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University Microfilms
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A Xarox Education Company IFEADI, Christopher Ngozi, 1939- QUANTXTATIVE MEASUREMENT AND SENSORY EVALUATION OF DAIRY WASTE ODOR.
The Ohio State University, Ph.D., 1972 Environmental Sciences
University Microfilms, A XEROX Company, Ann Arbor, Michigan QUANTITATIVE MEASUREMENT AND SENSORY EVALUATION
OF DAIRY WASTE ODOR
DISSERTATION
Presented In Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy In the Graduate School of The Ohio State University
by
Christopher Ngozi Ifeadi, B.S., M.S,
The Ohio State University 1972
Approved by
^ ■m l E. Paul Taiganides Adviser Agricultural Engineering Department PLEASE NOTE:
Some pages may have
i nd i s t inet print.
Filmed as received.
University Microfilms, A Xerox Education Company ACKNOWLEDGMENTS
Dr. E, Paul Taiganides is gratefully acknowledged for his
counsel and encouragement during my doctoral studies.
Dr. R.K. White is acknowledged for his useful suggestions in the design of the experiments.
Dr. R. Foltz of Battelle Memorial Institute, Columbus, Ohio, is acknowledged for the chemical ionization mass spectrometer analysis and interpretations.
Dr. K.S. Shumate and Dr. T.L. Sweeney are acknowledged for their interest, advice, and suggestions during my study.
Acknowledgment is made of the financial support from the
Ohio Agricultural Research and Development Center, and from the Live" stock Engineering and Farm Structures Research Branch of the United
States Department of Agriculture, R.G. Yeck, Chief. VITA
1939 ...... Born at Ubulu” Okiti, Nigeria
1955-1959 ...... Okongwu Memorial Grammar School, Nnewi Nigeria
1962-1963 ...... Greenville College, Greenville, Illinois U.S.A.
1963-1967 ...... B.S. Agricultural Engineering, University of California, Davis, California
1967-1969...... Research Assistant, University of Nebraska, Lincoln, Nebraska
1969 ...... M.S. Agricultural Engineering, University of Nebraska, Lincoln, Nebraska
1969-1972...... Graduate Research Associate, The Ohio State University, Columbus, Ohio
PUBLICATIONS
Ventilation systems for controlling gases produced in confined swine housing. M.S. Thesis.
Effects of slotted floors on airflow characteristics in swine confinement housing. ASAE Paper No. 68-9^5* American Society of Agricultural Engineers, St. Joseph, Michigan. 1968 .
A model study to determine the Effect of ventilation systems upon NHj concentrations in swine confinement housing. ASAE Paper No. MC-71-103. American Society of Agricultural Engineers, St. Joseph, Michigan. 1971•
iii FIELDS OF STUDY
Major Field: Agricultural Environmental Engineering
Studies in Waste Management: Drs. E. Paul Taiganides and Richard K. White
Studies in Air Pollution: Drs. Thomas L. Sweeney and Ross D. Brazee
Studies in Water Pollution: Dr. Kenesaw S. Shumate TABLE OF CONTENTS
Page
ACKNOWLEDGMENTS...... ii
VITA ...... iii
LIST OF T A B L E S ...... V
LIST OF F I G U R E S ...... vii
INTRODUCTION ...... 1
OBJECTIVES ...... 8
LITERATURE REVIEW ...... 9
Nature of Odor and Theories of Sense of Smell
Odor Measurement Methods
Odor Production from Anaerobic Decomposition of Organic Wastes
EXPERIMENTAL PROCEDURES...... 60
Instrumentation Set-Up for the Objective Measurement
Instrumentation Set-Up for Sensory Evaluation
Equipment Calibration and Standardization of Procedures
Test Procedures
v Page
EXPERIMENTAL RESULTS, ANALYSIS, AND DISCUSSION ...... 100
Equipment Performance
Chemical Ionization Mass Spectrometer of Dairy Waste Volatiles
Diffusion Cell Calibration and GC Calibration Curves for (CH3 )2S and (C2H,-)2S
Quantitative Measurement of Dimethyl Sulfide and Diethyl Sulfide in Dairy Waste Volatiles
Sensory Evaluations
Dairy Waste Odor Units
SUMMARY ...... 144
CONCLUSIONS ...... 149
RECOMMENDATIONS FOR FURTHER R E S E A R C H ...... 151
APPENDICES ...... 152
A. Equipment Diagrams
B. Calibration of Equipment
C. GC Calibration of Pure Compounds
REFERENCES ...... 3,77
vi LIST OF TABLES
Table Page
1 Reported Legal Cases of Odor Nuisance from Animal Production Units 2
2 Theories on Odor“Producing Attributes of Odorous Molecules 11
3 Properties of Ammonia aitl Hydrogen Sulfide and Their Physiological Effects 26
4 Mechanical Devices for the Detection of Odors 28
5 Methods of Sampling and Concentrating Odorous Gases 34
6 Composition of Typical Rations Fed to Swine, Poultry, Dairy, and Beef Animals from which Waste Samples for Tests Were Collected 51
7 Percentage Composition of Dry Material in Animal Manure 53
8 Compounds Identified in Decomposing Animal Wastes and Their Odor Characteristics 57
9 Serial Dilution of Dimethyl Sulfide 94
10 Serial Dilution of Diethyl Sulfide 97
11 Identification of Peaks Produced by Contaminants in the Equipment of the Sampling Train 101
12 Retention Times for (CH3)2S and (C2 H 5 )2 S 104
13 First Series of the Quantitative Measurements of (CH3)2S and CC2h 5^2® D a *ry Waste Volatiles 119
14 Second Series of the Quantitative Measurements of (CH3 >2 S and (C^,-)^ in Dairy Waste Volatiles 122
15 Third Series of the Quantitative Measurements of (0 1 3 ) 2 1 5 and ( C ^ ) ^ in Dairy Waste Volatiles 126
vii Table I*age
16 Rotameter Setting for Serial Dilution of Dairy Waste Volatiles .
17 Odor Intensity Ranking for the First Series of Tests 133
18 Odor Intensity Ranking for Second Series of Tests 134
19 Odor Intensity Ranking for Third Series of Tests 135
20 Odor Units Associated with Dimethyl Sulfide in Hundreds of Odor Units 138
21 Odor Units Associated with Diethyl Sulfide in Hundreds of Odor Units . - 140
22 Summary: Average Concentration and Odor Units Associ“ ated with Dimethyl and Diethyl Sulfides 141
23 Dimensions of the Diffusion Cells 161
24 Diffusion Cell Calibrations for Diethyl Sulfide 165
25 Diffusion Cell Calibration for Dimethyl Sulfide 168
26 GC Calibration Table for Dimethyl Sulfide at 25°C 172
27 GC Calibration Table for Diethyl Sulfide at 40°C 172
viii LIST OF FIGURES
Figure Page
1 Approximate Amounts of Air that Must Be Exhaustively Sampled to Supply Enough Odorant for Identification by Various Techniques 29
2 Schematic Representation of the Main Parts of the Anaerobic Digestion Process 56
3 Block Diagram for the Objective Measurement 60
4 Instrumentation for Odor Generation and Collection 62
5 Diffusion Cell 63
6 Sample Collector 65
7 Apparatus for Purifying and Conditioning Sample Collector 67
8 Apparatus for the Transfer of Sample into the Injection Needle 68
9 Cold/Hot Block Scissors Mechanism 69
10 Initiating Injection Process - Transfer of Samples from the Needle to the GC 72
11 Odor Analysis Instrumentation 74
12 The Sniffing Hood 76
13 Sample Transfer to Injection Needle 88
14 Serial Dilution of Diffused Pure Compounds 95
15 Evaluating the Dairy Odor 99
16 Similarity of Peaks Produced Using Pure Compounds 106
17 Mass Spectrometer Figures 107
18 Chemical Ionization Spectra of Reference Compounds 108
ix Figure Page
19 Dairy Waste Volatile Chromatogram 110
20 Chemical Ionization Spectra of (ClO-S and ^2H5^2S *n Dairy Waste Volatiles 111
21 GC Calibration Curve for Dimethyl Sulfide (Semi~Calculated) 114
22 GC Calibration Curve for Diethyl Sulfide (Semi"Calculated) 115
23 GC Calibration Curve for Dimethyl Sulfide (Measured) 116
24 GC Calibration Curve for Diethyl Sulfide (Measured) 117
25 Chromatogram Obtained from Sampling Undiluted Waste on the 5th Day of Incubation for 1st Series of Tests 120
26 Chromatogram Obtained with a Sample from the Diluted Waste on the 5th Day of Incubation, 1st Series 121
27 Chromatogram Obtained from Sampling Diluted Waste of Second Series of Test on the 4th Day of Incubation 123
28 Chromatogram Obtained from Sampling Undiluted Waste 4th Day of Incubation, 2nd Series of Tests 124
29 Chromatogram Obtained from Flask I on 4th Day of Incubation, 3rd Series of Tests 127
30 Chromatogram Obtained from Sampling Flask 2 of the 3rd Series of Tests on the 4th Day of Incubation 128
31 Chromatogram Obtained from Sampling Flask 3, 3rd Series, on 4th Day of Incubation 129
32 Sample Collector 154
33 Diffusion Cell 155
34 Cold Blocks of Injection System in Contact with the Injection Needle 156
x Figure Page
35 Detailed Diagram of Parts of the Injection System 157
36 Solenoid Attachment to the Cold Block 158
37 Partial Vapor Pressure of Known Compounds 160
38 Diffusion Cell No, 1 Calibration Curve for Diethyl Sulfide 166
39 Diffusion Cell No. 2 Calibration for Diethyl Sulfide 167
40 Diffusion Cell No. 1 Calibration Curve for Dimethyl Sulfide 169
41 Diffusion Cell No. 2 Calibration Curve for Dimethyl Sulfide 170
42 Calibration Chromatograms for (C^^^S 173
43 Calibration Chromatograms for (CHg^S 174
44 Calibration Chromatograms for 175
45 Calibration Chromatograms for 176
xi INTRODUCTION
Odor nuisance is a threat to the animal production industry.
As larger numbers of animals are raised in confined units and urban sprawl encroaches on the agricultural areas, offensive odors from the animal wastes prompt neighbors to legal actions. Table 1 gives eight cases of such legal actions. Of the cases reviewed, in only one case did the court find the defendant not guilty. Since that time there have been several other cases with favorable decisions for the producer.
However, the number of suits brought on against producers is on the increase.
To avert costly law suits and stringent air pollution regula" tions, there is a need for intensive effort to develop malodor control methods. There are two concepts employed in odor control: minimization or elimination of the initial production of the odorous compounds; or the control of the odorous material after it is produced. For the for" mer concept, the most common and perhaps the most effective method available is aeration. For the latter, many methods are available, such as, dilution with air, adsorption with activated carbon systems, . catalytic or direct flame“burners, scrubbers, and masking or counter" acting agents. Good management, like frequent cleaning, may be used to control both the former and the latter concepts.
This study was initiated to quantitatively measure and organo leptically evaluate the odorous emissions from decomposing animal wastes. TABLE 1. Reported Legal Cases of Odor Nuisance from Animal Production Units
Case Details/(Reference) Legal Case or Complaint Dispositipn of Case
CASE 1
Persons Involved: Warden vs. Civil action seeking damages and Hung jury and was dismissed. In a Sinning, Marshall County, Iowa. injunction relief because of a post trial agreement, the Sinnings (Willrich, et al., 1971) private nuisance caused by offen agreed to clean the anaerobic sive odors. storage pit, and to stop using con Facility: 160 acre farm. finement housing within mile of Housed 500 hogs on a partially the Warden home. slotted floor, with a liquid manure pit helow the floor. Sinning property was ahout 525 ft. S.W. and acrss the road from the Warden's house.
CASE 2
Persons Involved: Winnebago A request for injunction since Court found defendant violating Co. vs. Flugel; Winnebago (l) Fugel property violated zoning ordinance for operating County, Illinois, 1970 zoning ordinance and (2 ) the commercial feedlot (or a stockyard) (Willrich, et al., 1971) cattle feedlot was both public in agricultural use district. Also and private nuisance because found the feedlot to be both public Facility: A circular, funnel of odors, flies, insects, and private nuisance. Defendant shaped beef feedlot area on bacteria in air and nitrates in was permanently enjoined from using about 1* acres was planned to ground water. the premises as a beef cattle feed confine about 2,800 heads. lot. Neighbors were living less than one mile from the feed lot site.
co TABLE 1. (Continued)
- - - — «_ _ __ — _ _ « — _ Case Details/(Reference) Legal Case or Complaint Disposition of Case
CASE 5
Person Involved: Hardin Co. Complained of public and private Court found odor and flies consti vs. Gifford Feedlots, Inc., nuisance from offensive odors, tuted public and private nuisance. Hardin County, Iowa, 1968 flies and noise from bawling An injunction was decreed by the (Willrich, et al., 1971) cattle, and possible contamina court. tion of shallow wells. Facility; 10 pens with a com bined capacity of about 1200 heads on 5 acres. Distance from barest neighbor was 800 ft. N.E. of Gifford Feedlot.
CASE
Persons Involved: 19 Urban Complaint of odor nuisance com The farmer changed the waste dis Neighbors vs. Dairy farmer, ing from the farm when the herd posal method from anaerobic lagoons State of Washington, 196? size was increased from 50 to to burial in long trenches, 15 ft. (Davis, 1967 ) 200 cows. deep.
Facility: Sixty acres, 200 dairy cows TABLE 1. (Continued)
Case Details/(Reference) Legal Case or Complaint Disposition of Case
CASE 5
Persons Involved: Bower Plaintiff sought a civil action Plaintiffs won the jury verdict for Families vs. Hog Builders, to recover damages and an injunc $46,200 actual, $90,000 punitive Inc., Missouri, 1970 tion because of a private nui damage, plus court costs. Injunction (Willrich, et al., 1971) sance, created by odor. was not granted. Decision was appealed. Supreme Court of Missouri Facility: 159 acre farm; upheld the district court decision. 11 hog buildings with a com bined capacity of about 3*800 head. Open lots for hog con finement about 30 acres. Nearest home location 800 ft.
CASE 6
Persons Involved: Neighbors Plaintiff sued the owners for Plaintiffs won eleven damage suits vs. Beef Cattle owner. •jdor nuisance. averaging about $15,000 each. Milford, Texas, 1968 (Anon, 1968)
Facility: 27,000 head of beef cattle feedlot. TABLE 1. (Continued)
Case Details/(Reference .Legal Case or Complaint Disposition of Case
CASE 7
Persons Involved: Biergans vs. Plaintiff sued far injunction and Court declared that the smell from Crandall, Clinton County, $250,000 damage from intolerable the hog facility was "normal", Michigan, 1972 (Wilmore, 1972) smells produced by defendant's refused the damage claim and hog facility. Claimed that the declined the injunction or movement Facility; 32* x 120* hog odors had lowered the value of of the facility as long as the finishing tarn, completely the plaintiff’s property and units were run in a husband like enclosed with partial slats, diminished the use and enjoyment manner and odor control products located ahout U80' from of them. and/or devices that were economi Crandall's (plaintiff) house. cally feasible were used.
CASE 8
Persons Involved: Patz vs. Patz1 complained of offensive Court found odors coming from the Farmegg Products, Webster odor and dust exhausted from the poultry building were offensive, County, Iowa, 1970 (Willrich, building and about manure spill injurious and dangerous to the et al., 1971) age on the public road in front health and comfort of the plaintiff. of their home. Also, the court found that the Facility; Two buildings to operation interfered with the plain grow chicken to laying age tiff's right to use and enjoy their on the 4 acre track. Each residence and that invasion of with 43,000 birds in cages private rights was substantial and suspended over 10 in. deep intentional. Court awarded plaintiff pits. Patz residence was $20,000 in damage. Enjoined the located 1,000 ft. W., N.W. defendant from spilling manure upon from nearest exhaust fan. highways, but denied injunction. An important step in the abatement of animal waste odors is to identify
the odor“producing components within a complex odorous mixture and then
measure their threshold levels and concentration. Such knowledge would
provide a basis for calculating the following abatement alternatives:
(1) The degree of dilution by ventilation or outside
dispersal, which would be needed to dilute a given
odor from a source below threshold;
(2) The amount or percentage of odorants that must be
removed from a space by methods such as activated
carbon adsorption to lower the concentration of
odorants below the threshold*
(3) The amount and kind of the substance that could
effectively counteract or mask the odors within the
confined unit.
For the total characterization of odor, both subjective and objective measurements have been suggested by several researchers (Lindvall,
1970* Wright, 1964; Dravnieks, 1968a). Subjective or organoleptic measurement uses the human nose, while objective or analytical measure" ment is by physical or chemical methods.
The human nose is very sensitive to many chemical compounds.
It has the ability to discriminate between a large number of odor types with extremely small differences in qualities. It has reasonable pre"
cision and reproducibility when under controlled and standardized pro" cedures. On the other hand, the nose gives a composite measurement of a mixture of odorous compounds. It may easily be fatigued, and its
response to odor intensity is non"linear. Analytical instruments, like gas chromatographs, may also detect and measure small quantities of compounds but the sensitivity is somewhat less and strongly dependent on chemical structure. Gas chromatography can separate complex mixtures of volatile compounds, can identify the separated volatiles, and when calibrated, can quan- tify the amounts present. With careful techniques, the gas chroma- tograph has good precision and reproducibility.
This study combines sensory evaluation with gas chromatographic measurement to analyze malodor from dairy waste volatiles. It is part of a long-term program initiated by the Agricultural Pollution Control
Research Laboratory of the Agricultural Engineering Department at Ohio
State University on the identification, quantification, and control of animal malodors. Other studies conducted in this program were pub lished by White (1969), Nordstedt (1969), and Cole (1970). OBJECTIVES
Odor control is a primary requirement for livestock production in an urban society. Identification and quantification of the odorous compounds would provide clues as to the best control measures. A combination of objective and organoleptic measurements would provide most of the necessary information needed.
The specific objectives of this study were:
(1) To develop instrumentation and methodology for
quantitative and organoleptic measurements of odor.
(2) To measure objectively concentrations of major odor
compounds which are released during the decomposi
tion of dairy wastes.
(3) To determine organoleptically odor thresholds of
dairy wastes.
8 LITERATURE REVIEW
The review of literature was confined to the nature of odor and theories of the sense of smell, odor measurement methods, and characterization of odors eminating from animal wastes. Particular emphasis was placed on quantitative techniques of odor measurements, since this most directly related to the main objective of the study.
Nature of Odor and Theories of Sense of Smell
Several definitions of odor are given in literature. The definition that incorporates most basic aspects of the sense of smell states that odor is a sensation characteristic of the property of a substance or substances perceived as a result of the stimulus of the olfactory area of the human nose. Various requisites for substance to produce odor have been postulated. According to Moncrieff (1967), the three criteria that must be complied with for a substance to be odorous are:
(1) It must be volatile at ambient temperatures so that it
will lose molecules to the atmosphere which then can be
breathed into the nasal passage,
(2) It must be capable of being adsorbed on the sensitive
surface of the epithelium,
(3) It must be a substance which is not already present
on the olfactory epithelium.
9 10
In recent years, however, the second criterion has been attacked by some prominent authorities in the field (Dravnieks, 1968c) leaving
the first and third criteria as the ones on which there is an agreement
among odor specialists. The opponents argue that some compounds can
still be smelled without meeting the adsorbability requirements. They
feel that the condition is too broad. For such compounds, other cri-
teria, like electron transfer and molecular vibration, could be used
to explain their ability to produce odors.
Theories of the Sense of Smell
There have been several comprehensive reviews of odor theories
(Moncrieff, 1967; Dravnieks, 1967; 1968a). The postulated theories are mainly concerned with the physio“chemical properties of the odorant and with the mechanisms of peripheral interactions. According to
Dravnieks (1968a), these theories could be grouped into four classes
(see Table 2): sterochemical, profile functional, interface adsorp"
tion, and vibrational.
Amoore's (1952, 1962) theory places emphasis on the theory that similar shapes and sizes lead to similarity in odor.
Beet's (1961) theory stresses that molecular shape and the nature of the peripheral functional groups are the odor relevant attri“ butes. One of the functional groups interacts with the receptor site more strongly than the other groups. This determines the orientation of the rest of the molecule with respect to the receptor site.
Davies* (1965) theory is similar to Beet's. The odor“relevant attributes are the molecular cross sections and the free energy of 11
TABLE 2. Theories on Odor Producing Attributes of Odorous Molecules
(taken from Dravnieks, 1968a, p. 28)
Theory (Proponent) Odor Relevant Attributes Strong Points
Stereochemical Molecule size and shape; Evidence that si (Amoore) nucleophilic and electro- milar shape and phi 1 ic nature size leads to some similarity in odor
Profile Functional Orienting groups, mole FlexibiIity in ac Group (Beets) cule shape and size in counting for odor certain orientation changes upon smell changes in molecules
Interface Adsorption Size, adsorption energy Measurement of profile (Membrane Puncturing) at water-oil interface functional group and (Davis) other shape, size, and functional effects experimentally; link to sensor mechanism
Vibrational (Wright) Certain (osmic) frequen Possibility of a uni cies In far infrared, fied code for size, correlation exists be shape and functional tween Infrared or Raman ity effects spectra and odor quality. 12
adsorption of the odorant at the lipid-water interface. The positions
of odorants in the two-dimensional field represented by these two attri
butes determine odor quality.
Wright (1964) theorizes that molecular vibration frequencies,
indicated by absorption in the far infrared spectra constitute odor
relevant attributes. Hence, if the frequencies occur, a certain odor quality may be present. This theory is directed toward a vibrational mechanism of odor sensing.
None of the aforementioned theories of smell has been univer sally accepted. However, all of the theories agree that molecular shape and size and the orienting of electron donor-acceptor strength of functional groups are related to odor quality (Dravnieks, 1968a).
Mqc^anisms of Smell and Basic Facts about Olfaction
Sensory perception of odor is experienced when nerve endings are stimulated and when the resultant impulses pass to the brain. The nerve endings are located in epithelium of the upper part of the nose.
The process of stimulation of nerve endings begins when air is inhaled.
In normal breathing, most of the main air stream passes through the nose without passing over the olfactory receptor surfaces which are high in the nasal passages. As the main air stream passes through the nose, eddy currents are formed and some of these swirl past the olfactory receptors, creating the sensation of odor. If in the process of breath ing, a sniffing action is made, then a larger portion of the air stream with the odor compounds is directed to the upper nasal passes, thus increasing a greater intensity of the odor sensation. 13
Basic facts about olfaction, as listed by Moncrieff (1967) and others, include these facts:
(1) All normal people can smell, while people suffering from
brain lesions, injured olfactory nerves, or obstructed
nasal passages may be anosraic. A normal person could be
anosmic to a particular compound.
(2) Substances of similar chemical structure usually have
similar odors, e.g., in a homologous series. Substances
of different chemical structures may have similar odors,
e.g., compounds with musk odor.
(3) The quality, pleasantness, or unplesantness, as well as
the intensity or strength of an odor may change by
dilution,
(4) Odor threshold, the lowest concentration of a compound
at which an odor is detected, varies between individuals
and with the time of the detection.
(5) The sense of smell is rapidly fatigued. In the presence
of strong stimulus, fatigue occurs in two to three minutes.
(6) Fatigue for one odor may not affect the perception of
dissimilar odors, but will interfere with the perception
of similar odors.
(7) Two or more odorous substances may cancel each other.
This principle is the basis for odor masking and counter"
actants.
(8) A number of odorous materials behave in an additive 14
manner, when present in sub-threshold concentrations in
a mixture (Guadagni, ££, , 1963).
(9) Odor character of a mixture of compounds is not neces
sarily proportional to the total amount of each in the
mixture, i.e., the greatest odor intensity may come from
the compound with the smallest concentration (Guadagni,
filal, 1966).
(10) Inorganic elements that are odorous are limited to the
halogens “ ozone, phosphorous, and arsenic~to compounds
with sulfur , CS2, and CSC12 , and H2S2“-to nitrogen
compounds NH^, N02 , and NH2C1.
(11) The greatest number of odorous substances are organic
compounds.
Odor Measurement Methods
Odor measurements may be categorized into two methods:
(1) Organoleptic or subjective methods that use the human nose
to analyse the odor.
(2) Objective or analytical methods that are based upon the
measurement of some physical or chemical property of the
odorant to be analyzed.
The human nose is a very sensitive, qualitative "instrument" but it is not a good analytical instrument quantitatively. The nose can distinguish between odorous and non-odorous compounds and between pleasant and unpleasant odors. Its sensitivity for some compounds is in the sub-part-per-billion concentration range. However, organoleptic , 15 have many difficulties and pitfalls as pointed out by Matheson (1955).
The most serious difficulties are (1) smelling ability of people differs between individuals and is said to differ within oneself at different times; (2) the sensation of smell is in response to a mixture of several odorous compounds, thus giving composite or summed effect, and (3) the response to odor sensation is not linear,but a power function of concen tration (Stevens, 1961).
Currently, no instrument or chemical analysis can fully replace the function of the nose, mainly because of two reasons; (1) many odor- ants are easily perceived by the human nose in dilute concentrations which are undetectable by the present physical or chemical methods, and
(2) the quality of an odor cannot be measured analytically. Furthermore, malodors have physiological effects on people, which an instrument or chemical analysis cannot measure. These effects are (Wohlers, 1967);
(a) Weak odors are not perceived by people in the presence of strong ones; (b) Odors of the same strength may blend to produce a combination in which one or both of the compounds is unrecognizable by people; (c)
The constant smelling of an odor causes an individual to develop odor fatigue and thus to lose the awareness of the odor sensation; (d) The like and dislike for an odor often depends on an individual's associa tion with pleasant or unpleasant experiences in the past.
Analytical methods have advantages in reproducibility and linear ity of response, but the ability of analytical methods to give quality measurements is not solved. Therefore, it is important to correlate 16
subjective measurements with analytical measurements for complete
characterization of an odor.
Organoleptic Measurement Methods
Organoleptic odor-measurement techniques rely on organoleptic
evaluation of the characteristics of the odors. These characteristics
are normally divided into four categories: threshold and intensity,
specific qualities or attributes, affective response, and irritant
properties.
Odor Threshold and Intensity.— Odor threshold is the minimum concentra
tion at which an odorous substance can be distinguished from odor-free
air ("detection threshold"), or at which its quality can be recognized
("recognition threshold"). The latter has the higher concentration of odorant. Odor threshold levels depend on the nature of the odorant, on the sensitivity of the person, and the manner in which the samples are presented to the person. Odor intensity is the magnitude of the stimulus produced when a person is exposed to an odorant at superliminal
levels of concentration.
Threshold concentration is generally considered a better value to use for expressing the level of odorant in the ambient air, rather than intensity values. This is especially true in atmospheric diffusion cal culations. For example, an odor intensity scaled as 5 (very strong) measured in one location does not give us much information as to how far
the parcel of air must travel or how much it must be diluted before its intensity is reduced to 2 , 1 , or 0 , whereas odor threshold could express 17
the number of dilutions necessary to arrive at odorlessness.
Relative strength of an odor may be described in terms of dilu
tion. The greater the number of dilutions with odorfree air that are
necessary to bring an odorous gas sample to the threshold, or barely
detectable level, the stronger the odor. Odor measurement has been
postulated in terms of odor units. An odor unit is defined as the
amount of odor necessary to contaminate one cubic foot of clean air
(odorfree) to the threshold, or barely perceptible level. The product
of flow rate times its odor intensity gives the odor units emitted
(Byrd, et al.r 1968). For example, 10 odor units/ft^ x 10 cfm = 100
odor units/min.
Other factors affecting threshold and intensity values are many
and some of these are:
(1) Exposure system of the odorant to the panelists,
(2 ) Method of odor presentation,
(3) Instructions to the subject, his criteria of judgment,
his attitudes, motivations, rewards, and punishments, etc.
(4) Relative frequency of presentation of various stimuli,
including blanks (Harper, 1968),
Panelists may be exposed to odor by either a static or dynamic
system. In static exposure, the gas mixture is stationary, except for movement due to diffusion, or the subject's inhalation, for example,
sniffing at the opening of a flaslc. Dynamic exposure, on the other hand,
requires a flow of air and facilitates the continuous dilution of the
odorant with odor-free air. In addition, the danger of variation in 18
the concentration and quality of the odor, due to the adsorption or chemical alterations are reduced. However, it has a disadvantage in that the movement of the odorous gas may affect the panelists* impres- sion of the odor (Lindvall, 1970).
For the static system, the commonest procedure is to have odor panelists sniff at the opening of the dosage system. Lindvall gave the disadvantages of this method as the effect of the surrounding odors, variation in temperature and humidity, or possible unintentional dilu tion of the gas.
Other researchers have introduced the odorant directly into one or both nostrils by stream or blast injection (Elsberg, 1937; Wenzell,
1948), or by tubes inserted into the nostrils (Kuehner, 1954; Schneider,
£jL aL . , 1963; Woedeman, 1955; Fox and Gex, 1957; Huey, £±. aJLr» 1960;
Stuiver, 1960). An advantage of the static method is that the pressure and volume may be more easily controlled, but it would seem that this procedure departs too much from normal breathing. Dynamic systems have been developed to expose the panelists to the odor in as natural manner as possible. The dynamic system avoids the disturbing influence of other smells and enables the use of normal inhalations. One type of dynamic system is where panelists sit completely surrounded by the odor ous atmosphere (Foster, e±. ai., 1950; Deininger and McKinley, 1954;
Schneider and Wolf, 1955; Kerka and Kaiser, 1958; Komuro, 1921). The advantage of a whole body exposure is that several panelists could be subjected to the same odor stimulus under identical conditions. However, it has the disadvantage of requiring large space and extensive 19 installation and a large volume of odorous gas, making changes in con" centration difficult.
A second type of dynamic system is the odor hood in which the face or head are exposed to the odorous atmosphere. This system is considered as the most acceptable because it combines the advantages of the odor chamber with simplicity of construction and operation.
Several arrangements of this have been presented (Nader, 1958a; Mrak,
£ i Si.* r 1959; Ough and Stone, 1961; Stone, si. sJL., 1962; Merrion, 1968;
Sullivan, £i. s i . . , 1968; Lindvall, 1970).
The method of odor presentation can be random, or ascending or descending order of concentrations. Presentation of odors in ascend- ing concentrations is preferable to descending or random order. Both the descending and random order of odor presentation to the panelists create odor fatigue.
Age, sex, profession, and attitudes of individuals affect the response of panelists to odor stimulus. It is best to select panelists from a cross section of the population, when possible.
With clear smelling instructions to the panelists, and with care- ful training and some practice, it is possible to obtain precise and reproducible judgment from the sniffers. Such procedure improves the attitude and motivation of the panelists (Harper, 1968), Other psycho physical factors are to be considered: the manner by which the response of the panelists should be recorded and the intervals in the intensity of the odorant to be used in the test. These factors are discussed by
Lindvall (1970). 20
Turk (1969) suggests the following qualification for members of odor panels: (1 ) that they can discriminate among the different odors at low or moderate levels of intensity, and (2 ) that they can focus their attention on more than one odor sensation. He suggested that standard odors with respect to intensity and quality be used to screen candidates for odor panels. The panelists would be asked to arrange the standard samples in ascending odor of intensity. With a careful evalu ation of each panelist, the potential value of each candidate may be assessed.
Intensity renresentation.— Odor, like other sensations, is a logarithmic or exponential function of the stimulus or concentration.
Several attempts had been made to mathematically describe the relation between the magnitude of stimulus (concentration of the odorant) and the strength of sensation produced.
The Weber-Fechner Law gives a logarithmis relation between the magnitude of the stimulus and the strength of the sensation. This law states that if x is the magnitude of the stimulus, as measured in some convenient way (pounds, or parts per million), add if A* is the amount by which x must be increased to give a Just Noticeable Difference (J.N.D.) then:
Ax s a constant for the J.N.D. x and is true also for other increments in the stimulus which is expressed mathematically by
dS a C X 21
dS = increment in the sensation generated by an increment in the
stimulus,
dx = superimposed on an already existing stimulus, x, and
C * proportionality constant.
Integration gives
S « a(log x " log xQ )
where: S = strength of the sensation,
log x = the logarithm of the stimulus that produces the sensation, S,
log Xq = the logarithm of the stimulus that produces a J.N.D.
and a = a constant.
The problem with this method is the difficulty of measuring S, i.e.,
the strength of the sensation.
Ratio Estimation or Scales (Stevens, 1961) is an attempt to
measure odor intensity. In this method, judges make subjective esti"
mates of magnitude between points that are represented by a reference
standard or standards. A number is attached to a sensation by assign-
ing a definite value of one sensation and then observers are asked to
say whether another sensation is one“half or one-third, or some other
fraction of it, or whether it is 2 or 3 times greater than the standard.
This is mathematically expressed as
whe re
S = the sensation measure by the method of Ratio Estimation,
x - the stimulus measured in some physical units such as pounds, ppm.,
n “ a constant of proportionality. Integrating the above gives
where: xQ is the threshold stimulus.
Instead of a logarithmic function, odor sensation may also be considered an exponential function of the stimulus, where the concen" trations of the reference odor standard is distributed along some exponential scale, say, on exponents of 2 : thus,
Dilution No. Concentration of Odorant in Odorfree Fluid
1
2 1/2C1 'a c x X 2_ 1
3 1/AC1 = C 1 x 2 “ 2
Odor intensity scales are yet another method. This method uses no specific reference standard, but instead, one defined by descriptions and rating numbers, thus:
Ratine Number P-escxiiLiipn
0 No odor
1 Barely perceptible
2 Distinct
3 Moderate
4 Strong
5 Overpowering 23
Odor intensity scale actually implied reference standards of a panel ist’s personal experience, and thus, would be imprecise.
_Qfl9_r 'quality.— 'The quality of an odor is the character described in terms of resemblance to some other odor. Standard Methods (1965) contains 2 2 descriptive terms like ’’putrid," "musky," "fragrant," which provide some consistent system for naming odors.
It has been postulated (Wright, 1963) that if a general descrip tion of odor qualities could be set up, it would be possible to describe any odor in terms of a number of primary odor standards. Amoore (1962; fiiaJL.* 1964), classified odors into the following primary odors. The number of compounds identified for each "primary odor" is also given.
Number of Erimary_Odor Chemical Example- Familiar Substance ggmppvmte.
Camphoraceous Camphor Moth Repellent 106
Musky Pentadecanolactone Angelica Root Oil 69
Floral Penylethyl Methyl Roses 71 Ethyl Carbinol
Pepperminty Menthone Mint Candy 77
Ethereal Ethelene Dry*Cleaning Fluid 53 Dichloride
Pungent Formic Acid Vinegar 95
Putrid Butyl Mercaptan Rotten Egg 49
Some other classification systems, comprising from four to over thirty basic odor qualities have been proposed. Unfortunately, there is no simple, universally accepted classification system of basic odor qualities. 24
Affective resnonse-Affective Response is the complex attitude produced by an odor, which is expressed in degree of like or dislike, acceptable or objectionable. A measure of affective response such as
the degree of acceptability, or the degree of objectionability inte" grates all other three odor characteristics~"intensity, quality, and irritant properties*— plus inherent or acquired attitudes towards par" ticular type of smells (Schutz, 1960). For example, the acceptability
Cor objectionability) of an odor is dependent on the intensity, fre" quency, and duration of the experience as well as the conditions under which exposure occurs.
Quantitative objective measure of acceptability (or objection” ability) by instrumental or sensory methods is not possible. However, the following categories in a linear arrangement ha.ve been suggested
(Turk, 1969);
1 . Like extremely 6 . Dislike slightly
2 . Like very much 7. Dislike moderately
3. Like moderately S. Dislike very much
4. Like slightly 9. Dislike extremely
5. Neither like nor dislike
Irritant Pronerties. The irritant properties of an odor are determined by the pain or irritation that it produces, not by stimula” tion of the olfactory receptors but rather by stimulation of nerve endings in the nasal passages. When one is subjected to high odor irritation level, it is frequently difficult to separate the 25 stimulation produced by irritation from stimulation produced by mere odor intensity.
The physiological effects produced by an irritant odorous com" pound depend on the concentration and the exposure time. For example,
Table 3 shows the properties of two odorous gases, ammonia and hydro" gen sulfide, at different concentrations (Dubois £jfc. aX., 1959;
Dreisback, 1966).
Objective Measurement Methods
Instruments used in measuring characteristic property of odor" ants are discussed under two categories: objective olfactometers and chemical instruments.
Objective olfactometers."" Objective olfactometers are combina" tions of techniques and instruments which analyze samples of odorous air and, without the use of the human nose, report the intensity and quality of odor. Essentially, the principle of operation is that when a simple odorant vapor is presented to the quality-measuring device, it is able to produce readings in proportion to some tangible odor-relevant attributes of the odorants, like Amoore*s (1962) molecular shape theory attribute.
Dravnieks (1968b) has discussed two basic types of objective olfactometers, namely: (1 ) a device with several detectors, each one specializing on some odor quality parameter and sufficiently sensitive to all odorants* and (2 ) a device in which the detector is sufficiently sensitive but not specialized and the discrimination of odor parameters is accomplished by additional devices. TABLE 3, Properties of Ammonia and Hydrogen Sulfide and Their Physiological Effects
Physiological Effects Gas Odor MIO1 MAC2 Q (ppm) (ppm) (conc. ppm) Exposure timeh Effects^
Ammonia sharp, 5.3 100 Irritant nh3 pungent 400 - Irritation, throat 700 - Irritation, eyes
1700 - Coughing 3000 30 min. Asphyxiating 5000 40 min. Could be fatal
Hydrogen rotten 0.7 20 Poison Sulfide egg 100 hours Irritation of eye & h 9s nose 200 60 min. Headache, dizziness 500 30 min. Nausea, insomnia 1000 — Unconsciousness, death
■SlIO = Minimum Identifiable Odor: the threshold odor.
T4AC = Maximum Allowable Concentration: the concentration set by health agencies as the maximum in an atmosphere where men work over an 8-10 hour period. 3 Cone, ppm = Concentration in part per million of air
^Exposure time = The time during which the effects of the noxious gas are felt by an adult human being. 5 Effects = Those found to occur in adult humans. 27
Some devices utilizing the phenomenon of the single-stage process are summarized by Dravnieks in Table 4, which lists selected artificial noses or olfactory analogue proposed by various researchers.
The possible properties of the odorant that appear to be most signifi cant in producing differences in the device response are indicated.
Dravnieks pointed that some devices are nothing more than detectors of large molecules without any relevance to odor quality parameters. Furthermore, Wright (Lundquist, 1968) warns that:
Unless or until we have definitely identified what I have called the specifically asmic properties of molecules, that is to say the properties which give rise to their specifically odorous sensations, then I don’t think we’re justified in referring to mechani cal or electrical devices as odor detectors. They are intrinsic pieces of apparatus for use in analytical chemistry, and as such they are both interesting, important and valuable.
Detectors (Chemical instruments).— Dravnieks (1968b) classified the detectors into three broad groups: descriptive articulate detec tors, semiarticulate or specific detectors, and nonspecific detectors
(Figure 1). The discussion that follows is intended to show the use of these detectors in odor research.
Descriptive articulate detectors provide much information about the nature of the odorant. Examples of such devices are the infrared spectrophotometer and mass spectrograph. The articulate detectors cannot, at the present day of their development, specify the odor quality.
Semiarticulate or specific detectors, provide much less informa tion. They are sensitive to certain features of the molecules, TABLE 4 . Mechanical Devices for the Detection of Odors (taken from
Dravnieks, 1 9 6 8 b, p. 3 8 6 )
Best Estimate of the Description of Principle Odorant Sensor System Effect Observed Properly Measured (Proponent)
Electrode Electrode Potential resulting Adsorbability of Interface from adsorption at odorant.Oxidizability electrode, of current or reducibility of Berton resulting from oxida the odorant. Wilkens & Hartman tion or reduction of Rosano odorant
Electrode/Gas Interface Surface potential shift Adsorbability of resulting from orien odorant. Polarity of Tanyolac tation of polar mole odorant. Ability of Phillips cules at surface or odorant to polarize Dawnieks partial exchange of and to form charge- Chapman charges between odorant transfer bonds with and surface surface.
Phase Under Electrode Electrical conductivity Adsorbability of the Gas Surface change in solid caused odorant, principally by interaction of through chemisorption charge carriers with mechanism adsorbed odorant mole cules
Films and Surface Surface tension change Adsorbability of gas- of liquid liquid interface Tanyolac & Eaton
Moncrief Heat of adsorption or Adsorbability of surface Friedman, Mackay solution solubility in liquid and Rosano or semi-liquid forms
Others Disarrangement of Solubility in film liquid crystal films
Change in viscoelastic Solubility in film properties of polymers
Change in Electrical Solubility in membrane or diffusional per meability of membranes 29
Liters of Air at Cone. 10^® moles/cm^
Detector 1CT1* IQ"? 10~2 1CT1 1° 10+1 10+2 10*3 i q +^ i q +5 i q +6 1" 1 1 1 1 1 — r 1 1 i ARTICULATE
Infrared
Mas s-spec trographic
SELECTIVE
Electron Capture
Microwave-Heated Plasma Emission Spectrometry
NON-SELECTIVE Argon Ionization w Hydrogen Flame Ionization m
Figure 1. Approximate amounts of air that must "be exhaustively sampled to supply enough odorant for identification by various techniques (taken from Dravnieks 19 68 b, p. 376). 30
e.g., electronegativity in the case of the electron-capture detector.
Another example of this device is microwave-heated emission spectrometry.
It has been observed that the basic problem with the specific detectors
is to distinguish whether, for a given signal size, we deal with a small
concentration of that substance to which the detector is very sensitive,
or with another substance to which the detector is relatively insensi
tive but which is present at high concentrations. Consequently, this
group of detectors is more useful in combination with additional, more
specific instruments.
Nonspecific detectors can detect either, all substances (e.g.,
vacuum-tube ionization detector), or broad groups of substances, such
as all organic molecules in the case of the hydrogen-flame ionization
detector. These devices always need discriminator stages for distin
guishing molecular features of the odorants. Gas-Liquid chromatography
(GLC) falls under this category. In Figure 1, it will be noted that when sampling a source of odorous compound for analysis by certain
detectors (e.g., electron capture, mass spectrometer, or hydrogen flame
ionization) the size of the sample that will effect identification of
compound in the sample will vary. The size of the sample will be in
the order of 1 0 0 0 , 1 0 0 , 1 0 for electron capture, mass spectrometer, and hydrogen flame, respectively. Since gas chromatography is about the most popular device in odor and taste research, more discussion on the
topic is relevant. . 31
Gas Chroma toe ranhv in odor research.-" Gas chromatography has been applied successfully in odor research. Two excellent general means of detection, thermal conductivity (physical) and flame ioniza tion (chemical) have been very useful.
The thermal conductivity employs a resistor (either filament or thermistor) which is heated from the element at some constant rate, but when a component passes through the detector, the rate of heat dissipation is changed with the subsequent change in resistance being reflected in a change in the voltage drop across the element which is transmitted to the recorder where it appears as a peak.
The flame ionization detector employs a hydrogen flame to com bust the sample and, thereby, produce ions. A DC potential is applied across the flame by means of electrodes which collect the charged ions generated during the burning of the sample. This current flows through a high resistor, and the resultant voltage is amplified by an electrom eter and the resulting current is displayed on a recorder as a peak.
Gas chromatography gives a relatively easy, quick, quantitative and objective measure of the odorants. A way of correlating sensory evaluation of odors with chromatographic data is by splitting the carrier gas flow during chromatographic analysis. Part of the flow is diverted outside the equipment where it can be sniffed at the time the other portion of the flow is analyzed by the flame ionization detectors. Gas chromatography, however, has some limitations:
(1) Retention volume data (i.e., volume of gas required to
elute the compound under study) cannot be used alone 32
for the identification of components in a complex mixture,
especially unknown mixtures which consist of a number of
heterofunctioral or isomeric components having the same,
or nearly the same, retention volumes.
(2) GC cannot detect some of the odorous components at the
very low concentrations at which they are odorous to
humans.
Analysis of a complex mixture could be accomplished by finding suitable stationary phases to effect resolution and identification.
The large number of stationary phases to be studied makes this approach time consuming. This problem has been solved in some cases by collect" ing the gas chromatographically elected peaks and employing infrared spectrophotometry (Beilis and Slowinski, 1956; and Heaton and Wentworth,
1959), or mass spectrometry (Drew, et al., 1956) for subsequent iden" tification. However, the supplementary instrumentation is very expen sive and the procedures for collecting eluted chromatographic peaks are cumbersome and not always reliable. In addition, many labora" tories do not have such instrumentation available and must rely on gas chromatography alone (Flath, et al., 1967). According to Pecsok (1959), the failure of the GC method to provide complete qualitative analysis is due primarily to the inability of the GC to determine or distinguish organic groups.
Since GC cannot detect very low concentrations of odorous com pounds, samples from an odor source need to be concentrated before injection into the GC. Furthermore, samples for chromatographic 33 analysis must possess suitable and desirable characteristics, namely:
(1) A minimum amount of water should be concentrated with
the odorous volatiles. The sensitivity of flame
ionization is not affected by up to about 3 //I water
(Dravnieks, 1969).
(2) The collected sample should be representative of the
volatiles in the environment sampled.
(3) The collected sample must be in a form suitable for
injection into the GC.
White (1969) discussed several methods for sampling and concen- trating organic volatiles. These methods are: direct sampling, salt ing out, selective chemical adsorption and regeneration, cryogenic col lection, and equilibrium sampling.
In Table 5, the sampling methods are discussed in terms of process, advantages and disadvantages of the method, while examples of application are also cited. Equilibrium sampling appears to have the greatest promise in sampling organic volatiles with low concentrations.
Its major disadvantage is that it produces artifacts.
Eouilibrium sampling.-" The equilibrium sampling technique is based on the adsorption of organic vapors on large surface areas of porous substances enabling the analysis of sub-ppb concentrations of organic components in air. Such porous substances are aluminum oxides, activated carbon, silica gels, organic polymers, etc. The sorptive qualities of a porous material depend on previous treatment, type of vapor to be adsorbed, etc. Dravnieks and Kratosynski et al. (1967) TABLE 5» Methods of Sampling and Concentrating Odorous Gases
Sampling/Concentrating Principle of Advantages and Process and Applications Operation Disadvantages
1. Direct Sampling Waste filtered and Advantage: Technique Burnett and Sobel (1967 , centrifuged to remove relatively simple. 1968) used technique to particulates. Clear Disadvantages : determine the odors from supernatants injected (i) Too small concen poultry wastes directly into the G.C. tration of significant odorant for good G.C. response, (ii) Rela tive concentration of components in the odor ous mixture likely to be different from that in the air above waste.
2. Salting Out Addition of Anhydrous Advantage: Requires Merkel (1 9 6 7 ) used the inorganic salts, e.g. , simple equipment and technique to enrich NagSO^, to increase can easily and quickly volatiles from hog the vapor pressure of be conducted. manure. Bassette, et odorants in an aque Disadvantages: al., (1 9 6 2 ) enriched ous solution. Mix (i) Proportion of the head space in sul ture shaken, heated odorant in the salted fides, esters and to 60°C to release out head space gas may alcohols. dissolved gases. not be representative Head space gases in of that without salting jected directly into out technique, (ii) G.C. Lack of knowledge of salting out effect on organics of different water solubility. (iii) Possible heat alteration and problem of water vapor in the Injected sample. 35.
TABLE 5. (Continued)
Sampling/Concentrating Principle of Advantages and Process and Applications Operation Disadvantages
3. Selective Chemical Similar functional Advantages: Separation Absorption and Regenera groups or atoms of of complex mixture into tion volatile organic com its functional groups Merkel, et al., (I96 Q) pounds are absorbed facilitates the analysis used the technique to from a gas stream of the complex odorous establish the presence mixture in solution compound. of functional groups in that react with the Disadvantage: Diffi hog waste volatiles. particular group of culty in correlating Hoff and Fe it (1964 ) compounds. (See the concentration of differentiated vapors Cheronis and the odorous compounds of carbonyl compounds Entrikin, 1957•) in the sample air with alcohols, ethers, ole The absorbed group the absorbed and regen fins, hydrocarbons, etc., is separately in erated quantities using this process. jected directly into analyzed by G.C. the G.C. for detail and analysis of the group.
4. Cryogenic Collection A cold trap technique. Advantages: Cold trap (Beroza, 1 9 6 4 ) for highly Dry ice in acetone ping produces no arti volatile compounds and/or a cold trap of facts, and collected liquid nitrogen is sample is representa used. Sampling valve tive of the medium may be used to trans sampled. fer the collected sam Disadvantages: ple directly into the (i) Water vapor is G.C. (Williams, 196 5 ) usually trapped and drying is difficult without introducing contaminants, (ii) Large molecules of or ganic compounds, espe cially if they are hy drogen bonded, aromatic or polar absorb on hydrates (Dravnieks, et al., 1 9 6 7 ). 36
TABLE 5. (Continued)
Sampling/Concentrating Principle of Advantages and Process and Applications Operation Disadvantages
Equilibrium Sampling This process operates Advantages: (i) Dravnieks, et al. (1 9 6 8 , on absorption/adsorp- Efficiency of adsorp 19 69, 1970). tion and desorption tion not diminished principles. Volatiles by increasing dilution, Kratosynski and from source are passed hence useful in the Dravnieks (1968) over stationary analysis of trace liquid (e.g., organic materials, (ii) Bear absorbents, like low ing no artifact forma volatility liquids tion and temperature and grease) or solid alteration, the sampled (e.g. non-polar mixture is reasonably polymer adsorbent). representative. Desorption is effected (iii) Minimum water by raising the vapor is collected in temperature, reducing the process. the pressure, etc. Disadvantage: The eluting or dis“ The technique is selec placing components are tive and likely to injected directly produce artifacts. into the GC. 37 developed an expression relating some of these properties. They showed that the amount of any one volatile component adsorbed in the collect” ing phase is directly proportional to the concentration of that compon- ent in the air stream passing over the phase, thus:
CNi) = 6 . 0 2 x 102 3 CQ) ^ ^ 4 * - = K(Ni)a c 7i (Ni )q where th (Ni) = the concentration of the i component in the collecting C phase (molecules/cm’3).
(Ni)0 = the concentration of i in air. a
(Ni)0 = the concentration of i in saturated vapor above pure i at
the same temperature.
Q - a coefficient that depends on the molecular weight (M) and
the density (d) of the collecting phase and is equal to d/M. y i - the activity coefficient of the i**1 solution in the station
ary phase; although for a given solution i, yi is relatively
constant at low concentrations, in principle can vary with
concentration.
K - is the partition coefficient and shows the ratio of the con
centration of i in the collecting phase to that in the air.
An important factor of concentration in equilibrium sampling is partition coefficient:
Ne Cone, (molecules/cm ) in collecting phase a Cone, (molecules/cm3 ) in air When many components are simultaneously present but at a low concen tration, each is collected without substantially influencing the col- lection of the others. If some components are present at high concen trations, the partition coefficient may become modified by interactions among the adsorbed components. According to Dravnieks, et. al.t (1970) the magnitude of the partition coefficient is inversely proportional to the vapor pressure of the pure component at the respective tempera ture (so that K decreased with an increase in temperature) and to the activity coefficient of the component in solution in the adsorbent.
When the collecting phase is non-polar, the activity coefficient for substances with equal vapor pressures is higher (so that K is small because N is small) if the substance is polar. Therefore, the maximum t* amount of a particular odorant that can be collected in a collector filled with a fixed amount of the collecting phase, from a polluted air containing a small amount W(g/cm^) of the odorant is determined by its partition coefficient. The length of time needed to pass a certain volume of air through the collector in order to collect the maximum amount depends on the geometry of the space filled with the collector powder and the air pumping rate.
A suitable collecting phase should have certain characteristics
(1) The collecting phase must not collect too much
water which will interfere with GC analysis.
(2) Characteristic low viscosity is desirable so that
diffusion of the volatiles in the collecting phase
is rapid. 39
(3) Decomposition of the collecting phase should be
negligible when the collectors temperature is
raised during desorption.
(4) The collecting phase should not produce artifacts.
According to Dravnieks and his associates, the amount of water
permissable in analysis by a gas chromatographic method is of the order
of 10**^g per injection. They stated that a sample of such size is
equivalent to an air sample of approximately 50 cm3 of air. Assuming
the routine analytical sensitivity of a typical hydrogen flame ioniza"
tion detector in GC systems for organic compounds is 10 g per compon"
ent, then a method in which water is collected together with organic
substances, is not useful below a concentration of approximately 1 0
to 10"Hg of an organic component per cm3 of air. This they found
insufficient if the criterion is set at 1 0 “*l3g/cm3 or lower, where
several substances can cause odor. On the other hand, water separa"
tion is cumbersome and can introduce its own contaminants and artifacts.
Hence, some collecting phases, such as Chromosorb 102 which does not
collect moisture, could become an important material in vapor collection
for GC analysis.
Activated carbon has great affinity for organic vapors. However
it collects water, cannot easily attain complete desorption, and pos
sesses catalytic activity which produces artifacts.
Methylsilicone fluid (SF-96) with a low partition coefficient
on a teflon support has been successfully used by White (1969). A
superior adsorbent with very high partition efficient i» Chromosorb 102. 40
This is a styrene-divinyl-benzene polymer with a greatly expanded surface. The properties of Chromosorb 102 are such that they make
it the most suitable adsorbent of organic vapor.
quantitative measurements using gas chromatography.— Gas chroma tography is a powerful tool for the separation of complex mixtures into a series of pure components. The individual constituents can then be analyzed quantitatively. Works on quantitative analysis have been cited in literature (Coffman, et al.t 1960; Cvetanovic, 1965).
Quantitative measurements using differential-type detectors depend upon the determination of the recorded peak area or peak height, and the relationship of these quantities to the amount or concentration of solute in the injected sample. Peak height measurements are recom mended when small peaks must be measured or when the band width is narrow. The measurement of peak height is easier than the peak area techniques. However, it is less accurate, especially when there is an unstable baseline.
Peak Area methods. — A number of techniques are used for the determination of peak areas, namely:
(1) Planimetry. In this method, the peak is traced manually
with a planimeter, a device which mechanically inte
grates the peak. The area of the peak is presented
digitally on a dial. This technique is time consuming,
and has the greatest variation between operators. Also,
there is a problem when the peaks are not resolved
properly. (2) Height times width at one-half height. This technique
works best when the peaks are sharp and symmetrical.
The height and half height are determined and the width
measured at one“half height. The area is the product
of the two. The measurement of the width of one-half is
very difficult to make, and it is best made from the
inside of one ink line to the outside of the other.
This method is not accurate for asymmetrical peaks be
cause of the difficulty of determining the true width
at one-half height.
(3) Triangulation. In this method a triangle is constructed
by drawing tangents to the sides of the peak. This tech
nique gives good accuracy for asymmetrical peaks, but
the reproducibility from person to person is poor.
(4) Paner dolls. To use this technique the peak is cut out
and weighed. Good reproducibility can be obtained but
too much time is spent in cutting the paper. The chroma
togram is destroyed in the process which is also unde
sirable.
(5) Disc integrator. This is an electromechanical device
attached to the recorder, A pen attached to the inte
grator travels over the bottom 1 Cff> of the chart; with
the speed of this integrator pen being proportional to
the displacement of the recorder pen. As in the manual
techniques, the peak must stay on the recorder scale. 42
It has advantage of speed, some accuracy, and has
correction for drifting baseline and for partially
resolved peaks.
(6 ) Electronic integrator. These instruments electron!"
cally integrate the peaks and may print out the peak
area and retention time. Has provisions for baseline
drift connection and unresolved peaks and operates
independently of recorder. The main disadvantage is
its high cost.
Standardization or calibration techniques.~wThe height and area of chromatographic peaks are affected by sample size and by factors which influence the sensitivity or response of the detector, such as fluctuations in the operating bridge current, the carrier gas flow rate, the column and detector temperatures (Dal Nogare and Juvet, 1962). For quantitative analysis, these variables must be carefully controlled or
the effect of a variation must be compensated.
Standardization compensates, in part, for the sample"column" detector variations for different sample species. Three standardiza" tion techniques are employed:
(1) Internal normalization. In this technique the quantity
of a compound present in a mixture is expressed as a
percentage of the total area of the chromatogram. For
example, if the total area (sum of peak areas) is equal
to 1 0 0 units and the peak being measured has an area of
1 0 units, then the area percentage is equal to 1 0 JS> of
that peak. 43
This technique has an advantage of not being dependent
on sample size, however, it has many disadvantages. It
is not applicable to this study because (a) of its require
ment that all peaks must be measured whether they are of
interest or not; (b) the response factor for all the peaks
in a chromatogram cannot be calculated since the identity
of some of the peaks is not known.
(2) Internal standard. In the internal standard method of
quantitative analysis, a known amount of a substance not
present in the original unknown mixture is added to the
mixture. The peak area of the added standard is deter
mined from the chromatogram and is compared with the
area(s) of the sample(s) of interest. An advantage of
the internal standard is that its weight proportion may
be made approximately equal to that of a compound of
interest and errors are thereby minimized. It has the
disadvantage of requiring more time for preparing for the
analysis. This method is not applicable to dairy odor
problem. Besides the time factor, an organic compound
to be used as a standard that is not already in the
dairy odor mixture will be difficult to select, since
not all the compounds are identified yet.
(3) External standard or absolute calibration. By this
method, samples of the standard and the unknown are
analyzed independently. It has a limiting factor of 44
requiring accurate control of the sample size for
reproducibility. It has the advantage of saving time
in that the response factor is not required in the
calculation and the standard is the identical compound.
In this study, the External Standard procedure is
employed because it is more amenable to analysis of a
composite sample of odorous volatiles where only some
of the peaks in the chromatogram are of interest.
Odor Production from Anaerobic Decomposition of Organic Wastes.
During the anaerobic decomposition of organic substrates,
odorous volatiles may be produced. The decomposition of the substrate
is effected by microorganisms during processes of cell multiplication
and growth. The odorous volatiles produced are part of the intermediate
and final end products of the decomposition processes. A number of
factors are known to affect the decomposition processes and odor pro-
duction.
Factors Affecting Odor Production
The quality and intensity of odor produced during the decom
position of organic wastes can be affected by aeration, temperature,
pH, electrode potential, microorganisms, and type of waste. In a
biological system, these factors are not independent on one another.
Much research has been done on the effects of these factors on methane production during anaerobic decomposition of sewage sludges. With the
exception of aeration, few studies have been done to determine their 45
effects on odor production. White (1969) studied the effects of temper"
ature, pH, electrode potential, and aeration on the production of prin"
cipal malodors from dairy animal wastes. The effect of aeration (i.e.,
oxygen transfer to the substrate) on odor production is well documented.
Aeration."" If an aerobic environment is maintained during the
decomposition of organic substrate inoffensive odors are produced. This
is one of the important reasons for aeration in waste treatment pro"
cesses. Under aerobic conditions, the end productions of. composition
of organic waste will be C O 2 , HgO, N0 3 , and SO^j, none of which is odor"
ous. However, under anaerobic conditions, that is, in the absence of
dissolved oxygen, offensive odors are produced. The process of decompo"
sition involves series of reactions. Many of the intermediate products
of these reactions are very odorous, while only a few of the end prod"
ucts are odorous.
The effect of aeration on odor production from decomposing
animal wastes has been studied by Ludington, al.. (1967), and White
(1969). Working with poultry wastes, Ludington and his associates
demonstrated the elimination of HjS at low aeration rates. White
worked with dairy waste and he observed the elimination of thiol, sul"
fides, and acetates in the dairy waste volatiles with aeration.
Temperature. ""Microorganisms have an optimum temperature range
for growth. The microorganisms are grouped as: thcrmophiles (45~60°C), mesophiles (25"40°C), and psychrophiles (20"30°C) (Pelczar and Ried,
1965). The optimum temperature for bacterial decomposition of organic waste varies between 30° and 40° (Alexander, 1961, p. 149). 46
Theoretically, bacterial activity doubles with every 10°C rise in temperature up to a limiting temperature. Above this temperature, bacterial enzymes begin to denature and the enzymatic activity slows down for microorganisms, excepting those which are thermophilic.
It is no surprise therefore, that more odors are apt to be produced during warm months than during cold or very hot months.
pH.***’Hvdrogen-ion concentration influences the activities of microorganisms and enzymes in organic waste decomposition. Species of bacteria and enzymes are very specific and have optimum pH ranges. For example, Escherichia coli. enteric bacteria, have an optimum pH range of 6.7 “ 7.0; Witrobacter species, bacteria that oxidize nitrites to nitrates, have a range of 7.6 to 8 .6 ; while Thiobacillus tftiooxidans__ bacteria that oxidize sulfur to sulfate, have a pH range of 2.0 to 2.8
(Pelczar and Reid, 1965). An example of an enzyme is glutamate decar” boxylase, which has an optimum pH range of 5.5 to 6.0 (Sawyer, 1960).
The pH of a biological system has a marked effect on the type of odorous compounds produced. For example, at low pH, the amino acids are ionized and decomposition proceeds by decarboxylation. The optimum pH for this reaction is between 4 and 5. The decomposition of amino acids releases carbon dioxide, and accumulates amines in solution. At high pH deamination occurs with the release of ammonia and acid products accumulated in solution. In a complimentary study, Dague, si. &X* (1970) observed a definite relationship between the pH and rate of gas pro” duction during anaerobic digestion. A pH of less than 6,5 was consid" ered inhibitory, regardless of the volatile acid concentrations. 47
Furthermore, pH level has some effect on the oxidation and reduction
of sulfur compounds. For example, at pH values of 8 and above, most
of the reduced sulfur exists in solution as HS“ and S ions and the
amount of H 2S is negligible. However, at pH levels below 8 , the equi“
librium shifts towards formation of un“ionized HgS.
In general, therefore, at low pH, due to carboxylation pro
cesses, the odors may be associated with amines and production,
while at high pH due to deamination processes, ammonia and volatile
acids may be the compounds causing the odor.
Electrode potential (E^).““Microbial populations oxidize the
complex heterogeneous organic material into more simple, stable com
pounds. This process of oxidation which enables the microorganism to
obtain the energy necessary for growth and reproduction is called
dehydrogeneration (Stainer jU.., 1959). This involves transfer of
electrons, and in the case of oxidation, a loss of electrons. When a
compound loses an electron it becomes positively charged. This posi
tive charge is passed onto a hy.drogen atom, and the loss of both the
atom and electron results in the removal of a hydrogen ion. In anaer
obic conditions, various reducible compounds act as hydrogen acceptors, while in aerobic systems molecular oxygen is the hydrogen acceptor,
resulting in the oxidation-reduction reactions of a biological system
(Dirasian, 1968a).
In biological systems, the electron transfer from one compound
to another results in a potential difference measurable by use of a
potentiometer. The direction in which a reaction proceeds is 48 dependent on the free electrons in the system. Grune et al.t (1958) observed that if the number of electrons is increased more of the
reductant is produced, and if the number of electrons is decreased, more of the oxidant is produced. Therefore, electrode potential can be used as a measure of the state of the biological process.
The E}, developed in a biological system can be determined by substituting for the glass electrode used in pH measurements, a noble metal, usually platinum or gold. The results are read on a standard vacuum tube potentiometer calibrated to read directly in millivolts.
E^and PK have inverse relationship to each other. The effect of raising the pH of biological systems lowers the E^. Since anaerobic reactions proceed in a "reducing environment," low E^ is characteristic of anaerobic conditions. Eckenfelder and Hood (1951) thus concluded that "in many cases, the intensity of an odor produced is proportional to the degree of negativity of the electrode potential." The E-h for anaerobic conditions has a range of +50 to "400 mv, while aerobic con" ditions range from +400 to "200 mv (Dirasian et al., 1963), The E^ for good methane bacteria activity in sludge digestion is "265 to “295 mv (Dirasian, 1968b).
Microorganisms.""Anaerobic decomposition is carried out by a wide variety of bacteria. In the study of microbiology of anaerobic processes of sewage sludges, many microorganisms have been implicated
(Toerien, 1970). A similar study on the anaerobic decomposition of animal wastes is lacking. It is logical to assume that the processes of digestion will be similar in both substrates under identical r
49
environmental conditions, especially with respect to the decomposition
of specific food types in the waste. The increase in population or
creation of more bacterial cells is a function of food concentration in
the substrate. Alternatively expressed, the food concentration in the
anaerobic tank decreases with increased active microbial population,
resulting in turn, with decreased rate of bacterial synthesis. Auto-*
digestion and autolysis may start if no fresh food is added.
There are three phases of anaerobic decomposition: liquefac- 4 *■ tion, acidification, and gasification. During liquefaction, complex
polysacharides are hydroxlyzed to simple sugars, and proteins are deaminized to peptides and amino acids. These processes are brought
about by the secretion of extra cellular enzymes. During acidification
processes, small molecular weight organic acids like acetic, propionic,
or butyric acids are produced. At the final stage, gasification, the organic acids are utilized as substrates by methane bacteria to produce gaseous end products of carbon dioxide and methane. These methane pro ducers are strict anaerobes, inactivated by the presence of dissolved oxygen, and sensitive to variations of pH, temperature, and other environmental changes.
Type of organic waste. The type of organic waste affects the
rate of decomposition process, influences the microorganisms for the digestion, and the decompose tioi products produced. Taking animal waste as an example, it is observed that it consists of partially digested plant and animal materials, and microbial tissues and synthesis products which have been formed in the animal’s digestive tract. Therefore, the 50
decomposition rates of various food components in the waste, and their
end products will vary.
Typical ingredients in swine, poultry, beef, and dairy rations
are presented in Table 6 . It will be noted that the percentage of
total nutrients which are digestible ranges from 50.7% for alfalfa hay
to 82.6% for ground c o m . Therefore, between 17 and 49% of the food
material in the animal's ration will be excreted in its waste.
These food materials in the wastes can be classified into
organic (proteins, lipids, and fats, carbohydrates, organic sulfur
compounds), and inorganic (compounds of Cu, Fe, K, Zn, Ca, Mn, etc.).
Analyses of the organic matter in manure samples have been conducted
by Benne (1961) and Wakeman (1952, p. 304). Stroshine (1970) summar
ized the results which are presented in Table 7.
According to Benne, easily digestible carbohydrates are sugars
and starch which also are reported by Waksman as cold~water soluble and
hot“water soluble. The carbohydrates re'sistant to digestion are com
prised of lignins and portions of the celluloses and hemiocelluloses.
According to Benne, carbohydrates comprise 40 to 55% of the dry matter
in the poultry and swine waste, while they comprise 60 to 75% of the
dry matter in dairy, beef, sheep, and horse wastes. Crude protein
comprises 17 to 2C% of the dry matter in the poultry and swine waste,
while it comprises 7 to 13% of the dry matter in dairy, beef, sheep,
and horse wastes.
It may be concluded that because of higher protein content in poultry and swine wastes, the end products of their decomposition will TABLE 6. — Composition of typical rations fed to swine, poultry, dairy, and beef animals from which waste sample for tests were collected.
Animal Ration5 Swine Poultry Dairy Beef Ingredient i of jo of of jt of Ration Ration Ration Ratio
Ground shelled corn 82.4 65.3 Cracked corn -- 15.8 27.3 Soybean oil meal 1 1 . 15.5 5.3 — Meat and bone scrap 2.0 2.5 - - Silage it* - 43.9 45.5 Oats or rolled oats -- 7.0 18.1 Dehydrated alfalfa meal 2.5 2.5 -- Wheat bran - - 3.5 - Linseed oil meal • — 1.8 «■» 34^ protein pellets -- 9.1 - Dicalcium phosphate 1.1 1.2 - - 'Premix trace minerals 1.0 2.5 1.1 _ Dried fish solubles _ 2.5 Dried whey - 2.5 - Alfalfa hay - - 21.1 -
Nations fed to animals on The Ohio State University farms,
cComposition unknown. TABLE 6. - - Continued
Average Total Composition3 Total Protein Fat Fiber N-Free Mineral Dry Digestible Digestible Ingredient $ TS Matter Protein Nutrients h> TS $ TS £ TS
Ground shelled corn 9.1 4,2 2.1 70.8 1.6 87.8 7.0 82.6 Cracked corn 8.3 J2.1 1.2 74.9 1.1 87.6 6.4 81.0 Soybean oil meal 45.7 1.3 5.8 31.4 6.1 90.4 42.0 78.0 Meat and bone scrap 49.7 10.6 2.2 3.1 23.1 93.7 40.8 65.3 Silage 2.3 .8 6.7 16.2 1.6 27.6 1.2 18.3 Oats or rolled oats 12.0 4.6 11.0 58.6 4.0 90.2 9.4 70.1 Dehydrated alfalfa meal 17.7 2.5 24.0 38.4 10.1 92.7 12.4 54.4 Wheat bran 16.4 4.5 10.0 53.1 6.1 90.1 13.3 66.9 Linseed oil meal 35.2 4.6 8.9 36.7 5.7 91.1 30.6 75.5 ytfo protein pellets 3 4 ** -- - -- Dicalcium phosphate ------Premix trace minerals _- •• __ — Dried fish solubles 71.3 8.5 .6 .8 10.1 91.2 63.5 79.2 Dried whey 12.8 .7 .2 70.1 9.2 93.0 11.5 78.3 Alfalfa hay 15.3 1.9 28.6 36.7 8.0 90.5 10,9 50.7
Value from Morrison, 1959
cComposition unknown. LPl CO TABLE 7. — Percentage composition of dry material in animal manure.
a Carbohydrates______Crude Crude TfiTAf Tunff 1 b Tv no' M 1'" Fat-8 0 __ . Type of Manure Reference Ash TOTAL Type 1b Type 11L Fat Prote % TS % TS % TS % TS % TS %TS
Poultry Benne, 1961 36.1 42.6 15.2 27.4 .8 20.5
Dai ry Benne, 1961 10.6 73.9 31 .4 42.5 1.8 13.6 Waksman, 1952 13.0 - — - -™ 2.8 14.9
Beef Benne, 1961 10.2 75.0 27.3 47.7 1.6 13.2
Swi ne Benne, 1961 28.8 53.0 19.1 33.9 1.6 16.6
Sheep Benne, 1961 10.0 74.6 35.6 39.0 2.2 13.1 Waksman, 1952 17.2 ------2.8 25.5
Horse Benne, 1961 27.9 61.1 31.8 29.3 1.1 9.9 Waksman, 1952 9.1 — —— 1.9 6.8
aMaterial remaining after ignition
^Difficulty digestible carbohydrates c Easily digestible carbohydrates
^Ether-soluble materials
Nitrogen content multiplied by 6.25 f Values are for sample of feces only
Ul w TABLE 7 . — Continued
Cold-Water Hot-Water Soluble Soluble Organ i c Organ i c Type Manure Reference Cellulose Hemicel 1ulose Lignin Matter Matter % TS % TS % TS % TS % TS
Poultry Benne, 1961 — — -- — —
Dai ry Benne, 1961 ------Waksman, 1952 25.2 18.6 20.2 5.0 5.3
Beef Benne, 1961 — — — — —
Swine Benne, 1961 —— — ——
Sheep Benne, 1961 — -- — — - Waksman, 1952 18.7 18.5 20.7 19.2 5.7
Horse Benne, 1961 0 a* - - _ - Waksman, 1952a 27.5 23.5 14.2 3.2 2.4
aVaIues are for sample of feces only 55 consist of more "ammonia-type odor," than is the case with the end products of the decomposition of dairy, beef, sheep, and horse wastes.
In Figure 2, the main paths of the anaerobic digestion of various food material in a waste is represented. The three phases of anaerobic digestion: liquefaction, acidification, and gasification are shown. Most of the odors produced during digestion are released during the intermediate process of acidification.
Characterization of Odor from Decomposing Animal Wastes
Studies of the odors produced from anaerobic decomposition of animal wastes have largely been devoted to the identification of spe" cific odor-producing compounds. Table 8 lists the compounds which have been identified as products of decomposition of poultry, swine, and dairy wastes. However, the quantitative analysis of odor inten sity and threshold measurements have been neglected.
Burnett (1969a) analyzed poultry waste odors using the gas chromatograph. He showed some correlation between odor intensity and concentration of volatile organic acids, ammonia and sulfides and with storage time of the wastes. Also, Ludington et al. (1969) found that dilute poultry manure produced more than undiluted manure. Further more, they observed that the diluted manure produced more NHg but that the undiluted manure released more NH 3 to the atmosphere. The high solubility of NH 3 in dilution water prevents the release of NH 3 from the diluted manure. It was also noted that agitation of waste produced a more offensive odor. 56 DAIRY WASTES
INORGANIC ORGANIC
SULFUEEOUS NITROGENOUS CARBONACEOUS
fe; s EH LIPIDSCARBOHYDRATES O ?• P=4 PROTEINS M l=> [ GLYCEROL SUGARS O’
PEPTIDES FIBER *3 « M M K IT o pq *=J CH o t *aj AMINO FATTYa t : ALCOHOLS JLCJ-QS 1C2HS f Cu fe; M O Q M HEH o < VOLATILE ACIDS •=J o
COMPOUNDS I OF Cu, Ff K, Zn, SULFIDES fe; Mn, Ca, Co, o hH Fe, H, 0
N NH, CELLULOSE LIGNIN H2S I
Figure 2 - Schematic representation of the main parts of the anaerobic digestion process. 57
Table 8. Compounds Identified in Decomposing Animal. Wastes and Their Odor Characteristics
C0H70UNM IDENTIFIED OCOfc Odor Threshold*
T y p . o l Ca ca (Co— on ' N o l. U f a r - Odor r m M p /l1 lo r y JU) laa 1 Vaata C laaa tt'FAC Niaa IIj n ) fo rm u la w t. ncn Q u a lity n T T i r 4 - "IS itO '7 -. Poultry, Suloa, Dalry_ S u lfid e s Hydro|in eullide 34 a (b , e , t < otteft «U« V h it r id -J P o u ltry Kcthyl aulflda : fl3s 32 b ( 22*10 fecayid cabbage 1U 10* ‘ «* “Urc e p ta o a Methyl Hercaptan : h ?* sh 48 b 6« 10* S ' decayed cabba|a | 6 * l 0 ' r Ethyl aarcaptao : 2Af*SH 62 b 2 3 *1 0 ' ,'nplaaaant M s*propyl sarraptao : 3H9*s h 76 b m Putrid, la rllc n-butyl oarcaptan : 4 h«-sh 90 b la lO ' 1 3 1 * 1 0 'J ? Qaeayed eabba|a D a iry rb lo a th a ra Olaethyl sulCld* (C H j)jS 62 I 2J . 1 0 "1 | 92 *1 0 ' 5 mI Diethyl aulllde ( C jH fljS 90 B taul, ta rllc D a iry " . I " 37x10 J 36*10 poultry, Swtna, Dairy A n org anic Aratonla HH3 17 e ,b ,d ,e P u nfan t # faultrjr, Suloa A lip h a tic H«thyl seine CHjKN 30 C pf Poultry, Swine, Dairy A clnea Ethyl seine C.H5MI 44 c , f ,B 6 » I0 * 1 9 6 .1 0 * * Flaby aavm lal D a iry Trleethyl salne ( O IjK N 59 8 5 Poultry, Swlna Trlechyl aelna lC 2f l f ) 3N 101 Cpf * * 1 Facal P o u ltry H a te ro - Ba nro{t>)-pyrrole 117 b . b , e y e llc ( In d o le ) 7 5 .1 0 " * 6 * 10'® Aaloea 3-aethjl- Indole 131 r a t a l f P o u ltry * . * 2 ( ik a t o le ) 6 1 Poultry, Swine A lc o h o ls E th a n o l :, h 5oh b ,a n «p rop aool cn3(CHj)jOH b ,e S M ■ teo-prnp«Dol CH3 (CH2) 2OH b *« c n m a -b u ta n o l b ,e ‘ " ^ chou S M M m lio-butanol s C h J > CHCK2° « a « lao-peatanol c S j > eH< ° la, 20H
» m Aldehydes Methanol (formaldehyde HjCO 30 b«e Pungent 20* 1 0 *1 6* 1 0 *1 m * Ethanol (aeetalde* 44 b .e Pungent -a hyde) I s » • ftopaBoldshyda b .« 3 u S»>n» a h Ieo-butenoldehyde ^ C K C D O b .e aig o a ■ e Haptaldahyda b , * ■ M ValeraIdehyde b .e 1 m * A ldahyd a* Decalrfthyda b ,« f o u l t t T , S'j Lm , D a iry O rg a n ic Acetic acid CH3COOH « ,b ,h Vlu*|*r Ilk. A c id s b - Poultry. Sw lu, Da try Propionic acid c h 3ch?coh a .b .b ptcklallka 1 P o u ltry 2-atthyl propionic CH3- c f£ 00H b.e Sw t*tllk*°2 a c id 21*10,-S U lO - Poultry Suln*. Dairy a»bucyric acid |CHj
• - Earth, «t si. <1971) 1 • SuarMt, (1961) b • Burnett and Dondoro, (1969) 2 • Valuta apply at 26 C and 760 m Hi hi- Burnett, (lib*:* a) bj- lufo«tt, (I9b9 M c - tfe ib rl, <1966) J 4 - ludington, «t al, (1967) • - IWki»l, nr a l, (la**) 4 - Htiw *i*J luavu Merkel (1969) analyzed the atmosphere of an enclosed swine building for the odorous components. He used analytical microchemical tests to identify mercaptans, sulfides, disulfides, amines, and amides. His findings are shown in Table 8. Volatiles from stored dairy waste under controlled anaerobic conditions were analyzed using gas chromatographic procedures (White, et al. 1971). The compounds identified were trimethyl and ethylamine, hydrogen sulfide, methanethiol, dimethyl sulfide, diethyl, sulfide, propyl acetate, and n“butyl acetate (see Table 8). Barth et al. (1971) also analyzed the volatiles from dairy waste. The analytical procedure they used was paper chromatography. The volatile acids identified in both aerobic and anaerobic conditions were acetic, butyric, and valeric, while propionic acid was found only in aerobic units. The amines they identified were methy”, dimethyl”, ethyl” and diethylamine. Hydrogen sulfide, mercaptans, and disulfides were found in the liquid portion of the storage units, while ammonia was identified in both liquid portions and gaseous exhausts from the storage units. It can be noted in Table 8 that: (1) A significant portion of the odorous compounds belong to sulfur and nitrogen compounds. The classes of sulfur compounds implicated are sulfides and mercaptans and the class of nitrogen compounds is amines. (2) More nitrogen compounds have been identified for chicken and swine than dairy waste. This may be due to higher percentages of proteineous material in swine and chicken waste than in dairy wastes. 59 (3) The relative importance and significance of the respec tive compounds with respect to odor nuisance may not be determined until some quantitative analyses have been conducted. EXPERIMENTAL PROCEDURES Instrumentation was developed both for objective and organoleptic measurement of dairy waste odors. Instrumentation Set**un for the Objective Measurement Components of the objective measurement system are shown in Figure 3. [r e c o r d e r !. A lODOR SOURCHl 1------PLATE [electromethi ^ *- f ILAMENT SAMPLE COLLECTOR VOLTAGE AIR POLARIZING CARRIER GAS SAMPLE ►) SAMPLE I---r» H INTEcf0R[ COLUMN TRANSFER INJECTION I s y s t e m I . SAMPLING TRAIN GAS CHROMATOGRAPHY TRAIN Figure 3. Block Diagram for the Objective Measurement 60 61 The sampling train consisted of the following components: odor source, cylindrical sample collector, transfer mechanism, and injection system. The sampling train was first discussed by Dravnieks al. 1968). Using the sampling train, White (1969) set up an instru" mentation for his qualitative study. In this study an instrumentation for a quantitative and organoleptic analysis was developed as dis- cussed beloiv. Odor Source__and C o llection Equipment The set up for odor generation and collection for gas chroma" tographic analysis is shown in Figure 4. It consisted of a set of three 5"liter, three neck, round"bottomed flasks (only one is shown in the figure). The three necks facilitated ease of feeding and sampling plus the introduction of instruments to measure pH, and other par" ameters inside the flask. Standard instruments were used to measure temperature, pH and E^ of the dairy waste in the flasks. Methane gas discharged into the head space of the flask built up pressure inside the flask and flushed out the dairy volatiles into the collector. This flask was connected to the collector by 0.3175 cm (1/8 in.) o.d. teflon tubing. The flow rate of the system was measured by a rotameter which was connected to the sample collector as shown in Figure 4, Diffusion Cell.""The external standard calibration for the quantitative measurement of concentrations of identified compounds was used. The diffusion cell apparatus, shown in Figure 5, was used to measure the quantity of compounds collected. The diffusion cell used was similar to that described by McKelvey and Hoelscher (1957). ■h T =c TEFLON TUBING T f l A J l SAMPLE COLLECTOR WATER RECIRCULATION TUBING WASTE METHANE MAGNETIC STIRRER TANK ROTAMETER FORMA TEMPERATURE REGULATOR Figure 4. Instrumentation for Odor Generation and Collection 63 Figure 5, Diffusion Cell 64 It consisted of two 50 ml round"bottomed flasks connected by a 10 cm long and 0.40 cm I.D. glass tubing. The inlet and outlet lines of the upper flask were designed to promote mixing and to minimize exit effects from the tube. The air“tight stopper prevented the loss of materials from the flask during weighing. The stop cock on the upper flask pro" vided a means of measuring the pressure in the cell. In operation, the lower flask was partially filled with the pure liquid chemical to be calibrated and the entire cell, submerged in a constant temperature bath. The space above the liquid in the lower flask became saturated with vapor. The vapor diffused through the tube into the upper flask, from where it is carried away into the col” lector. Knowing the physical dimension of the flasks and of the other components of the diffusion cell apparatus, it was possible to calculate the quantity of odorous volatiles collected into the collector. Samnle collector.”wrhe concentration of the odorous compounds in the flasks are too low for direct gas chromatographic analysis. It is necessary to concentrate the compounds first. A collector that was developed by Dravnieks et al. (1970) is shown in Figure 6. It con" sisted of 1.8 cm (3/4 in.) O.D. thin-wall stainless steel cylinder, 10 cm (4 in.) long, equipped with 0.3175 cm (1/8 in.) O.D. stainless steel tubing ends. The adsorbent material was Chromosorb 102, a com" mercial styrene”divinyl"benzene copolymer, which has a high surface area, and has been shown to be a suitable adsorber of organic vapors. Five grams of the Chromosorb 102 powder, with an estimated surface area of 1500 m , were put in the collector cylinder. 65 SPRING CHROMOSORB 102 \J SCREEN Figure 6. Sample Collector 66 The powder in the collector cylinder was held in place by two lOOnnesh stainless steel screens, one fixed and the other spring loaded. The spring loading provided reasonable compaction and eliminated channel formation, and permitted use of the collector in either vertical or horizontal position. The outer parts of the collector were joined by silverbrazing. The collector filled with powder was first purified by heating it in a nitrogen gas stream at 200“220°C overnight, then was conditioned at 120°C in a pure helium gas stream at a flow rate of <■2 30-60 c m v m i n for two hours. The apparatus used to condition the Chromosorb 102 is shown in Figure 7. It consisted of an oven, flow meter, and gas tank. The oven was constructed with 7.62 cm (3 in.) O.D. cylinder packed with sand and closed at both ends by a plate that supported one 1.905 cm (3/4 in.) O.D. copper tube. A heating tape of 100“watt capacity was wound round the copper tube, which was connected to a variable transformer that heated the oven above ambient temperature. A dial thermometer was in serted into the oven to monitor its temperature. After conditioning, the collector was closed with teflon caps until vapor collection was to be made. Sample Transfer and Injection System The set up for the sample transfer from the collector to the needle positioned between the cold blocks of the cold/hot block mech anism is shown in Figure 8. During sample transfer, pure nitrogen was passed through the collector which was placed within the oven, maintained at a constant To Variable Transformer DIAL THERMOMETER SAMPLE COLLECTOR OVEN NITROGEN TANK ROTAMETER Figure 7. Apparatus for Purifying and Conditioning Sample Col lector O' To Variable Transformer TEFLON TEFLON TUBING TUB ING SAMPLE COLLECTOR INJECTOR SYSTEM OVEN AT 120° C (SEE FIGURE 9) NITROGEN TANK ROTAMETER Figure 8. Apparatus for the Transfer of Sample Into the Injection Needle o 00 LEVER COLD BIOCK' INJECTION NEEDLE HOT BLOCK SOLENOID TO CPEK COLD BLOCKS Figure 9. Cold/Mot Block Scissors Mechanism 70 temperature of 120°C, by variable transformer. The heat broke the bonds of adhesion of the odorous volatiles onto the Chromosorb 102 adsorber. Hence, when the nitrogen gas was passed into the collector it flushed the desorbed volatiles into the injection needle, held between the jaws of the cold blocks immersed in liquid nitrogen. The roameter measured the flow rate used in the transfer. Injection system.-"The injection system consisted of the in" jection needle, the cold/hot block scissor mechanism and the bellow mechanism. The injection needle and the scissor mechanism are shown in Figure 9 and the details of construction are given in Appendix A. An essential feature in the construction of the injection needle was the restriction at the base of the injector needle. During the injection process, the 0,16 cm Cl/16 in.) O.D. end is inserted into the injection port of the gas chromatograph through a teflon seal, while the base end is connected to a bellows mechanism by 0.16 cm (1/16 in.) O.D. stainless steel tubing. The cold/hot block mechanism was designed to cool the injection needle during sample transfer. This enabled the trapping of the odor" ous volatiles on the sides of the injection needle as the odorous vola tiles were desorbed and flushed from the collector. It also has a feature for heating the injection needle during sample injection into the gas chromatograph. The heating caused the volatilization of the trapped compounds and allowed them to be flushed into the gas chroma" tograph. The cold/hot block scissor mechanism consisted of two hot copper blocks, each of which contained a 100**watt heating cartridge which was connected to a variable transformer and maintained at a constant temperature of 200°C. Two cold copper blocks, welded to copper plates, immersed in liquid nitrogen, were positioned by two tension springs, and attached to 0.5 inch stroke solenoids. In Figure 10, the injection process is being initiated. The detailed diagram of the bellows mechanism and the circuit diagram of the electrical controls on the injection panel are given by White (1969). The bellows mechanism is used to withdraw 0.5 ml of helium carrier gas from the gas chromatographic column through the needle while the cold blocks remained closed. It is equipped with a 3 rpm motor to rotate the cam, a pressure gauge to monitor leakage should it occur in the injection system, and a control panel. The operation is initiated by switching to retract position. The bellows mechanism is actuated by the 3 rpm motor driving the cam. During the process, the cold blocks remain closed while the bellows withdraw 0.5 ml of helium gas from the GC column. To inject, it is switched to the inject position. The cam" controlled microswitches actuate the two solenoids opening the cold blocks and bring together the hot blocks by means of the scissors mechanism. The needle is heated and the cold blocks remain open for the ten seconds that the cam rotates 180°. The cam rotation provides the injection stroke of the bellows. The 0.5 ml helium, previously withdrawn from the GC column flushes the volatilized compound from the injection needle into the gas chromatograph. Figure 10. Initiating Injection Process “ Transfer Samples from the Needle to the GC 73 Gas chromatogranh.-~The gas chromatograph used in the study was a Varian Aerograph, Model 1520-3C with two columns equipped with two flame ionization detectors, and an automatic linear tempera" ture programmer. A twin"channel Honeywell Electronic 19 recorder was used to record the detector response. Instrumentation Set-Up for Sensory, Evaluation The general instrumentation set up for the odor evaluation is shown in Figure 11. The essential features for sensory evaluation are odor sources, the dilution train, and the sniffing hood. Odor sources.— Odor was metered into the dilution train on the basis of the type of analysis to be conducted. For known compounds, the diffusion cell was used. Dairy waste volatiles were metered directly into the train for dilution and GC analysis. By this means, the number of dilutions required to reach odorlessness Cor threshold level) could be measured. Dilution.-" The dilution train consisted of air filters and a series of rotameters. Dilution air was obtained from the laboratory pressure system, which was connected to a commercial air dehumidifier" filter which dried and filtered the air. The purified air was connected to a pressure regulator which was set to deliver outlet pressure to a cross, fitted with three brass metering valves. These provided con trollable flows in the dilution train. One outlet valve was connected to the primary dilution through 0.635 cm (1/4 in.) teflon tubing; another outlet valve connected the secondary dilution section. f | ODOR |“ I ^ DILUTION 2 ^ DILUTION , SENSINGi 1 I HOOD CHARCOAL FILTER I DIFFUSION CELL MIXING CHAMBER WATER BATH FLOWMETER ODORANT- NITROGEN INJECTION SYSTEM WATER BATH FILTER REGULATOR GAS —WASTE CHROMATOGAPH Figure 11. Odor Analysis Instrumentation The bleeding ports and exhaust ports in the dilution train were connected to carbon filter cannisters which filtered the outgoing odorants, thus preventing the contamination of the air in the room. Odorants from any of the sources were diluted with the filtered air. The rotameters with control valves enabled the odorants to be diluted. By this means, several concentrations of the odorants were metered to the sniffing hood and to the collector for the gas chroma- tographic analysis. Sniffing Hood.— The sniffing hood is shown in Figure 12. It was constructed of wood lined with mylar plastic film, which helped to reduce the adsorption of the odorous compounds into the walls of the hood. The odorous air was introduced into the hood through an opening in the lower part. The opening on the front was shaped to permit one’s face to be placed in the flow of odorous air. The ex“ baust outlet was located in the upper part of the hood with a small fan installed to maintain a reduced pressure, preventing leakage of the odorants to the room. The odorous mixture was exhausted through a carbon filter on the top. Equipment.Calibration and Standardization of P rocedures A quantitative study requires the calibration of the equipment used and standardization of procedures before the tests are conducted. In the calibration of the diffusion cell and the dilution train, a steady** state condition was assumed, which implies a continuous supply of odorants Figure 12. The Sniffing Hood 77 through the apparatus at steady conditions of temperature, pressure, concentration, and flow rate at any one point in the system. The general equation describing the diffusion of one gas through a second stagnant gas was used by McKelvey and Hoelscher (1956) to calibrate their diffusion cell. The equation is based on the theory that the net transfer of component A in the Z direction is the rate of diffusion, plus the transfer of A due to the bulk flow (Sherwood, 1965, p. 5). Na - lit" * CNa * Nb> T- “> Where P = Total pressure (atm) N& = Diffusion rate of Component a 2 (g moles/sec/cm ) Pa = Partial pressure of Component a (atm) D = Diffusivity or diffusion coefficient (cm /sec) = Diffusion rate of Component b 2 (g moles/sec/cm ) C = Concentration (g moles/cm ) cL Component a - odorant " b - odor“free air The first term on the right side of the equation is for diffusion, while the second term is for bulk flow. For the diffusion of odorous molecules from the bottom flask of the diffusion cell through the capillary tube into the upper flask where odor-free air is assumed to be stagnant (i.e., - 0), Equation 1 reduces to the following equation: dCa " a » " ? pb - dz f < 2 > but C = p /RT a a then N = ^ — -k (3) a RTp dz RTp dz b d Nb is assumed zero because the odor-free air in the upper flask approaches stagnant conditions due to small flow rates entering and leaving the larger space of the flask. Integrating between the limits Zy and Zg in the direction of the diffusion: -BE in a RT( ^ " Z’y ) Pal Where p ^ and pb;l are the partial pressure of the gas b at the planes Z2 and Z y f respectively, but P ~ p& ■* pb> then: PP 1 R*"Pa2 . . Na = l ln C5) rtCZR P“Pai But the partial pressure of the odorant at the upper plane of the capillary tube is assumed to be zero (i.e., pa 2 = 0)* Then 79 where M = molecular weight of odorant A = cross-sectional area of tube (cm ) L = length of the capillary tube (cm) = (Z2-Z^) P„ = partial pressure of odorant (atm) a r = diffusion rate (g/sec) In Equation (6 b) the only term which needs to be experimentally deter- mined or theoretically approximated is diffusivity, D. The other terms can be obtained or measured during the experiment. Diffusion Cell Calibration Two calibration methods were used: one was semi-theoretical and the other was purely experimental. In the semi“theoretical method, the diffusion coefficient in Equation 4 was estimated using Gilliland’s diffusion equation (Gilliland, 1934): D = 4 3 x 10" 3 T. _ — L 4, — 1— (7) P(Va1/3+ Vb 1 / 3 )2 M a M b where D = diffusion coefficient (cm^/sec) P = total pressure (atms) Va = molar volume for Component a (cm3/g) Vb = molar volume for Component b (cm3/g) Ma = molecular weight for Component a (g/g mole) a molecular weight for Component b (g/g mole) T = temperature (°K) Using Equation 7, the diffusion coefficients for the various compounds were calculated at 1 atmosphere pressure and their boiling point 80 temperature. Diffusion values at other temperatures and pressures were estimated using the following equation based on kinetic theory: p t 3/2 D = D0 -fzS/i (8) o where D = Diffusion coefficient at temperature T and pressure P D0= Diffusion coefficient at 1 atm, and b.p. temperature. Combining Equations 6 b and 8 : „l/ 2 Do PnM A T______r R L T n 3/2 n p " P a The data and the computer program used for the calculation of diffusion rates are given in Appendix B. The purely experimental method was based on measuring the loss in weight of the odorants in the diffusion cell over an extended time. Into the lower flask of the diffusion cell were measured 20 ml of the odorant. The two cell parts were assembled and weighed, then sub merged in a constant temperature bath, A steady flow of 2000 cc/min odor-free air was passed through the upper flask and the loss in weight of the liquid was determined after fifteen hours. The calibration was done for each odorant at various temperatures. Certain precautions were necessary when weighing the cells since the loss in weight was very small. Therefore, finding the exact loss in weight was critical. The sides of the diffusion cell were cleaned of any material accumulated on them during the period the cells were immersed in the constant temperature medium and also dried of the moisture setting on the sides before weighing. The sides of the cells were first cleaned with chloroform, then with acetone, and then vacuum o dried for five minutes at 103 C. Dilution Train Flow Rates With the knowledge of the quantity of odorants diffused from the diffusion cell into the dilution train, a series of rotameters were used to effect the dilution of the odorants to the required con centration level. The dilution train had two main purposes. It en abled the determination of the number of dilutions of an odorant with odor-free air to reach odorlessness (threshold level), and it was used to quantify the compounds in the complex dairy waste volatiles. There were two sections of the dilution train (Figure 11). The following equations were used to calculate the concentration at the respective section. The notation is the same as that used in the computer program (see Appendix B). FR x in6 PPM 1 PPM Concentration of odorants coming out of the Where 1 diffusion cell (part per million) FR Volume diffusion rate of the odoranvs from the cell (cc/min) F Flow rate of odor**free air passed into the 4 cell (cc/min) where PPM2 = concentration of the odorants leaving the 1 st section of the dilution train (parts per million) « flow rate of odor“free air (cc/min) PPM = g.7. x FR IQ? 3 (f4 * Fl> F11 where PPM3 " concentration of odorants leaving the 2 nd section of the dilution train (parts per million) F7 =* flow rate of a fraction of 1 st section dilution, metered for further dilution (cc/min) Fj^ = flow rate of odor“free air (cc/min) Standardization of Procedures Factors generally used to establish standard procedures for the quantitative study are sample size, flow rates, temperature, attenuator, and range settings. Sample size."* Experiments on the sample size were focused on finding: (1) The maximum sample size that could be collected in the collector before breakthrough of the Chromosorb 102 was reached. (2) The minimum transfer time of the adsorbed volatiles in 83 the collector to the injection needle which would enable a complete transfer of the volatiles. (3) The number of injections to enable the complete injection of all the trapped samples in the in jection needle into the gas chromatograph. Both the sampling time and flow rate would determine the sam pling volume and thus, the sample size collected by the collector. Another factor of the sample size was the waste load in the dairy odor generating flask and the daily feeding. The waste load and feeding were kept constant while other factors were varied. To determine the maximum sample size to be collected before saturation or breakthrough was reached, dairy waste volatiles were collected at the following flow rates: 15, 20, 25, 30, 35, and 40 cc/min. Each flow rate was used to collect volatiles for 5, 10, 15, 20, 25, and 30 minutes. Breakthrough was determined by sniffing at the exit end of the sample collector to determine the presence or absence of odor. The maximum volume sampled before the breakthrough was 2 0 0 cc (e.g., 2 0 cc/min for 1 0 min.). To determine the minimum transfer time from the collector to the injection needle, at a constant collector temperature of 120°C, the effect of flow rate and transfer times were tested. For 10, 15, 20, 25, and 30 minutes, pure nitrogen was passed through the collector at 15, 20, 25, and 30 cc/min. The combination of flow and transfer time that enabled complete trapping of the volatiles in the injection 84 needle was obtained by sniffing at the injection end of the needle. . The combination that gave the best result was 20 cc/min. for 15 minutes. Tests were made to determine whether lowering the temperature around the injection needle has any effect on the chromatograms pro" duced. Previously, experiments had been performed (White, 1969) by filling the cooling flask, into which the cold blocks were placed, with liquid nitrogen and the injection needle was kept cold by con" duction. In this study, the temperature around the injection needle was lowered by a constant flow of liquid nitrogen applied directly on the needle throughout the transfer period. Chromatograms produced with and without constant flow of liquid nitrogen on the injection needle showed larger peaks on the chromatograms for those tests where constant flow of liquid nitrogen was applied on the injection needle. Tests were made to find whether a single injection was enough to volatilize all the trapped volatiles in the needle. The controlling factor was the temperature of the hot blocks. It must be hot enough to volatilize all the volatiles yet not too hot to decompose the compounds. A constant temperature of 200°C was used. The second injection showed less than 5 per cent of height of the peaks of the compounds. A single injection with the hot blocks at 200°C was used throughout the study. Flow rates."^The behavior of chromatogram migration as a func tion of flow rate is approximated by the following general rule: doubling or halving the flow rate, halves or doubles the retention time. There are limits for detector flow requirement, noise, and .85 column efficiency. Beyond the flow limits, the column efficiency is seriously affected, with a resulting loss of resolution. The carrier gas flow rate of 30 cc/min, hydrogen 30 cc/min, and air 300 cc/min was found satisfactory for the tests. Temrv rature. -“’Temperature programming was considered neces- sary for the study because it has the advantage of analyzing samples of wide boiling ranges. Samples which normally elute over a long period of time could be analyzed in reduced time. Furthermore, it gives a fairly constant peak symmetry. With the initial temperature of 60°C, tests were run with following programming rates: 4, 6 , and 8 °C/min. The rate that gave the most satisfactory resolution of the peaks was 4°C/min, Attenuator and ranee settings.— A constant range setting of 10*"H amp/mv was used during the study. Since the areas under the curves were calculated with a planimeter, it was important that the recorder should not run off the chart paper so that the peaks of the chromatograms should be established on the paper. Therefore, constant supervision was required so that when the recorder pen was to go off scale, the attenuator was switched to a higher multiplier. 86 Test Procedures Gas Chromatogranhic Analysis of Dairy Waste Volatiles Samples of dairy waste feces and urine were collected from the Ohio State University farm. The feed rations for the cows were dis- cussed earlier and described in Table 7. The waste was refrigerated soon after collection so that each treatment was conducted from the same batch of waste. Three series of tests were conducted. The first series of tests consisted of two flasks containing 600 g of dairy waste (70 per cent feces and 30 per cent urine). The contents of one flask were diluted with distilled water to 3000 ml. Fifty grams of waste were fed to each flask daily. For five days, daily samples were collected and analyzed by gas chromatography. The second series of tests consisted also of two flasks as in the first series but extended to a period of seven days. The final series of tests consisted of three flasks, each containing 600 g of wastes and each diluted to 3000 ml. Each flask was fed with 50 g of fresh waste, daily. Volatiles from each flask were collected and analyzed daily for twelve days. In all tests the flasks were continuously stirred with a magnetic stirrer and daily measurements of pH were taken. Sampling head space volatiles.— To enable the calculation of relative retention times for the peaks of the compounds, methane was used as the standard. Methane was chosen as the standard because it has no odor and also eluted before any peaks in the dairy waste volatiles under the conditions of the experiment. Sampling the head space from a flask under pressure containing the dairy waste was con-* ducted by discharging methane into the head space of the flask. The collector, after being purified in the oven, was connected by means of teflon tubing to a glass tube which was connected to the flask through a rubber cork. Samples were collected at 20 ml/min flow rate over a period of 1 0 minutes. Sample transfer and injection.--Figure 13 shows a sample being transferred to the injection needle. The needle, held by the jaws of the cold blocks which were immersed inside the flask of liquid nitro” gen, was first cooled for ten minutes while the collector was being heated to 120°C. Then the collector and injection needle were con- nected in series. While a constant flow of liquid nitrogen was main tained at the needle, purified nigrogen gas under pressure was passed through the collector at a flow rate of 20 cc/min for 15 minutes. The direction of transfer was opposite to the direction the volatiles were collected. The flow rate was measured by a rotameter connected in series with the needle. At the end of 15 minutes, the injection needle was inserted into the injection port of the gas chromatograph through a teflon seal. The electrical heaters of the hot blocks of the injection mech anism were switched on, while the GC pressure was allowed to equili brate to the initial back pressure of 50 psi. With the cold blocks 88 Figure 13. Sample transfer to the injection needle 89 still in place, 0.5 ml. of helium carrier gas was withdrawn from the GC column into the bellows. The gas chromatograph and the recorder settings were adjusted to the pre-established operating conditions. The injection stroke was initiated when the temperature of the hot blocks reached 200°C. Gas chroma tog ranhic procedure. —‘The sample was injected into 1/8 in by 12 ft stainless steel column, packed with 10 per cent Carbo wax 20 M on Chromosorb P, A W D M C S , 80/100 mesh. Carbowax 20 M has been successfully used in separating alcohols, carbonyls, esters, aromatics, hydrocarbons, and sulfur compounds. The helium carrier gas back pressure was 50 psi, while the flow rates for helium, hydro gen and air were 30, 30 and 300 cc/min respectively. The door of the GC was closed manually half a minute before the injection stroke was initiated. When injection was completed, the GC temperature programmer which had been set previously to give a 4°C/min rise in temperature, was started. The GC range was set at 1 0 "’^ amp/mv, while the attenuator was initially set at 1 , and the recorder speed at 1 min/in. Identification and Confirmation of Chromatographic Peaks Identification is based on the comparison of the retention time of the unknown compound with that obtained from a known compound analyzed under identical conditions. Retention time (volume) is the time (volume) it takes to elute a compound. 90 The use of adjusted retention time (ART), that is, the time measured from the air peak to the peak maximum, was found to be un" satisfactory for identification in this study. This was because ART corrects only for instrument dead volume; it does not correct for the following factors: column dimensions, liquid phase (type and a" mount), column temperature, flow rate, type of carrier gas, and pres“ sure drop. Therefore, relative retention time (RRT), i.e., time measured from the leading edge of a reference standard to the peak maximum, was used in the identification. The reference standard used in this study was methane (CH^). Certain compounds in the dairy waste volatiles had been ten" tatively identified previously (see Table 8 ). It was necessary first to reestablish the presence of (or re"identify) the compounds under the conditions of this study. Studies on identification and confirm" ation were conducted for dimethyl sulfide, diethyl sulfide, n-propyl acetate, and n“butyl acetate. Therefore, to analyze these compounds which are in liquid state at room temperature and pressure, the liquid compounds were diffused from the diffusion cell. The cells enabled the diffusion of known concentration of the compound to the collector. The sample in the collector was transferred to the injection needle, then injected into the GC, using the same procedures and conditions as were used for the dairy volatile analysis. 91 Mass Snectrometric Analysis of Dairy Waste Volatiles The confirmation of the presence of a compound within the dairy waste volatile is necessary before any quantitative measurements are conducted on the compound. The mass spectrometer was used to obtain such confirmations. The GC mass spectrometer experiments were conducted at the laboratories of the Battelle Memorial Institute, Columbus, Ohio. The type of mass spectrometry used is called chemical ionization (Cl) mass spectrometry. Chemical ionization mass spectrometry is a tech nique for the production of mass spectra of compounds in which the ions from the molecules of interest are formed by ion-molecule re actions (Munson, 1971). The advantages of Cl mass spectrometry over other types of mass spectrometry, electron impact ionization (El), and field ionization (FI) are: (1) the production of simpler spectra which are easier to interpret (the Cl mass spectra contain a rela tively small number of ion types, and for many compounds, the spectra contain significant amounts of ions with m/e values approximately equal to the molecular weight of the compounds), and (2 ) the reflection of the different aspects of the structure of a molecule by the Cl mass spectrum. This is very useful in the molecular structure determination and compound analysis. (Field, 1967) Briefly, the method of chemical ionization mass spectrometry is as follows: Within the source of the mass spectrometer there is a high pressure of methane (1 torr) and a low pressure of an additive (approximately 10-^torr). Under these conditions the primary ionization by the electron beam 92 will be predominantly of methane. The major ions of methaneproduced by electron impact, CH^, CH^, and CH2, react rapidly with the very large excess of methane to give CH*, C2 H£f and C-jH*. These three ions which com prise approximately 95% of the total ionization of methane at 1 torr, react very slowly with methane, but rapidly with most other compounds. Consequently, the reactions of these ions with the additives will pro duce ions from the additives which form characteristic mass spectra. (Munson and Field, 19663 These spectra are therefore used to determine the molecular weight and the structure of the additive or the unknown compound, thus con firming the presence of the compound. For the chemical ionization mass spectrometric study, two tests were conducted. The first test was on pure compounds like diethyl sulfide, n-butyl acetate, and n"propyl acetate, and the second test was on dairy waste volatiles. The volatiles were collected from a flask initially fed with 600 g of waste, diluted to 3000 ml and with daily feedings of 50 g. The collection was on the fourth day of incu bation. In each of the experiments the standard procedure established for sample collection, transfer, and injection was used. The GC Calibration Curves for the Known Compounds Of the four compounds studied, the chemical ionization mass spectrometric analysis of diluted dairy waste volatiles confirmed the presence of only dimethyl sulfide (CH^^S and diethyl sulfide Calibration curves were then developed for the two compounds dimethyl sulfide and diethyl sulfide. 93 In order to prepare a calibration curve for dimethyl sulfide, 2 0 ml of the liquid compound were measured into the bottom flask of the diffusion cell. The bottom flask and the top flask of the cell were assembled in a constant temperature water bath maintained at 25°C. The rate of diffusion of the compound from the cell at 25°C and total pressure 769.91 mm Hg was calculated to be 23.11 * 10 ^ g/rain using the semi-theoretical approach, and 2,95 x 10 ^ g/min using the direct measurement method. Table 9 gives the rotameter settings for the serial dilution to various concentrations, and Figure 14 shows the dilution train. The flow rates were converted to standard conditions, thus: V = where V - Volume flow rate of gas mixture in cc/min measured at standard conditions but flowing at pressure, P, and temperature, T. VQ « Volume flow rate of gas mixture in cc/min measured at standard conditions (as given on the rotameter). P = absolute pressure of gas mixture flowing (mm Hg) T = absolute temperature of gas mixture flowing in °K (°K = °C*273) # 1 94 Table 9. Serial Dilution of Dimethyl Sulfide Dilutions Concentrations Semi" Rotameter Settings, cc/min Calculated Measured corrected to standard conditions (ppm) (ppm) F ^1 4 1 F? Fu 1993 1369.8 175.0 2989 913.3 116.7 1993 1993 684.9 87.5 1993 5978 342.5 43.8 1993 5978 897 1993 154.1 19.7 1993 5978 747 4982 51.4 6 . 6 1993 5978 299 5973 17.1 2 . 2 3487 6476 49.8 6476 2 . 1 0.26 Onerating Conditions Water bath temperature: 25°C; Gas mixture temperature: 27°C; Total pressure: diffusion cell pressure ■* barometric pressure = 769.91 mm Hg. Semi-Calculated volume diffusion rate = 2.73 cc/min; measured volume diffusion rate = 0.3488 cc/min. 95 Figure 14. Serial Dilution of Diffused Pure Compounds 96 Starting with the maximum dilution (smallest concentration), the flow rate calculated for each rotameter was set, and after wait- ing 30 minutes for steady-state condition to be established, samples were collected at the rate of 20 cc/min for 10 minutes. The same procedures and conditions for sample collection, transfer, and in jection into the GC used for the dairy waste volatile analysis were also used for the pure compound analysis. Three replications of each dilution level were analyzed. Similar procedures used in preparing the calibration curve for dimethyl sulfide were used to prepare a calibration curve for diethyl sulfide. The number one diffusion cell was also used for the calibration. From the previous calibration of the diffusion cell, the rate of diffusion of the compound from the cell at 4 0 ° C and total pressure, 771.31 mm Hg was 1,554 x 10 ^ g/min for the semi-calculated, and 8.06 x 10 ** g/min for the measured calibration. Table 10 gives the rotameter settings for the serial dilution to various concentra tions. Sensory Tests on Dairy Waste Volatiles The purpose of the sensory tests was to determine the dairy waste odor threshold. The intensity of each stage of the dilution was rated on the basis of a six-point rating system: (0 ) no odor, (1) barely perceptible, (2) distinct, (3) moderate, (4) strong, and (5) overpowering. 97 Table 10. Serial Dilution of Diethyl Sulfide Dilutions Concentrat ions Rotameter Settings, cc/min Semi** Measured corrected to standard conditions Calculated (ppm) (ppm) F 4 F1 F7 F 11 2 0 0 1 6004 233.6 1 2 . 0 2 0 0 1 6004 1 0 0 1 3002 77.9 4.0 2 0 0 1 6504 650 5503 26.0 1.3 2 0 0 1 6504 1 0 0 6504 3.4 0.17 Operating Conditions Water Bath temperature = 40°C; Gas mixture temperature - 25°C. Total pressure = diffusion cell pressure + barometric pressure = 771.31 mm Hg. Semi-calculated volume diffusion rate = 1.87 cc/min. Measured volume diffusion rate = 0.09631 cc/min. 98 The author was the only person conducting the sensory tests because of time, personnel, and equipment limitations. An odor panel is essential in odor studies. However, for this study gross indica” tion of the threshold level would suffice. It was felt, therefore, that the author alone could give such gross estimates. Nitrogen gas under pressure was used to flush dairy waste volatiles from the flask. The gas flow was at the rate of 400 cc/min. The displaced odorous volatiles were diluted serially, using odor**free air and were sniffed by the author at the sniffing hood (Figure 15). In order to prevent odor fatigue, the tests were conducted in an increasing order of concentration (a decreasing number of dilutions). These tests were conducted for each of the three series of tests on dairy waste volatiles. Sniffing of the gases was done once on the first series of tests and twice on each of the others. 99 Figure 15. Evaluating the Dairy Odor EXPERIMENTAL RESULTS, ANALYSIS, AND DISCUSSION Equipment Performance In a gas chromatographic experiment where some components of the instrumentation are directly used in the collection and trans fer of the sample, artifacts are apt to be produced. It is necessary, therefore, that these background peaks be identified. The identifi cation will help in isolating background peaks from peaks produced by the sample being analyzed. Once the isolation is completed and peaks in the sample are well separated from other peaks in the chroma togram, then qualitative and/or quantitative study can go ahead satisfactorily. In addition, equipment performance is considered in terms of reliability and reproducibility of the chromatograms. Identification of Background Peaks In order to identify the peaks produced by contaminants in the individual instruments in the sampling train, the following scheme in Table 11 was developed. Injection Needle and Nitrorren Gas.-- The needle was cleaned first with chloroform, then with acetone and dried. No peaks were produced, showing no artifact in the needle. When pure nitrogen was passed through it in an amount equal to that used when transferring a sample from the collector to the needle, three peaks were produced, 100 101 Table 31 — Identification of peaks produced by contaminants in the equipment in the sampling train Factor in the Sampling Train Information Sought in the G.C. Analysis Blank Needle To know whether the needle pro duced artifacts. Needle + Blank Collector To identify the peaks produced by the collector only. Needle + Collector + Odor-free Air To identify the peaks produced by odor-free air. Needle + Collector + CHi To identify the peak produced by CH ^ . Needle + Collector + CH. + known T o identify the peaks and find k compounds the retention volumes of the k n o w n compounds. Needle + Collector + CH^ + Dairy To identify the peaks produced Waste Volatiles. by the dairy waste volatiles. 102 each of which was about 30 per cent of full scale at attenuator 1 and range 10"11. The use of helium gas as the transfer gas also produced peaks. This showed that both nitrogen and helium gases were not suf" ficiently pure. The peaks produced by contaminants in helium gas were absent only when the gas was first passed through a cold trap before entering the needle. A cold trap was not used in the experi ment because of the problem encountered with frequent closure of the « lines due to freezing of some compounds, possibly carbon dioxide or some moisture, at the trap. For a quantitative study the blocking of the transfer line was very critical, since the actual collected vola tiles may not be transferred when the line was blocked by frozen compounds. On analyzing a collector after conditioning, only two pro nounced peaks, each of which was less than 15 per cent the full scale height, were noted at attenuator 1 and range lO"1^. However, con tinued use of the collector produced more and bigger peaks. This was due to the fluctuation of the oven temperature above 130°C, the maxi mum recommended operating temperature for the collecting material, Chromosorb 1 0 2 . Odor-free air.--The use of a mixture of molecular sieves and activated carbon in the ratio of 2 : 1 to purify the air used in the dilution of the odorous compounds helped to remove most of the con taminants in the air. The small peaks produced were insignificant at the operating GC settings. 103 Methane (CH4 ) .““Methane was used as the standard for deter” mining the relative retention times of other compounds. In order to identify the peak produced by methane, it was collected, transferred, and injected into the GC using the established standard procedures and conditions. Methane did not produce very pronounced peaks, show“ ing that it was only partially adsorbed to the Chromosorb 102, Seven samples of methane were collected and analyzed. Each sample gave an uncorrected retention time (i.e., time measured from point of injec” tion to the peak maximum) of 1.6 minutes. Dimethyl sulfide and diethvl stil fide.“"In order to identify the peaks produced by dimethyl sulfide and determine its relative retention time, it was diffused from the diffusion cell. The diffused vapor line was connected to the methane line and the mixture was col" lected, transferred, and injected into the GC using the established conditions. The relative retention time was measured from the leading edge of methane to the peak maximum of dimethyl sulfide. Similar tests were conducted with diethyl sulfide. The retention times estab" lished for the compounds are presented in Table 12. The coefficient of variation (i.e., standard deviation ex" pressed as a percentage of the mean) for both uncorrected and relative retention times is small. All are less than 3 per cent. The low variability in these results would indicate that the injection system is reliable. Also, it is assumed that the flow rate, temperature pro" gramming, pressures, and other experimental conditions were operating stably. 104 Table 12 -- Retention Times for (CH,)„S and (C-H ) S. 3 2 2 5 2 Test Number (Chh)?S______(C^Hg)2S Uncorrected Relative Uncorrected Relat i' Retention Time Retent ion Retention Time Retent (min.) Time (min.) (min.) Time (1 1 4.4 2 . 9 0 8 . 4 0 6 . 9 0 2 4 . 5 0 3 . 0 0 8 . 4 0 6 . 9 0 3 if.5 5 3 . 0 0 8 . 5 0 7 . 0 0 4 4 . 4 5 2 . 9 5 8 . 0 5 6 . 5 5 5 if.6 0 2 . 9 0 8 . 2 5 6 . 7 0 6 4 . 7 0 3.10 8 . 2 0 6 . 6 5 7 if.6 0 3 . 0 5 8 . 2 0 6.65 8 if.6 0 3 . 0 0 — — Meant min if.55 3 . 0 0 8 . 3 6.8 Standard Deviationt 0 . 0 9 0 . 0 7 0.15 0.16 min Coefficient of Variation, percent 1 . 9 4 a 4 2 \ 8 6 2 * 4 4 105 The contaminants in the equipment having been controlled, and the peaks of those difficult to be controlled having been isolated from the peaks produced by the dairy waste volatiles, identification procedures were initiated. Reliability and Reproducibiiitv The reliability of the apparatus was checked by the repetition of the chromatograms for the various components of the system. For example, when two collectors were separately used to collect pure com" pound and dairy waste volatiles, the chromatograms produced in each case were identical to each other. Using known concentrations of pure compounds in each case ,the peak chromatograms were proportional to the concentration, occurred at a pre"calculated retention time, and had similar shape. Examples of typical peak chromatograms ob" tained are shown on Figure 16. Chemical Ionization Mass Spectrometer of Dairy Waste Volatiles In order to establish a reference standard, known compounds, diethyl sulfide, n-propyl acetate, and n-butyl acetate were analyzed first. Figures 17 and 18 are the results of the analysis. The top graph in Figure 18 is the mass spectrometer reproduction of GC chroma" togram in terms of spectrum numbers. In order to obtain the chemical ionization mass spectrum, the mass spectrometer was programmed to scan the mass range from 44 to 300 at the significant peaks of the J.06 COLUMN - 12'* 5s"SS LIQUID PHASE- CARBCWAX 20 M 10 V. SOLID PHASE - CHROMOSORB P. AW-DMCS COLUMN TEM R-60 tolSCf C AT 4*C/M!N X n i - j q o Q n mocpr-olo FLCW RATE- 30.30:300■ H .He'AIR QOjOr PRESSURE DROP - SOpsi CHART SPEED - I min/m z5n5> CHEMICAL 5Sgo7 n i q i D i (A - TRANSFERRED —ISmin at 20 cc/min 1 DU.H 1 n* HOT DLOCK TEMP - 200*C -To ' 'eg 7=5^? c 3 i, .urrnT CONCENTRATION - 24 ppm i 3 - "* 2 myoit.ui 3 •O °°° X lSg«3 3 pj ■ T >tDO 1-| ro -’ t/I aiQft ll9 s |A MIU. GC CONDITIONS COLUMN - 12'*5S"SS LIOUID PHASE - CARBOWAX 20 M 10V. SOLID PHASE - CHROMOSORB R. COLUMN TEMR-CO tOlSCf C AT 4*OMIT COLUMN - 12'* te" S S FLOW RATE- 30: 30:300■ H He.AIR LIQUID PHASE - CARfiO.VAX 20 M 10V. PRESSURE DROP - SOptji SOLID PHASE - CHROMOSORB P, AW-DMCS CHART SPEED - 1 m in/ln COLUMN TEMP - 60 tolSCf C AT 4*0MIN FLCM/ RATE- 30 30:300=H .He AIR PRESSURE DROP - 50psi 5 chemical- Pure (CH-)9S CHART SPEED - 1 m m/ln COLLECTION - 10 mm. at 2<3cc/min TRANSFERRED —ISmin at 20cc/m in SAMPLE HOT BLOCK TEMP — 200*C CHEMICAL- Pure (C2H5)2S ^ _ CONCENTRATION- 5Q ppm COLLECTION —.10 mm at 20ec/mm TRANSFERRED — ISmin at 20cc/m in HOT BLOCK T E M P -200* C X C O N C E N TR A TIO N - 24 ppm 4 5 8 2 3 M|N. MIN. Figure 16. Similarity of Peaks Produced Using Pure Compounds r* t n m t i t u m i f f Gc-rt co+ii iue1. as pcrmtr Figures Spectrometer Mass 17.Figure a r oc ar S M ra a cr ro rm (a) b Ceia Inzto Seta f S of Spectra Ionization Chemical (b) C - GC (C2H5>?S MS f o Diethyl eeec Compounds Reference 2 1 0 2 2 0 2 3 0 * 1 0 S O 2 C 0 Acetate Propvl Acetate Butyl 10 ? r nrjvn isr is c* U-LlCT to* M Df'tR ^ U T l r m.otii r I I L I N w m p t - i IR t C F M - | J ' suPTU>cr U'.lt l| *vvloft NOi*«,r{r. cm cnrmfiMifloi \st Propyl Acetate Go-rc UHti cr a e j u o c c c m o N x yrurM I** tw ft* SftlC T *Akt O'HH prtNiii H - I 'J T I Y ntr r -*i-» 1FICIIU*- .fj-rn If • *, APIt.! f‘i'» K*-r trtrsuiH j«nzso Butyl Acetate g C(HC (art) tf KTTHKE CBfOKS « ns t o l I } J. . '' Figure 18. Chemical Ionization of Reference Compounds 109 chromatogram. The scanning produced the chemical ionization spectra- depicted in the bottom graph in Figure 17 and Figure 18. When the spectra were compared with the characteristic spectra for pure com” pounds, the lower graph in Figure 17 was identified as diethyl sul” fide, the top graph of Figure 18 as propyl acetate, and the lower that of butyl acetate. The successful results obtained with known compounds showed that the instrumentation used in the study was functioning properly, that the experimental procedures and conditions reproduced satisfac” torily. Mass spectrometer reproduction of GC chromatogram of dairy waste volatiles is indicated in Figure 19. Chemical ionization spectra were obtained when selected peaks in the chromatogram were scanned at their spectrum numbers. The spectra obtained which con” firmed the presence of dimethyl sulfide, (CH^^S; and diethyl CC2H^)2S are presented in Figure 20. Using the chemical ionization spectra, the molecular weights of some compounds were determined on the basis of their fragmentation patterns. The compounds which were identified as present in the sample were pyruvaldehyde, caproaldehydc, benzal” dehyde, acetophenone, and pentyl mercaptan. The chemical ionization spectra for these compounds are in the author’s files. l ILRJ v s t « m w P t m w a t n i « i Figure Figure -19., Dairy Waste Volatile Chromatogram Volatile Waste Dairy -19., m s t m 110 Ill RITtX IM II ON « n .r r r »* p* i ■ tv MIMIl I V M H < r i t i * ' ^ i f i-" MM t I -' ■ H t S I fcMMTvn >•..i • i- j y »» - i»r ii St'* ti ( P*rrt.m. *>».! U 1 ‘ t 7-1 Jr'«Cl • i ' *« -• • l-.'C.- ... I ul M < Alt <*’* UiVL 7XCK ( I I I I •CMiMJll Qr/i sncipui t u r n a com cf cram WLmius (C H o)-jS fi. 210 223 230 2V 2S9 oisnui m« ut •o-rs (wn or a m vucnus WL 120 120 110 ISO 100 170 110 130 200 210 229 220 24 2SB 2Gi Figure 20. Chemical Ionization Spectra of (CH^)p S and (°2H5 ^2S in Dairy Waste Volatlle Diffusion Cel] Calj.b.rfttj.pn Two cells, identified as Cell No. 1 and No. 2, were calibrated for diffusion rates using dimethyl sulfide at temperatures from 10° to 30°C, and for diethyl sulfide at temperatures from 20° to 60°C. The temperature ranges selected were below the boiling points of the com" pounds to prevent a massive convectional transfer through the capillary tube of the cells. The slope of the semi“calculated curve was consistently steeper than the slope of the measured curve for each cell and compound cali" brated. The curves obtained from both calibration methods are given in Appendix B. The difference between the two methods of calibration could be attributed to the extreme difficulty encountered in measuring very small diffusion losses with the available equipment. In the sue" ceeding paragraphs, data obtained from both calibration methods will be used in an analysis of the results. QC Calibration Curves At 25°C, three replications of the concentration calibration curves for dimethyl sulfide were prepared, and at 40°C, three repli" cations of the concentration calibration curves for diethyl sulfide were also prepared. The temperatures were chosen, such that it was high enough to cause the volatilization of the liquid compounds to take place, yet low enough (i.e., below the boiling point) to prevent massive convectional transport of the compound. 113 The concentration calibration curves for dimethyl and diethyl sulfides using the semi"calculated diffusion rates are shown in Figures 21 and 22, respectively, while the calibration curves obtained with the measured diffusion rates are shown in Figures 23 and 24. The experimental results used to obtain Figures 21 through 24 and the chromatograms obtained from one replication of the calibration curves for diethyl and dimethyl sulfides are shown in Appendix C. The shape of these figures is due to absorption of the sample by the stationary phase (Carbowax 20M) of the GC column or instrument and the shape of these curves is typically "S" shaped. The molecules of the compounds migrate into the matrix of the stationary phase. Being held by strong forces, desorption was not easily obtained. However, in absolute calibration methods as used in this study, direct elution is not essential, provided standardized procedures are used. Quantitative Measurements of Pain/ Waste Volatiles_ In order to quantify dimethyl and diethyl sulfides produced by the decomposition of dairy waste, three series of tests were con" ducted to establish the production of these compounds in both diluted and undiluted waste. The tests were designed to show the differences, if any, in concentrations of the compounds released from each of the . treatments. The final series of tests consisted of three identical treatments, all diluted, and all fed daily with wastes. The purpose of the last series of tests was to verify whether any differences in production of these compounds existed when the same dairy substrate was equally treated. 1100 1000 800 700 600 300 10Q .114 250 500 75o iloOo 1250 Concentration ration of (Cl^)^ in Air Sampled in ppmConcent Figure 21. GC Calibration Curve for Dimethyl Sulfide (semi~calculated). \ I t 80 ■ 60 20. 50 1 0 0 ; iio 200 Concentration of CC-,Hr)-S .in Air Sampled in ppm * * Figure 22. GC Calibration Curve for Diethyl Sulfide (serai-calculated) 1000 - 900- 800" 400- 300- 200. 50 100 150 Concentration of in the Air sampled in ppn Figure 23, GC Calibration Curve for Dimethyl Sulfide 80 . 70 - 60 _ ,50 '40 - 30- 2 0 . 10 - 117 Concentration of (CgHg)^5 *n the air sampled in ppm Figure 24. GC Calibration Curve for Diethyl Sulfide (measured) First Series of Tests Data for the first series of tests of five days are given in Table 13 and shown in graphs, Figures 25 and 26. The undiluted dairy waste produced both dimethyl and diethyl sulfides. The amount of diethyl sulfide measured in undiluted waste was consistently larger than the amount of the same compound measured in diluted waste. The smaller amount of diethyl sulfide which was measured in the diluted waste might be due to its slight solubility in water. Solubility might have caused only a fraction of the compound produced to be re- leased for collection. Since dimethyl sulfide is insoluble in water, it is assumed that any differences in concentrations between the diluted and undiluted waste must be due to other factors. Second Series of Tests The second series of tests were conducted for seven days. The presence of dimethyl and diethyl sulfides was confirmed by the chromatograms from the treatments. On the first day of the test, no measurable peak of diethyl sulfide was observed in any of the chroma" tograms.. Table 14 and Figures 27 and 28, show respectively the con centrations measured and the chromatograms obtained from each of the treatments. Again, the diethyl sulfide concentration was observed to be higher in the undiluted waste than in the diluted form. Third Series of Tests The third series of tests was conducted for twelve days. The purpose of this series was to find whether there were differences in TABLE 13. First Series of the Quantitative Measurements of Dimethyl and Diethyl Sulfides in Dairy Waste Volatiles Date Dimethyl Sulfide (CH^^S Diethyl Sulfide ( C ^ ^ S Diluted Undiluted Diluted Undiluted Peak Area Peak Area Peak Area Peak Area Cm2 P P ^ PPm2 Cm2 PPm-L PPm2 Cm2 ppmx PPm2 Cm^ ppm^ ppm2 * 8/17/72 9.81 17.4 2.24 1.35 2.4 0.14 NMP — — NMP — 8/18/72 224.32 520.0 65.63 31.81 56.5 7.31 1.22 4.0 0.21 3.61 12.0 0.60 8/19/72 880.84 1210.0 154.67 NA — — 3.50 11.5 0.58 NA — 8/20/72 865.00 1190.0 153.13 NA — — 3.23 10.1 0.53 NA— 8/21/72 322.06 645.0 82.00 771.35 1093.0 142.20 1.00 3.3 0.17 12.65 .66.2 3.25 NA = Not Analyzed NMP = No Measureable Peak ppra = Concentration obtained from the semi-calculated calibration curve £ 1 o PPm2= Concentration obtained from the measured calibration curve (CH3 )2S G-C- CONDITIONS c o u w n - t t v t e - s s liouid phase- carbon ax 20 m io *a SOUD PHASE- CHROMOSORB R, AW -O-CS COLUMN T E M R -6 0 tolSCP C A T^O M M FLCW RATE- 3 0 :3 0 :3 0 0 .H :He AIR PRESSURE D R O P -50psl _ C HA RI SP E E D - 1 min/|n C H E M I C A L - Dairy Waste COLLECTION — 10 nun at 20cc/mm TRANSFERRED“ 15mjn at 20cc/m in * * HOT BLOCK T E M P - 200* C CONCENTRATION- (C^feS P 142.20 PpBl (C2H5)2S =3iJ25 10 12 13 14 15 16 17 18 19. 20 21 22 23 "la MIN. Pigure 25. Chromatogram Obtained from Sampling the Undiluted Treatment on the 5th Day of Incubation, 1st Series e. M “O °° II -p|l cn SSs ^ ^ % o £352",e b , , o„ - KtJ i a. •-SwS.'.SS 15 16 17 18 19 £0 21 22 23 — Figure 26. Chromatogram Obtained with a Sample from the 1st Test Series Diluted Treatment 5th Day of Incubation 121 i TABLE 14. Second Series of the Quantitative Measurements of Dimethyl and Diethyl Sulfides in Dairy Waste Volatiles. Date Dimethyl 1Sulfide (CH3)2S Diethyl Sulfide, ( ( ^ H ^ S Diluted Undiluted Diluted Undiluted Peak Area Peak Area Peak^Area Peak Area 2 cm2 P P ^ Ppm2 cm ppm PP™2 cm ppmL ppm2 cm^ ppmt PPm2 8/31/72 12.84 22.7 2.95 22.58 40.0 5.2 NMP — — ’’ NMP —— 9/1/72 25.10 44.5 5.75 17.55 31.1 4.04' NMP -- — 14.97 90.0 4.0 9/2/72 82.90 145.0 20.8 102.55 209.0 37.5 1.48 4.8 0.294 2.39 7.4 0.394 9/3/72 ' 150.69 375.0 48.4 89.10 177.0 22.4 2.10 7.0 0.346 10.60 41.0 2.02 9/4/72 96.64 170.0 24.3 13.61 24.2 3.1 NMP — — 13.50 84.0 3.63 9/6/72 NA -- -- 389.29 619.0 92.0 NA 22.54 118.0 5.25 NA = Not analyzed. NMP = No measureable peaks. ppir^ = Concentration obtained from the Semicalculated Calibration Curve, ppnu 51 Concentration obtained from the measured Calibration Curve. w to G.C. CONDITIONS COLUMN - 12'xl8"S.S. LIQUID PHASE-CARBOJVAX 20 M 10°/» SOLID PHASE - CHROMOSORB R. AW-DMCS, C2V 2S ■fr 1 10 1*2 13 14 16 17 123 8 M I N. Figure 27. Chromatogram obtained from Sampling Diluted Waste of 2nd Series on the 4th day of Incubation G.C.. CONDITIONS COLUMN - 12’x te" S.S. k!9^ D PHASE_ carbqvax 20 M 10'/,. ISHR PHASE~ CHROMOSORB R AW-DMCS e^V ,nN TEMR-60 to 150° c AT^C/MIN. FLCMI R A T E ' 3 0 :3 0 :3 0 0 = H He AIR PRESSURE D R O P - 5 0 psl ' He AIK CHART SPEED - 1 min/in SAMPLE c h e m i c a l - Dairy Waste COLLECTION — 10 mln. at 20cc/min TRANSFERRED —15mfn at 20cc/nnin HOT BLOCK TEMP - 200" C CONCENTRATION-(eg-) S =22>4 ppm ( f I « H f* i O n n n m era cn i. I U ’I 125 the release of dimethyl and diethyl sulfides when the wastes were similarly treated. The average concentration of dimethyl sulfide estimated during the days of incubation for the three flasks was 552 ppm when using the semi“calculated calibration curves and 74 ppm when the measured calibration was used. For diethyl sulfide, the average concentration was 4 ppm when evaluated with the semi“calculated cali” bration curve, and 0.2 ppm with the measured calibration curve. A statistical test of the treatment means at five per cent level showed the average concentration of..dimethyl sulfide in the second flask to be significantly less than the mean concentrations in the other two flasks by 30 per cent. The mean concentration of diethyl sulfide in the third flask was significantly greater than the means measured in the other two flasks by about 30 per cent, also. The differences might be due to differences in biolobical decomposition rates. The results of this series of tests are given in Table 15 and the chromotograms are shown in Figures 29 through 31. Sensory Evaluations Sensory tests were conducted in order to determine the odor threshold of the decomposing dairy waste, or the number of dilutions that would render the dairy waste volatilcs odorless. Since most of the odorous compounds in the dairy volatile mixture are unknown, and their concentrations or emission rates are * Table 15. Third Series of the Quantitative Measurements of Dimethyl and Diethyl Sulfide in Dairy Waste Volatiles Date Dimethyl Sulfide Peak Areas, Cm2 Concentration, Peak Area, Cm2 Concentration, 1 2 3 Mean ppm1 PPm 2 1 2 3 Mean PPn^ PP”2 10/1/72 NA 49.46 NA 87.6 172.0 26.6 NA NMP NA — -- - 10/2/72 NMP 49.46 93.94 51.1 100.0 14.1 NMP 0.06 1.61 0.55 1.8 0.09 10/3/72 160.00 507.87 336.52 334.8 643.6 84.4 0.52 1.55 1.35 1.14 3.8 0.188 10/4/72 474.84 494.48 377.80 448.7 776.3 100.0 0.90 1.29 0.64 0.94 3.1 0.155 10/5/72 491.35 228.13 243.93 321.1 570.1 82.2 1.61 NMP 1.42 1.01 3.3 0.165 10/6/72 425.29 243.22 549.16 405.7 725.7 93.75 2.26 1.03 0.90 1.40 4.6 0.23 10/7/72 565.68 NMP 280.77 282.2 499.6 76.6 1.03 0.43 0.64 0.71 2.7 0.116 10/8/72 478.97 470.71 355.35 435.0 767.6 99.2 1.48 0.77 0.69 0.98 3.2 0.165 10/9/72 186.58 358.00 208.52 284.3 551.3 76.8 NMP 2.97 0.69 1.22 4.4 0.20 10/10/72 429.42 169.69 396.39 331.8 634.3 84.2 NMP 1.16 0.32 0.75 1.9 0.123 10/12/72 402.58 NA 838.71 620.6 628.6 75.5 2.00 NA 5.03 3.51 10.5 .575 NA = Not Analyzed to NMP = No Measureable Peak ppm = Mean Concentration Obtained from the Semi-Calculated Calibration Curve ppm25= Mean Concentration Obtained from the Measured Calibration Curve G.C. CONDITIONS COLUMN - 12‘x Is" S.S. LIQUID PHASE-CARBCWAX 20 M 10*/» SOLID P H A S E - CHROMOSORB R t AW-DMCS. COLUMN TEMP. —60 to 150* C AT^C/MIN. FLCW RATE- 30:30:300= H :He;AIR 0*16 PCP_. 29 •.Chromatogram obtained from Sampling Flask I of the 3rd Series of Test, on the ifth. Day of incubation t It'w Figure 30. Chromatogram obtained from Sampling Flask 2 of the 3rd. Series of Test on the 4th day 4th the Test on of Series 3rd.the 2 of Flask Sampling from obtained 30. Chromatogram Figure of incubation* of .. CONDITIONS G.C. OLCIN- 0 i. 2c/ tn 20cc/m t a min. 10 - COLLECTION cal- - l a ic m e h c in/in m 1 - SPEED CHART OUN - COLUMN RNFRE 1mn t 0cmin 20cc/m at —15min TRANSFERRED RATON- f - N TIO A TR N E C N O C LW 30: 300 ;He;AIR = 0 H 0 :3 0 :3 0 3 AW-DMCS. - E T R, A R FLOW CHROMOSORB E- S A H P SOLID IUD HS-ABMA 20 M 10*/# M 0 2 PHASE-CARBCM/AX LIOUID HOT BLOCK TEMP - 200° C 200° - TEMP BLOCK HOT 50psi - DROP PRESSURE 12'xlS"S.S. Dairy Waste 17 128 18 day 129 I 7 •' 7•' 118 I 4th, 4th, I 6 I I 4 I Series of Tests on the the on Tests of Series 3^* ppm of the the of 3 3 MIN. Waste (c^3>zs *■ 90*4 90*4 *■ (c^3>zs CC2H5)2S =0.105 ppm LIQUID FHA5E-CARBCWAX 20 M 10“/, • COLUMN - 12'x )8" S.S. SOLID PHASE- CHROMOSORB R, AW-DMCS. PRESSURE DROP - 50psf FLO.V RATE- 30:30:300= H :He:AIR COLUMN TEMP.-6 0 to150° C AT4°C/MIN CHART SPEED - 1 min/in HOT BLOCK TEMP - 2 0 0C “ TRANSFERRED—I5min at 20cc/minCONCENTRATION- CHEMiCAL-Hairy COLLECTION - 10 min. at 20cc/min G.C. CONDITIONS SAMPLE 3*2S CCr of Incubation, of Figure 31, Chromatogram Obtained from Sampling Flask Flask Sampling from Obtained Chromatogram Figure 31, hu it* sV 130 also unknown, certain assumptions were made in calculating the dairy odor threshold. It was assumed that the odor of individual components in the mixture behaved in an additive fashion. In other words, if the concentration of the individual fractions in the mixture are known, the total concentration of dairy odor can be calculated, thus: N i=l where C. = concentration of the individual ith odor relevant i fraction in the mixture, in ppm, or grams, or in terms of emission rate, g/min. N = number of odor relevant compounds in the mixture CT = total concentration of odorants in the mixture, in ppm or grams, or in terms of emission rate, g/min. Another assumption was that no interaction occurred in the dairy volatile mixture, for such process could decrease the total odor intensity which might also alter the quality of the odor. For the study, the "total’* concentration of the dairy waste was approximated by the sum of dimethyl and diethyl sulfide concen- trations obtained in the quantitative experiment, thus: CT = C (C1I3)2S * C (C2H5 )2S To dilute the volatiles liberated by the decomposing dairy waste, nigrogen gas under pressure was delivered into the dairy flask 131 at the rate of 400 cc/min. The flushed volatiles from the flask were serially diluted, while the odor was evaluated at the odor“sensing hood. Table 16 gives a series of rotameter settings corrected to standard conditions for the dilution of the dairy volatiles. Necessary adjustments were made either to increase or decrease the dilution during the tests. The same serial dilution was used to find the odor threshold of the pure compounds. Table 16. Rotameter Settings for Serial Dilution of Dairy Waste Volatiles Dilution f 5 f , f 7 Concentration * F 11 ppm Rotameter settings in sec/min 1 400 - 400 - 32,868 2 400 3003 400 3003 1.28 3 400 6006 400 6506 0.31 4 400 3003 50 3003 0.16 5 400 6006 50 6506 0.03 ♦Concentration calculations based on the measured calibration data The concentration column in Table 16 was calculated in the follow” ing manner. From Table 13, the sum of concentrations measured for di methyl sulfide and diethyl sulfide for the diluted waste was 82.17 ppm on the basis of measured calibration data. 132 The concentration of the first dilution in Table 16 was then approximated using the following equation: 82.17 cc/min of ndorants 1,000,000 Scc/min of odor“free air 400 cc/min of C = 0.032868 cc/min of odorants or 32868 ppm. The concentrations of the remaining dilution levels were calculated using the following equation: .6 F7 x C x 10 PP™,3 - CF^FjJx where ppm^ * concentration of dairy volatiles delivered to the sniffing hood in parts of odorants per million parts of odor“free air (volume concentration). C ~ approximated flow rate of odorants from the dairy flask (cc/min). F? - flow rate of the fraction of the first dilution mixture (cc/min). F^ = flow rate of used to displace the dairy volatile into the dilution train (cc/min). F^ and F ^ = 1st and 2nd odor“free air flow rates (cc/min). Forexample, the 2nd dilution in Table 16 becomes: 400 x 0.032868 x 10^ _ , 3 - (400 . 3003) x 3003 ' ^ 133 First_Series of Tggts. The sniffing tests were conducted for both the diluted and the undiluted wastes on the fifth day of incubation. Table 17 shows the concentration of the diluted and undiluted waste calculated, using the rotameter settings of Figure 16. Odor threshold of volatiles from the diluted waste was between 0.03 and 0.16 ppm, while for the undi" luted waste, it was between 0.28 and 0.07. Hence, it took larger volumes of air to render the volatiles from the diluted waste to odor" lessness than it took the undiluted waste volatiles to reach threshold. Table 17. Odor Intensity Ranking for the First Series of Tests Diluted Waste Undiluted Waste Concentration Intensity Concentration Intensity ppm Rank ing ppm Ranking 0.03 0 0.07 0 0.16 3 0.28 0 0.31 3 0.56 3 1.28 4 2.28 4 32,868 5 58,180 5 Note: Odor Intensity Ranking 0 No odor 3 Moderate 1 tBarely perceptible 4 Strong 2 Distinct 5 Overpowering The quantitative measurements gave higher concentrations for diethyl sulfide in the undiluted waste than in the diluted waste. Therefore, ,134 the cause of lower odor threshold for the diluted waste than for the undiluted waste must be due to the production of other compounds, like mercaptan and aminos, which have lower thresholds than that of diethyl sulfide which is 25 x 10 3 ppm. Second Series of Tests During these series of tests, sensory evaluation was conducted on the fourth and seventh day of incubation. Table 18 shows the con centrations and the intensity ranking evaluation for the concentrations. Table 18. Odor Intensity Ranking for the Second Series of Tests Diluted Undiluted Concentration Intensity Concentration Intensity ppm Ranking ppm Ranking Dav 3rd 0.01 1 0.02 0 0.04 1 0.07 1 0.08 3 0.15 2 0.33 4 0.59 3 8,437.60 5 15,157.80 5 6th N.A. - 0.05 1 0.19 2 0.37 2 1.52 4 38,900 5 During the third day of incubation, the diluted wastes produced volatiles that had lower thresholds than the undiluted waste. 135 Third Series of Tests Sensory evaluation was conducted on the fifth and ninth day during incubation. Table 19 shows the concentrations and the intensity ranking for the treatments. The results of the odor ranking show a similar trend to the previous diluted wastes (i.e., lower threshold for the diluted wastes than the undiluted waste). Table 19. Odor Intensity Ranking for Third Series of Tests Concen. Intensity Concen. Intensity Concen. Intensity Day Ranking ppm Ranking ppm Ranking 5 th 0.05 0 0.03 1 0.03 1> 0.22 3 0.13 2 0.13 3 0.43 1 0.25 3 0.20 2 1.77 4 1.03 4 1.07 4 45,106 5 26,240 5 27,445.76 5 9 th 0.03 0 0.04 0 0.03 0 0.11 1 0.17 2 0.12 2 0.22 2 0.33 1 0.24 1 0.90 3 1.37 4 0.98 4 23,120 5 34,892.08 5 25,005.54 4 Sensory evaluat ion of the pure compounds gave the following threshold levels: 0.26 ppm for dimethyl sulfide, and 0.017 ppm for diethyl sulfide. These values which were calculated on the basis of measured calibration data are much higher than the published threshold values for these compounds, which are 2 x 10**^ ppm for dimethyl sul fide, and 25 x 10 ** for diethyl sulfides. A note of warning is given in the interpretation of these odor threshold values obtained for the pure compounds of dimethyl 136 sulfide and diethyl sulfide, and for the dairy odor. An odor threshold is a statistical value, and determination by a single person (as was the case in this study) is far from being accurate. Not all of the odor relevant compounds in the mixture are identified yet, and not even all of the identified compounds have been quantified. However, the investigation is useful in that it is a step in the right direction. Dairy Odor Waste Units The quantitative measurement and the organoleptic measure" ments were correlated in an attempt to postulate the number of dairy waste odor units. The quantitative technique was used to measure the concentration of dimethyl and diethyl sulfides within the dairy waste odor, while the organoleptic measurement provided the determination of the odor threshold of the known compounds and the dairy waste odor. An odor unit is postulated to be the amount of odor, that after dilution in a cubic foot of air, brings it to threshold or barely perceptible level. Since threshold is a random value with respect to individuals, the definition has an additional requirement, i.e., that the threshold will be established by half the observers in a panel of eight or more persons. Therefore, knowing the flow rate of the odorant from a source, the odor emission, in terms of odor unit/min, is simply the product of odor unit and flow rate. This approach to odor analysis is more directly applicable to a source consisting of one predominant com" ponent odor. For a complex mixture of odor source, like dairy waste, 137 where there are many odor-relevant components, a more applicable definition of odor unit is that suggested by Guadagni et al. <1966). Ui = Fi/Ti where Ui = number of odor units attributable to th i odor relevant component Ti = threshold concentration of the i*"*1 component in ppm Fi = concentration in ppm of the i ^ component in the mixture. Threshold concentration, Ti, in volume parts of odorant per million volume parts of odor-free air is arbitrarily defined as representing one odor unit. The total odor units of the odorous mixture could then be obtained by summing the odor units of the individual odor relevant components in the mixture, thus: N Ui = U, T i=l The above summation equation is based on the assumption that the components of the mixture of dairy odor are additive at the threshold levels. In Table 20, the odor units associated with the daily releases of dimethyl sulfide is tabulated for all the series of tests. The odor units were calculated using the odor threshold based on the measured TABLE 20. Odor Units Associated with Dimethyl Sulfide in Hundreds of Odor Units ""L*. Day Diluted Waste Undiluted waste la 2b 3C la 2b o.u.d o.u.e o.u.(1 O.U.e o.u.d O.U.6 o.u.d o.u.e o.u.d O.U.8 1 0.09 1.15 O . U 11.50 1.0 13.30 0.05 0.07 0.20 2.60 2 2.52 32.80 0.22 22.90 0.54 7.10 0.28 3.65 0.16 2.02 3 5.95 77.40 0.80 10.40 3.25 42.20 — — 1.44 18.75 4 5.89 76.50 1.88 24.40 3.85 50.00 — — 0.86 11.2 5 3.15 41.00 0.93 12.10 3.16 41.10 5.46 71.10 0.12 1.55 6 — — 3.61 46.90 — — — 7 —— 2.95 38.30 3.54 46.0 8 3.82 49.60 9 2.95 38.40 10 3.24 42.10 11 —— 12 2.90 37.80 _ a _ _ 1 = 1st series of tests. b 2 - 2nd series of tests, c 3 = 3rd series of tests. U f — Z i O.U. - Odor units based on odor threshold measured during this study (i.e., 26x10 ppm) e -2 O.U. = Odor units based on odor threshold given in literature (i.e. 2 x 10 ppm). 139 calibration curve, and the odor threshold given in the literature. Similar odor unit tabulations for diethyl sulfide is given in Table 21. It will be observed in Figure 20 that the dimethyl sulfide odor units based on literature thresholds were higher by approximately a power of ten than those obtained on the basis of odor threshold evaluated during the study. In Figure 21, the differences were even higher. All these differences were to be expected, since the odor threshold of individuals for any one compound is not a constant value but a range of values. The odor units for dimethyl and diethyl sulfides give the odor components which the compounds contribute to the total odor of the dairy odor mixture. As Guadagni and his associates pointed out, the odor units calculated say nothing about the odor quality of the final mixture, and it does not say anything about the relationship between stimulus or con“ centration and intensity of sensation above threshold. Table 22 is a summary of the average concentrations and odor units measured during the study, which were associated with dimethyl and diethyl sulfides in dairy waste odor. The Table reveals the importance of proper calibration in determining the concentration of an unknown compound, and the use of a reliable odor threshold value in the calcu lation of odor units. The concentrations of dimethyl sulfide obtained with semi-calculated calibration was about seven times higher than those obtained with measured calibration curve, while for the concentration of diethyl sulfide, the semi-calculated calibration gave values that were higher by over twenty times. However, the odor units calculated for Table 21. Odor Units Associate^ with Diethyl Sulfide in Hundreds of Odor Units Diluted Waste Undiluted Waste Tests: 1st Series 2nd Series 3rd Series 1st Series 2nd Series Day 0Ud OU6 OU2 OUe 01^ 0Ue______OU^______OU^______OU^______OU® 1 1 1 :• > — o 2 0.03 0.01 3.60 0.07 24.00 0.47 12.0 3 0.07 23.20 0.03 17.76 0.02 7.52 - 0.05 15.0 4 0.06 21.20 0.04 13.84 0.02 6.20 - «• 0.24 80.0 5 0.02 6.80 - 0.02 6.60 0.38 130.0 0.43 145.2 6 0.03 9.20 — — 7 0.01 4.64 0.62 210.0 8 0.02 6.60 9 0.02 8.00 10 0.01 4.92 11 — — 12 0.07 23.00 Note: OU^ = Odor units based on odor threshold measured during this study (85 x 10 ^ppm) OH 0Ue = Odor units based on odor threshold given in literature (25 x 10“5ppm) Table 22, Summary: Average Concentration and Odor Units Associated With Dimethly and Diethyl Sulfides Dimethyl Sulfide Diethyl Sulfide Diluted Waste Undiluted Waste Diluted Waste Undiluted Waste Measured Semi-Cal. Measured Semi-Cal. Measured Semi-Cal. Measured Seml-Cal. Calibration Calibration Calibration Calibration Calibration Calibration Calibration Calibration Average Concentration 65.41 495.65 34.87 250.24 0.26 6.78 2.73 59.8 for all Tests, ppm Odor Units x 10^ (Based on odor Thresh- 2.51 2.37 1.35 1.19 0.31 0.04 3.2 0.35 holds determined during the study) Odor Units x 10 (Based on odor Thresh- 32.70 247.83 17.44 125.14 10.72 263.0 109.39 2390.0 hold from literature) Note: Odor Threshold levels (0 8 3 ) 2 8 (C2H5)2S ■ Measured Calibration 0.26 ppm 85 x 10“3 ppm Semi-Calculated Calibration 2.1 ppm 1 .7 ppm Literature 2 x 10"2 ppm 25 x 1 0 “5 ppm 142 dimethyl sulfide, were in the same range for both methods of calibration, the measured calibration odor units being only slightly higher. The odor units calculated for diethyl sulfide using the measured calibration were about ten times higher than those obtained from semi-calculated calibration. Using the odor threshold values in literature as standard, high odor units were obtained for each compound. The values obtained were higher than those obtained on the basis of the author's sensory evaluation of the odors. This difference, as stated earlier, was due to higher odor thresholds the author obtained from his sensory evaluation . of the pure compounds (see footnote to Table 22). Therefore, using the concentrations of the compounds obtained ly measured calibration, the following points are evident in Table 22: (a) The diluted dairy waste released more dimethyl sulfide than undiluted waste; 65.4 ppm versus 34.9 ppm. (b) The undiluted dairy waste released more diethyl sulfide than the diluted waste; 2.7 ppm versus 0.3 ppm. (c) The amount of dimethyl sulfide released from either the diluted or undiluted dairy waste was always higher than the amount of diethyl sulfide released from the waste. Using the published odor threshold as standard, together with the con centration obtained from the measured calibration, the following points about the odor units produced by the compounds are evident in Table 22. 143 (a) Diethyl sulfide released from the undiluted dairy waste produced the highest amount of odor units. The amount of odor units produced was higher than those produced by dimethyl sulfide from either diluted or undiluted dairy waste. (b) For the diluted waste, more odor units are associated with dimethyl sulfide than with diethyl sulfide. As stated earlier, the lower released of diethyl sulfide in the diluted waste might be due to its slight solubility in water. Finally, from the preceeding, it would seem that a system of using the number of odor units emitted from a source as the basis of odor control will be not only a valid one but a workable one. It has implied odor threshold definition. An establishment of a maximum number of odor units, say 500, which a plant or an industry must not exceed, will help solve the growing odor problem in animal or otherindustry. However, the biggest problem is enforcement, because very little is known about the odor-relevant components in the source mixture, their concentrations, and their odor thresholds. 144 SUMMARY Odor nuisance is a serious environmental problem facing animal production industry. Many developments are being made in con finement animal production, making it possible for larger numbers of the animal to be crowded on small land areas. Urban areas are encroaching on the agricultural facilities, and offensive odors from the animals com pel neighbors to take legal action. Odor control is therefore a primary requirement for livestock production in an urban society. For the basis of engineering design for odor control, both identification and quantification of the odorous com pounds may be needed. A combined use of objective and subjective measure ments of the odors are the most adequate approaches to this end. This study was undertaken to develop an odor analysis instru mentation for both objective and subjective measurements. The odorous volatiles liberated from decomposing dairy manure was analyzed using the instrumentation developed. A gas chromatograph was used in both identifi cation and quantification of dimethyl and diethyl sulfides present in the liberated dairy waste odor. Chemical ionization mass spectrometer was used to confirm the presence of these two compounds in the dairy odor mixture. Threshold measurement of the dairy waste volatiles were conducted. The odor analysis instrumentation was designed, assembled, and operated in the Agricultural Pollution Control Research Laboratory of The Ohio State University. The objective (or analytical) instrumen tation consisted of a cylindrical sample collector, a mechanism for 145 transferring the sample concentrated in the collector into a specially designed injection needle, a mechanism for injecting the sample into the GC for both GC and mass spectrometer analysis, and diffusion cells for the quantitative GC calibration of the amount of odorous compounds in the unknown odorous mixture. The organoleptic instrumentation consisted of a dilution train and a sniffing face mask. A polymer adsorbent, Chromosorb 102, was the collecting material put into the specially designed cylindrical collector. At room tempera ture samples were collected from the head space of the dairy waste in the flask at the rate of 20 cc/min for 10 minutes. After collection, the collector was placed in an oven maintained at a constant temperature of 120°C, while the samples in the collector were transferred to a specially designed injection needle. Nitrogen gas was used to flush the desorbed samples in the collector into the injection needle at the rate of 20 cc/min for 15 minutes. When the sample was being transferred into the injection needle which was supported between the cold blocks of the cold/hot block scissor mechanism, it was cooled by the cold copper blocks, dipped into liquid nitrogen. This enabled trapping of the volatiles as they were desorbed from the collector. During injection of the sample into the GC for analysis, the cold/hot block scissor mechanism was operated by solenoid to bring the hot copper blocks, maintained at a constant temperature of 200^C, into contact with the injection needle. The temperature allowed 146 the volatilization of the samples. The injection into the GC was com pleted by means of the bellow mechanism. The diffusion cell, an apparatus for measuring very dilute gas mixtures, was used to prepare GC calibration curves for the known odorous compounds. When an odorous compound was put into the bottom flask of the cell, which was placed in a constant temperature medium, calibrated amounts of the odorant diffused from the cell into the dilution train. The dilution train consisted of air filters and series of rota meters. Filtered air from the laboratory pressure system, was used to dilute the calibrated amount of the odorants diffused from the cells to the required concentrations. The sniffing face mask was used to evaluate the odor from the dilution train. The study was designed to analyze the volatiles from decompos ing dairy waste. Two conditions of the waste were analyzed. One was stored in flasks, diluted and stirred; the other was also stored in flask, was not diluted and was not stirred. The loading rates for both treat ments were the same, and both were stored at constant temperature of 25°C. From the GC and chemical ionization mass spectrometer analysis of the diluted and undiluted dairy waste volatiles, the presence of di methyl and diethyl sulfides were identified and confirmed. Using the GC, the concentration of dimethyl sulfide and diethyl sulfide were measured. This was made possible by the combined use of the diffusion cells which diffused a calibrated known amount of the compound into the dilution train. 147 The absolute method of calibration was used in developing the GC calibra tion curves for dimethyl and diethyl sulfides. From the chemical ioniza tion mass spectrometer results the following compounds were tentatively identified; pyruvaldehyde, caproaldehyde, benzaldehyde, acetophenone, and pentyl mercaptan. The quantitative measurement using these known com pounds showed that the collection, transfer, and injection system were reliable, and samples were reproduced within plus or minus 10 percent on the average. During the days the stored dairy waste was sampled and analyzed, both and were released from the diluted and undiluted treatments. The concentration of (CH^^S released from either the diluted or the undiluted waste was higher than the concentration of (0 2 ^ ) 2 ^ from either the diluted or the undiluted waste. The concentration of diethyl sulfide released from the undiluted waste was about ten times greater than that from the diluted waste. During the first day, no measurable peak was measured for (C2 H^)2 S from either of the treatments. The average concentration of about 0.3 ppm and 2.7 ppm were measured forCC^H,.)^ for the diluted and undiluted waste, respectively, and the concentration for was 65.4 ppm for the diluted and 34.9 ppm for the undiluted waste. The sensory evaluation of the dairy waste odor was conducted on selected number of days, for each treatment during the test runs. The threshold measured for the diluted waste was lower than that obtained for the undiluted waste by about 10 fold on the average. 148 On the basis of defining one odor unit as the concentration of the odor at the threshold, the objective measured values of this study were correlated with known odor thresholds of the pure compounds. 3 3 Thus it was calculated that 3.3 x 10 and 1.7 x 10 odor units were attri buted to dimethyl sulfide released from diluted and undiluted waste res- 3 3 pectively. For diethyl sulfide, 10.9 x 10 and 1.1 x 10 odor units were obtained for the undiluted and diluted waste, respectively. 149 CONCLUSIONS An instrumentation for quantitative measurement and sensory evaluation that would prove useful in analyzing animal waste odor has been developed. When decomposing dairy anaimal waste was analyzed using the instrumentation, the results obtained were reliable and repro- ductibility was satisfactory. Main conclusions from the study are the following: (1) An adsorbent material, Chromosorb 102, was satisfactorily used to collect volatiles from decomposing dairy waste. From the reproducible results obtained, it proved useful in quantitative study whena constant volume of the sample was collected at room temperature. (2) The combined use of GC and chemical ionization mass spectrometer in analyzing the dairy waste volatiles identified and confirmed the pre sence of dimethyl sulfide and diethyl sulfide. Other compounds tentatively "identified" as present in the mixture were pyruvaldehyde, caproaldehyde, benzaldehyde, acetophenone, and pentyl mercaptan. (3) The quantitative measurement of the diethyl and dimethyl sulfide released from stored diluted dairy waste gave an average value of 0.3 ppm • for diethyl sulfide and 65.4 ppm for dimethyl sulfide for days that tests were conducted. For the undiluted waste, the values were 2.7 ppm for diethyl and 34.9 ppm for dimethyl sulfide. Thus, higherconcentrations of diethyl sulfide were released from undiluted waste than dilute waste, while higher concentation of dimethyl was found to be released from diluted than from the undiluted waste. 150 (4) The sensory evaluation showed that the diluted dairy waste had lower odor threshold level than the undiluted waste. (5) Even though the concentration of dimethyl sulfide which was measured was greater than that of diethyl sulfide, yet the number of odor units associated with diethyl sulfide was found to be greater than that associated with dimethyl sulfide. For the diluted waste, the number 3 3 of odor units was 3.3 x 10 for (CH^^S and 1.1 x 10 for (0 2 ^ ) 2 $ and 3 for the undiluted waste, the number of odor units was 1,7 x 10 for (CH3)2S and 10.9 x 103 for ^ H ^ S . 151 RECOMMENDATIONS FOR FURTHER RESEARCH The odor analysis instrumentation designed, developed and tested during this study was successfully used in quantitative and organo leptic measurement of dairy waste odor. It is hoped that further studies in analyzing not only the dairy waste odor but also other animal waste odors will be initiated and continued. This will involve laboratory sample testing and field sample analysis. Being able to define odor in terms of amounts, for example, odor units, is very essential for odor control and possible legislative odor control laws. For future tests using the instrumentation, it is recommended that finer temperature control for the oven be acquired; that another compound other than methane be used as a standard, for example, a higher molecular weight hydrocarbon, and that a GC column that would separate amine and mercaptans be used. APPENDICES 152 APPENDIX A Equipment Diagrams Figures in this section give the detailed construction of the cold/hot block scissor mechanism, the collector , and the diffusion cell. 153 154 5m m 1.5 cm 5cm 10cm KEY A. 3.2 mm. (1/8 in.) 0.0. B. Spring 1.19 mm. (3/16 i n.) DIA. 3.81 cm. (1£ in.) FREE LENGTH C. SCREEN 100 MESH D. STAINLESS STEEL PIPE 1.905 cm (3A in.) O.D. , 1.57 cm. 5 mm (0.62 in.) I.D. 2cm Figure 32 Sample Collector 155 STOP COCK vmm J AIR TIGHT STOPPER INLET OUTLET GROUNDED CONNECTION SPRING 3.33 cm 10 cm CAPILLARY TU9E 0.50 cm I.D. 3.33 cm 50 ml ROUND-ROTTOMED FLASK Figure 33 Diffusion Cell 156 3,2 m m ■Heating Cartridge 1.6 mm \ 1.6 mm 2.4 mm 2.4 mm coid Biock- 2.4 mm Injection Needle Scissor Mechanism Levers 3.2mm 32m m 1.3 1.3 cm cm 2 4 mm 4.8m m FIGURE 34 Cold Blocks of Injection System in Contact With the Injection Needle 157 2.4 mm ' 3.2 mm 1.3cm -5.7 c nrr 6.4 mm 3.2 mm\ HOT BLOCK HEATING CARTRIDGE 9.5 mm 24m m •3.2 mm t _L1.9 cm 5 .4 c m T 3.2 mm COLD £LOCK 1.3 cm 11.4 cm :zr.D X X 3.2 mm OD. 1.6mm O.D. INJECTION NEEDLE FIGURE 35 Detail Diagram of Parts of the Injection System 158 cm SOLENOID 4cm 1.6crT 2.5cm Radius 2.5cm Radius COLD BLOCK 17 8cm FIGURE 36 Solenoid Attachment to the Cold Block APPENDIX B Equipment Calibrations 159 Pressure, mm Hg 600 500 400 200 300 100 0 10 IUE3 Partial VaporPressure ofKnown Compounds FIGURE 37 20 ■DimethylSulfide 30 40 Temperature, °C DiethylSulfide 070 50 60 Toluene 80 90 100 161 TABLE 23 Dimensions of the Diffusion Cells Cell No.______Length of Capillary______I.D. of Capillary 1 10.025 cm 0.385 cm 2 10,200 cm 0.460 cm 3 10.040 cm 0.385 cm 4 10.040 cm 0.385 cm 162 Computer Program The computer program contains the calculation of diffusion coefficients using Gilland Theory, the diffusion cells calibration curves at various temperatures, and serial dilution of a calibrated Diffusion rate at a chosen temperatures TABLE 20 Identification of Symbols for Computer Program M=MOLBCULAR WEIGHT ,GM/GM HOLE D=OE NSI T Y » G M/CM3 T=AbS0LUTE TEM,KELVIN, WATER BATH TEMPERATURE PA=PARTIAL PRESSURE ,ATMOSPHERES MV=M0LECULAR VOLUME,CM3/GM P=BAROMETKIC PRESSURE,ATMS DG=DIFFUS1ON COEFF. FROM GILLIAND THEORY DR= DIFFUSION RATC= DIFFUSION CELL EQUATION (G/SEC) MFR = MOLECULAR FLOW RATE,MOLE/MIN FR= FLOW RATE,CM3/MIN PPM1= PARTS PER MILLION FIRST DILUTION PPM2= PARTS PER MILLION SbCOND DILUTION PPM3=- PARTS PER MILLION THIRD DILUTION FA= AIR FLOW RATE TO DIFFUSION CELL,CC/M1N Fl= AIR FLOW RATE FIRST DILUTION,CC/MIN F7= FIRST DILUTION FLOW RATE-,CC/MIN FI 1= AIR FLOW RATL SECOND DILUTION ,CC/M IN cl = uoiiing point temperature DCP = Diffusion Cell Pressure, atm PT = Total Pressure = P + DCP, atm TD = Diffusion Cell Capillary Tube Diameter, cm TL = Diffusion Cell Capillary Tube Length, cm k : AL M jMV TrlrK (b ) U.'U i k (d ) tPR (b ) ,PPMl(b ) ,P P M 2 ( b ),PPM3lb),C(b) *L’LP I b ) » PI { b i * "' il jibJ »lL(b),«K(b)iyFil(bi L - L tL G R L m i.- {b » 7 * ) M 11( jl'iV t L-I »Pmi T i? Re A,. [ b j 12 ) l. L P ( i ) » T G ( i )» I l ( i ) 11 r* LhM/t i .l j.g • A ) 12 huK'-!Al Ijhiu.H ) ob r-i-Ki-.Al I' .\=N--l L g u La k hii&HT,G*/GM MOLL'//' D = DE NS1T Y» GM/CM3 ' // i 1 t = « : . ^ L o k ikMjKt.LVl.'i'//* PA= PARIi AL PRESSURc j ATI-iuiPHL Rt i>'// i* MV-Hl L l LLLk K V g .-U.*1L fCMb/GK'// 1 P=BAROMETKIC PkEbSuRt ,ATMS ■// 1* ^v.-uihFULI-.Jj Lj-.cPF. FKoM GiLLIAMg TPiEURY'// i' t, J F FL)E1L.KA"I L FkLM MCKL l UEY A N u HLC LSCliLR ,Grt/SEC'// i' /iC k = . icLtCLLAK FLLiW RATE , ML1 Lt/MIN */ / ' PR- FLCW KA Tt.tCNJ/Kik' / / A' P P 1 — kAkTE PtK ;-.ILL1gR FiKbT ul LG1 loN •/ / l, * r'pr,2= PART b PlK Mi LLIlN £:_CGi«u ulLol i UN '// C* PF'Rb^ FAR lb P i K THIR g Gi LU11 DU •// L' k ^ — Aik FLg « k*-i L I g ul FF'JS 1 LLLLtLC/filfl1// ii ' 1-1= A-lK rLLW K m I*. FiRSl L 1 lG i 1LR rCC/i'.IM'// pi r 't- rikoT lILgTIgU FLu.. RA T I. T CC/Ri N '// g' Fli- AIR FLUrt RAIl EICgM: b 1LUTI gR »CC/RiFl1 // ) c2 PGM'iAl {• /•■ = ' tr 10.h ,2X, "U = *» .lb.GT2X» ';iV=' tFlC.A,2X , ,bT=' ,FiU.4,2X, 1* f h - 1 »t-j.G.Af2X» '1=' jFiL. AfE/t , P=, tF1u«Aj *iA » At.i>=' * » E lb • o / ) G A F^K.'.AT ( 1 uo= 1 » L 1 b • ~ »b A» * L> k = * TElb.»- ? b A» 1 riFR= * »Ei:>«6»bX» ,PR = *jt:ib»t>t 1 b A f * C — * L;Ki* * yi.iL* u/ / ) I Eg PuKnA r t * F*y 1 tr / .iii jXf 'r i 1 i H .2 » jA t * rPMa ' »Llb.fat_»X» * PPM 2 i k Fg k Rm I ( * F A ,TF7.E»_A»*t-l,»F/.GfjA»,F V ,tF7.2»GXj,FljL,»FY.2*3Xr I *RP,L ib.t/V) K tL “• i_ • I O t*1 U = i • \J c0 K.HH/aT I 1 l>CP= ' »F a G .*♦ f ga * 1 TUB E b 1 A= '« F10. A » gX * * TiJbE LENT= ' »Flo .At AGAt *T^rML PRcSSuKl = 11 fFlO.A-»2X» 'AcSi1-* »PiL »o//) l = i m R(a ) = J . Ia^ T ^ ( I) =?**:/A . PT (I ) = p ^21>.A + GC P (I ) 163 C(IJ=ALG g {PT(i )/(PT(I)-PAJ) G^ = tJo^ ( ( 1/g J +2*? • ( 1/2 ) ) **2 l.'H * ’ ’ ’ ' on nj no " ...... " T + 0 ~ 1 0 PI, ~n (0*10*1) 4T 0 T **<; m no nr ni no -j) jt -ld 7' r C n I ■"* 0 ,(T)?WdcJ‘ n H 4H*Td**»H(^0T *">) U T>IO ? .r- OJ. no c o 'ir* 7TTTJ H i ;______T T jni-vd PO. • OOpn =■ T t-j hr .*o o o t =/_ 1 • ' rr. -u no * noo^+Td - T-) . { • m < jd * (T )TWdd‘ Td‘ P-!( 07 T ‘ m IT '* 1*. no -oi no (* non°" j n * t n nr m+v-n/fo+TT-ov-n = mzvMd t < •non.7 = T d VJ/CJ+-TT*(r)\n) = (T )T!'dd „ •n.-jo^7 = *’d C I )T>iO* ( i n * ( T )Sd* (I) ¥=»M* (T )>m*0,- (70*0 ) TLIMM IS07* (T )ld * (T )1L* { I ) rtl* ( T )r» T ' (00*0) i« T»vt 00 V* d ‘ J, * Vd*10‘ AV.’ *n*i.! (.:o*:)]iTvy (■7o*o)T) ; \ . " t ( D D ^ ’^ l = ( 1 ) 0*1 - , O/'on* ( x )MC=( r ) V-? !'/( T )}J ’= { rj >KjW ■no*(T)Mn = ( t ) t y n ini*?M)/K***i*m'}*'1d * ( m v * ,,s!:00) = (T)V’ 3.T/\'p-0 0 c-**((w /'T+’ ^<’/',T)*^ **n )*^ ^ no-o^Njp TABLE 24 Diffusion Cell Calibrations for Diethyl Sulfide Experimental Water Bath 20°C 30°C 40°C 50°C Calibration Temperature______. Diffusion Cell Number 1 2 1 2 1 2 1 2 Total Pressure 773.43 775.08 773.58 775.23 771.31 773.06 762.68 764.43 (mm Hg) Air Flow Rate 2,000 2,000 2,000 2,000 2,000 2,000 2,000 2,000 (cc/min) Weight Loss .0180 .0277 .0503 .0440 .0595 .0479 .0755 .0680 (gm) Time Expired 1066 1079 983 973 738 734 582 532 (min) Diffusion Rate (gm/minfX 1 0 J 1.69 2.56 5.12 4.52 8.06 6.53 12.97 11.66 Diffusion Rate a (gn/minjao^ 5.53 7.74 9.47 13.25 15.54 21.81 25.72 33.95 Calibration Volume Diffusion .66 .93 1.131 1*58 1.87 2.6l 3*07 4.30 Rate (cc/min) T 3.15 3.14 5.39 5.38 8.85 8.85 l b . # * 14.59 (p - p ) K O' 166 30- o ♦H Calculated 10- Y = 6.3x10 + 9.5x10 X “f- 5 X = T iln / _ P _ V noKJ10 Figure 38. Diffusion cell >. 1 Calibration Curve for Diethyl Sulfide Diffusion Rate in (g/min x 10 ) iue 39.FigureDiffusion c-.:il No. d Calibration For DiethylSulfide in °K2 in Measu,re Calculated 167 TABLE 25 Diffusion Cell Calibration For Dimethyl Sulfide I Experimental Water Bath 10°C 1'5°C 20°C 25°0 30° C Calibration temp. C Diffusion Cell Number 1 2 1 2 1 2 1 2 1 2 Total Pressure 763.11 765.88 762.85 765.70 763.56 766.46 769.91 772.79 772.25 775.08 (mm Hg) Air Flow Bate 2,000 2,000 2,000 2,000 2,000 2,000 2,000 2,000 2,000 2,000 (a/min) Weight Loss .1300 .1355 .1270 .1665 .1798 .2049 .2135 .2370 .2750 .3161 (gm) Time Expired 747 760 734 737 851 854 723 727 675 675 (min) Diffusion Rate 1.74 1.78 2.14 2.26 2.113 2.400 2.95 3.26 4.074 4.683 (gm/min^XlO4) Diffusion Rate 12.26 23.88 47.22 Calculated 7.89 11.03 17.11 17.11 23.11 32.54 33.95 Calibration (Ratee (c“ min)°"-93 1 *3° ^ 2-°2 2-02 2*82 2'73 3-83 “ -01 5.58 Diffusion Rate in (g/min x 10 ) 10 Figure 40. DiffusionCellFigure iJoi CalibrationCurvefor 1 Dimethyl Iff )5 2 alculated Sulfide 169 Calculated 2Q. Measured- X - in ^ J — j in °kTp Flguie 41. Diffusion Cell No. 2' Calibration Curve for DircetNyl Sulfide APPENDIX C Tables 26 and 27 contain the GC calibration data for dimethyl and diethyl sulfides, respectively, for diffusion Cell No. 1. Figures 41 through 45 are the GC calibration chromatograms for (CH and for one of the three calibration replication. 171 172 Table 26. GC Calibration Table for Dimethyl Sulfide at 25°C Area (Cm2) Concentration (ppm) Replications 1 2 3 Semi“Calculated Measured 1060.0 - tm 1369.8 175.0 586.32 486.27 652.38 913.3 116.7 425.29 282.84 - 634.9 87.5 - 113.29 155.87 342.5 43.8 76.39 96.52 61.94 154.1 19.7 25.29 26.58 39.32 51.4 6.6 9.29 10.19 - 17,1 2.2 3.48 1.55 2.1 0.2 Table 27. GC Calibration Table for Diethyl Sulfide at 40°C Area (Cm2 ) Concentration (ppm) Replications 1 2 3 Semi“Calculated Measured 78.45 79.03 83.10 233.6 12.0 10.97 18.13 8.90 77.9 4.0 10.19 6.52 8.71 26.0 1.3 0.90 1.10 - 3.4 0.17 173 x * p r c o n d i t i o n s , C, c C O N D ITION S , . , , c o l u m n - j o m 'O’'. , C O L U M N T E M P - b O lO'SCf C AT .CC/MIN FLOW BAIE- 30 30 300.H He AIK PRESSURE DROP -50051 ? ^ WR A t V - 3 0 30:3Q0.H .Hr All* c h a r t S P E E D - 1 min/ir> PRESSURE DROP - 50PM ^AR T 5PCED- 1 I"'"'*" SAMPu.C^)cftL COLLECTION - 10 m m JT ?0cc7rmn SAMPLE , (CHt JdS TRANSECRRED-lSm.n .it 20cc/m,n CHEMICAL- V ^ jocc/'P'0 h o t b l o c k t e m p CONCENTRATION - 1 5 . D ppHl HOT OLOCK T*-1*1/ Jt.. /I CONCEflWT ®N- ©i o , ppm Q C CONDITIONS lIo u i d V'h a s e c /:r p o v w t o m to -/. G C CONDITIONS SOUD PHASE- CHROMOSORBP. A.V-OI^S C O L U M N - 12 * Is' S S c o l u m n t e m p .-g o wis er c LIQUID PHASE - CARF'TWAX 20 M )C". FLOW RATE- 30 30:300.1! He AIR SOLID PHASE - CHRO'-OSOR.'J P. A.V-OMCS PRESSURE DROP - SODS' C O L U M N 1 E M P - G 0 lOlSCf C A U O ' - l l I CHART SPEED - 1 m m /m FLCW RATE- 30 30 300. H He A m PRESSURE DHCI’- S O p m CHART SPEED - 1 mm/in ^fiLEcffbN - 1 ^ ^ SAMPLE TRANSFCRRCD-ISmm At ..Otc/m.n C H E M I C A L._ - < o W HOT BLOCK TEMP - 200 C C O L L E C T I O N ~ 10 min j: ZOcc/mn CONCENTRATION- 3 . 2 p p m TRANSFERRED-iSmm .-.t J O c c / m n HOT BLOCK TEMP - OOC'C CONCENTRATION- Q 2 ppl* MIN Figure 42. Calibration Chromatograms for (C^s ) ^ OTnOyirn y p r n H i ajcOgoQ a in 5 C o = ?; •j cd tom ± ■HV] « H i mzOn r ' n n n . . C 73 £^2 QT O ~Qn ^ o i m"nr5m‘:S‘s-Ssi-' a nj i QD OJOfifc W M I plso MIN. QiHnn. ^TJIDl/rrt U n i j Q n v O tj -lOiorn O O ? 0 l I ajr-QOso 3 X aj r- o o n o zn];rnh >nprCPr|.V iS5RS5 J» fti Qi^r-U r- noil-Cr»yc3J35S57 m2 1 > nz ^-+o | S g T S W H h O 1 8S'S«**i 051 1 0 W v ' h * zJ.'lO09 I j* J s - r ^ 3 7S ^ o « * * n X 1 "33 iigsso" ? o^? =r ^ CO iu 3 ? -uyEQJ3 W 3 -0 Oil 3 i? gui9 Irv Ps** iBBSir' Ia fViSS O 2 P * rt n •O *0 Q« I^OlQ •a 3 3 9 3 ; ^ 2 3 5 3 *1333 TJ 5i£° *j§9 a. *o* s fi V. "0 I 2 3 4. v MIN. ; vj * 4 Figure 43. Calibration Chromatograms for (CH3)2S G C. CONDITIONS G.C. CONDITIONS COLUMN - 12'xlS"SS. COLUMN - 12,x }8 " S .S . LIOUID PHASE-CARBOWAX 20 M 10 V. LIOUID PHASE - CARBOWAX 20 M 10*/. SOLID PHASE - CHROMOSORB R, AW-DMCS SOLID PHASE- CHROMOSORB R. AW-DMCS COLUMN TEM P,-60 tOl5CP C AT-‘TC/MIN. COLUMN TEM R-60 10150’ C ATtfOMIM FLOW RATE- 30:30:300* H :He:AIR FLOW RATE- 30:30:300*H :He:AIR PRESSURE DROP - 50psi p r e s s u r e d r o p - sopsi CHART SPEED - 1 min/in CHART SPEED-.1 min/in SAMPLE CHEMICAL— C H EM IC A L — COLLECTION - 10 min. at 20cc?min COLLECTION — 10 min, at 20cc7min TRANSFERRED —15min at 20cc/m in TRANSFERRED —15mln at 20tC/m in HOT BLOCK TEMP — 200*C HOT BLOCK TEMP - 200*C CONCENTRATION- ^ 3 CONCENTRATION- Q.17 ppm x ii MIN. 'j Ln > FIGURE 44. Calibration Chromatograms for (C2H5)2 S A, G.C. CONDITIONS G.C. CONDITIONS COLUMN - 12‘x IS" S5 COLUMN - 12'xl8"S5. LIOUID PHASE-CARBOWAX 20 M 10*/. PHASE- CARBOWAX 20 M 10V. SOLID PHASE- CHROMOSORB R, AW-DMCS, SOLID PHASE - CHROMOSORB R AW- DMCS COLUMN TEMP.-eOtQlSO" C ATiPOMIN COLUMN TEMP.-60to15Crc AT^C/MIN FLCM/ RATE- 30:30:300*H :He:AIR o b ^ R A T E ~ 30;30:200*H :He;AIR PRESSURE DROP - 50 psi PRESSURE D RO P-5 0 psi CHART SPEED - 1 m in/ln CHART SPEED - 1 m ln/jn SAMPLF CHEMICAL— C H E M IC A L — ^OLUECTION - 10 min at 20cc/min COLLECTION — 10 min. at 20cc7min TRANSFERRED—15mir at 20cc/m in t TRANSFERRED—15min at 20cc/m in HOT BLOCK TEMP - 200*C HOT BLOCK TEMP - 200*C CONCENTRATION- 1 2 # Q p p m CONCENTRATION- 4 #Q ppm UIN. FIGURE 45. Calibration Chromatograms for S REFERENCES Alexander, Martin. 1961. Introduction to Soil Microbiology,. New York: John Wiley and Sons, Inc. Amoore, J.E. 1952. Stereochemical Specificities of Human Olfactory Receptors. Perfum. Essent. Oil Rec., 43: 321. Amoore, J.A. 1962. The Stereochemical Theory of Olfaction, 1. Identification of the Seven Primary Odors. Proc. Sci. Sec. Toilet Goods Assn.. 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