oils and fats versus petroleum based products. Figure 11. Selected fingerprint IR region IR spectra from influent samples.

Figure 12. Selected fingerprint IR region IR spectra from effluent samples.

Figure 13. Selected fingerprint IR region IR spectra from influent samples and a comparative triglyceride samples.

Figure 14. Selected fingerprint IR region IR spectra from effluent samples and comparative triglyceride samples.

Special studies In addition to studies of the nature of influent and effluent samples, other studies were conducted to examine (1) calibration process and standards, (2) the nature of FOG moving through a WWTP, (3) the difference of FOG collected from different WWTP facilities, (4) the potential application of FTIR to examine environmental FOG impact and (5) the potential to use the FTIR protocol to provide a micro-method for gravimetric assessment of FOG.

1. Calibration The FTIR instrument remained stable from the beginning of the project through the present. Examples of the calibration data are shown in figures 15a, 15b and 16. Calibration data has remained consistent and linear for a range from 0 to 400 mg/L for the EPA Method 1464 and other standards. Figure 15a. Calibration data, 0-500 mg/L, dual log scale.

Figure 15b. Calibration data, 0 to 100 mg/L.

Figure 16. Calibration data, 0 to 400 mg/L, 2003.

One other aspect of calibration concerns the nature of standards and IR response. EPA method 1664 prescribes a specific standard made of 50% stearic acid and 50% hexadecane. While this standard may work well for USEPA Method 1664 gravimetric analyses, it is ill suited for wastewater and environmental waters contaminated with natural oils. Standard calibration series were made from motor oil and canola oil, and compared with the calibration curve produced by the USEPA 1664 standard; each material produced a different relationship between sample mass concentration and absorption (Figure 17). Samples containing calculated concentrations of motor oil and canola oil processed by complete sample extraction produced expected results.

Figure 17. Calibration regressions produced by USEPA standard 1664, motor oil and canola oil. (EPA 1664 slope = 273, motor oil slope = 387, canola oil slope = 486)

2. WWTP process effects on FOG Samples of FOG were collected from various locations within the LEWWTP treatment process and analyzed by FTIR for concentration and composition. The quantitative spectra are presented on a common scale in Figure 18; the reduction in concentration is clearly evident as wastewater is treated by facility processes. Figure 18. Spectra collected after sequential stages of treatment.

The samples were also analyzed for composition by HATR. Figure 19 illustrates the composition in the influent, post primary clarifier effluent, post secondary clarifier effluent and final effluent. It is observed that little, if any, change occurs in the composition of FOG as it moves through an activated sludge facility other than a reduction in concentration. Figure 19. Fingerprint spectra from various processes.

3. FOG from different facilities Samples from other facilities in the Denver metropolitan area were analyzed to see if the compositional results observed at our facility were consistent with other influents. The results are presented in Figure 20. Overall, it would appear that triglycerides are the dominant component of influent wastewater, at least in this region. Figure 20. Fingerprint IR spectra from three wastewater treatment facilities in the Denver region.

4. Potential environmental applications Because of the chemical nature of organic hydrophobic chemicals that might contaminate urban waters, the IR signatures of various chemicals of concern were examined. The fingerprint IR spectra of effluent FOG and various petroleum based compounds are presented in Figures 21a (overlapping full scale) and 21b (separate full scales). It is obvious that the nature of these compounds is notably different; while it is not possible to differentiate typical FOG products, it is easy to differentiate natural oils from petroleum products and determine the composition of various petroleum products.

Figure 21a. Fingerprint spectra from various natural oil and petroleum products, overlapping spectra.

Figure 21b. Fingerprint spectra from same suite of natural oil and petroleum products, presented as discrete spectra.

5. Micro gravimetric measurement potential To test the possibility that the method developed might be amenable to a micro gravimetric approach, we evaporated 10 ml of selected sample extracts in a 57 mm aluminum weighing dish and compared the resulting weight to the values predicted by quantitative IR spectral analyses. The correlation between extract sample mass and measured IR absorption is presented in figure 22; with a correlation coefficient of 0.98, the covariance is excellent. The relationship between the gravimetric measured mass and IR estimated mass can also be calculated and is presented in Figures 23a and 23b. Note that IR estimated mass values are presented as canola oil value equivalents to illustrate the excellent agreement. Data collected in 2003 agrees nicely with previous measurements. Figure 22. Correlation between observed IR absorption and gravimetric measurements, 1996-2000.

Figure 23a. Correlation between observed IR estimates and gravimetric measurements, 1996-2000.

Figure 23b. Correlation between observed IR estimates and gravimetric measurements, 2003.

DISCUSSION AND CONCLUSIONS Quantification and calibration The IR approach to measuring the FOG component in wastewater can produce acceptable results with the advantages of (1) high sensitivity and wide linear range, (2) limited environmental exposure to extract solvent release, (3) more rapid analytical results, and (4) the availability of chemical information about the nature of the FOG components. The one possible problem is that the IR calibration method is dependent on using an appropriate standard; if one is working with wastewater, a natural oil should be used as a standard. Because the IR method produces a linear calibration relationship for a wide range of concentrations, a simple slope adjustment can be used to produce equivalent mass estimates. Table 2 presents the relationship, as multiplication factors, of various standards observed in our research. Table 2. Translation table to convert standard equivalent FOG values.

Table 3 presents a summary of our quantitative data, using both EPA method 1664 and canola oil standard equivalent estimates; it was observed previously that the microgravimetric estimates correlate more accurately with the canola oil calibration. Table 3. Summary of LEWWTP FOG data derived from FTIR analyses form 1996 through 2000, and 2003. Sample Location Englewood Influent Littleton Influent Effluent
1 2

FOG (mg/L)1 1996-2000 33.6 9.9 27.6 7.5 0.62 0.51

FOG (mg/L) 1 2003 25.2 4.8 33.9 18.8 28.8 2.82 NA

FOG (mg/L)3 1996-2000 59.8 17.6 49.1 23.4 1.10 0.51

FOG (mg/L) 3 2003 44.9 8.5 60.3 33.5 51.3 5.02 NA

EPA 1664 standard equivalents Without outlier value of 86.6 3 Natural oil equivalents From this table, it is possible to conclude that the Pretreatment FOG Program in the Englewood collection district may be having a measurable effect on FOG entering the facility. Quantitative studies have also demonstrated that a micro-gravimetric protocol is feasible and would significantly reduce solvent release into the atmosphere. Compositional analysis The ability to observe the fingerprint region of the IR spectra has clearly indicated that the nature of FOG observed in wastewater facilities is composed mainly of triglycerides. It has also been demonstrated that FTIR can be used to differentiate natural oils from petroleum products in waste or environmental waters. It can be used with ease to identify the nature of slicks on still waters and determine if they are produced by a specific point source. With more research, FTIR can also be used to identify chemical changes that may occur to natural oils as they move through collection systems and facility processes.

REFERENCES Nicolet. __ Baseline sampling kit users manual. Thermo Electron Corporation, Madison, WI. Smith, Brian C. 1996. Fundamentals of Fourier transform infrared spectroscopy. CRC Press LLC, Boca Raton, FL. Smith, Brian C. 1999. Infrared spectral interpretation: a systematic approach. CRC Press LLC, Boca Raton, FL. USEPA. 1983. Method 413.2, Revision A: Oil and grease, total recoverable, spectrophotometric, infrared. Pages 413.2-1 to 413.2-3 in Methods for chemical analysis of water and wastes. EPA/600/4-79/020. Environmental monitoring and support laboratory, Office of research and development, Cincinnati, OH 45268. USEPA. 1999. Method 1664: N-hexane extractable material (HEM; oil and grease) and silica gel n-hexane extractable material (SGT-HEM; non polar material) by extraction and gravimetry. USEPA national center for environmental publications and information, Cincinnati, OH 45242.