bETERGENCY OF CHLORWATED THSODQUM PHOSPHATE ON MILK-PROTEEN 5:3st FRGM S‘QAENLESS STEEL AND GLASS Thesis éor H10 Dag!» cf Ph. D. MICHEGAN STATE UNWERSITY Evamfi‘ R Merrill €961 This is to certify that the thesis entitled Detergency of Chlorinated Trisodium Phosphate on Milk Protein Soils from Stainless Steel and Glass presented by Everett Perkins Merrill has been accepted towards fulfillment of the requirements for Food Science Ph.D. degree in Date 00 ”If? / (’i/ /76/ [ 0-169 LI BRA R Y Michigan Ststc University ! DETERGENCY OF CHLORINATED TRISODIUM PHOSPHATE ON MILK-PROTEIN SOILS FROM STAINLESS STEEL AND GLASS By Everett P. Merrill AN ABSTRACT OF A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Food Science 1961 ABSTRACT DETERGENCY OF CHLORINATED TRISODIUM PHOSPHATE ON MILK-PROTEIN SOILS FROM STAINLESS STEEL AND GLASS by Everett P. lerrill Chlorinated alkaline detergents are used widely in the food industry for cleaning of stainless steel equipment where the benefit is manifested.in brighter appearance and absence of spotting after cleaning and drying. Data on the cleansing function of chlorinated alkaline detergents were scarce and inconclusive when the present study was initi- ated. Recent postulations have been in general agreement with.the data of this study, showing that chlorinated alka- line detergents increase protein solubility. This investi- gation of chlorinated alkaline detergency is believed sig- nificant for the methods of study used, as well as for the results obtained. Measurements of skimmilk film deposited on glass and stainless steel surfaces were made. A gravimetric method was found to be superior to an interferometer, an ellipso- meter, light transmission, and an electron microscOpe pro- cedure for this purpose. When glass slides were dipped into skimmilk, the gravimetric method showed that after the first dip, the weight of film formed on the surface of the slide was a linear function of the number of times the slides were dipped. Everett P. Merrill The electron micrographs revealed marked unevenness of skimmilk films and showed why wetting ability and pene- trability were desirable in removing milk-film.soils. Tenacious milkeprotein films were made by dipping micro cover glass slides successively and momentarily into suspensions of clarifier slime and into solutions of cal- cium chloride, trisodium phosphate, and sodium hypochlorite. Quantitative measurements of cleaning were made by a combination gravimetric-microkjeldahl method. This method was very effective in.measuring residual films after a de- tergent treatment and was useful for making qualitative observations of cleaning during the washing procedure. Chlorination of trisodium phosphate solutions, by addition of sodium hypochlorite, significantly increased the solubility of protein films on glass and stainless steel when the surfaces were washed for 10 minutes with the solution at a temperature of 65° C. and a pH of 11.5 I 0.1. The beneficial effect was obtained, using either distilled or hard water. The hard water contained 540 p.p.m. of to- tal hardness. The trisodium phosphate solutions did not show a beneficial effect from chlorination when held at less than 65° 0., and pH 11.5 3 0.1. The effect of chlori- nation in solubilizing protein was not attained until after one minute of exposure, but advanced rapidly thereafter Everett P. Merrill with increasing time. A build-up, rather than removal, of film resulted when the slides were exposed to the detergent for less than one minute. The ability of the solution to remove protein in- creased in an almost linear manner until 226 p.p.m. avail- able chlorine was reached. Therefore, under the conditions of this study, an.0ptimum level of chlorination was in the vicinity of 200 p.p.m. available chlorine. A.modified diphenylcarbazide method for determining chromium VI in "used" detergent solutions was successful in evaluating loss of metal from stainless steel by action of chlorinated trisodium phosphate. Loss of chromium from type 504 stainless steel, washed with a trisodium phosphate solution for 48 hours at 47° 0., was found to be of small magnitude. Even in solutions containing 500 p.p.m. avail- able chlorine, the loss remained negligible. Very little chromium was removed from stainless steel piping cleaned automatically with a chlorinated proprietary alkaline de- tergent containing up to 234 p.p.m. available chlorine. DETERGENCY OF CHLORINATED TRISODIUM PHOSPHATE ON MILK-PROTEIN SOILS FROM STAINLESS STEEL AND GLASS By ’(\l‘ \h Everett Pf Merrill A THESIS Submitted to ' Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Food Science 1961 _T ;%///5 /7/5: Cepyright by ETMEHTJEEKHWSMHEHLL 1962 ii To my wife Phyllis and to my uncle and aunt, Mr. and Mrs. Hardy Merrill, for their invaluable encouragement iii ACKNOWLEDGMENTS The author wishes to express his appreciation to J. M. Jensen, Associate Professor of Food Science, for directing this research and for his suggestions and coun- sel. The sincere appreciation of the writer is also exe tended to S. L. Base, of Agricultural Chemistry, for his advice and direction in the chemical analyses and for the electron.micrograph. Grateful acknowledgment is also due to Dre. C. M. Trout and L. G. Harmon, Professors of Food Science, for their assistance in the preparation of the manuscript; to E. J. Benne, Professor of Agricultural Chemistry, for his counsel and equipment; to R. W. Luecke, Professor of Agri- cultural Chemistry, for the use of microkjeldahl apparatus; and to the members of the graduate committee for their time and cooperation. The writer is, indeed, most grateful to Dr. B. S. Schweigert, Professor and Head of the Food Science Depart- ment, and to Michigan State University and the Michigan Agricultural Experiment Station for providing the funds and facilities necessary to make this study possible. iv TABLE OF CONTENTS Page INTRODUCTION . . . . . . . . . . . . . . . . . . . . 1 REVIEW OF LITERATURE . . . . . . . . . . . . . . . . Alkaline Detergents . . . . . . .-. . . . . . Definition of detergency . . . . . Materials used . . . . Preperties of alkaline detergents. Dairy soils. . . . . Physical factors influencing efficiency of detergents on dairy soils. . . . . Limitations of detergents. . . . . . . . 0000 mm VIWNN N N p...- O cmorina O O O O O O O O O O O O O O O O O O 0 Compounds of chlorine used in detergents and sanitizers. . . . . . . . . . . . 10 Chemistry of hypochlorite compounds. . . 12 Benefits of chlorine compounds as de- tergent aids. . . . . . . . . . . . . 16 Corrosion by chlorine-bearing compounds. 17 Evaluation of Corrosion of Food Processing Equipment..................18 Methods of Evaluating Cleaning. . . . . . . . 20 Creation of Synthetic Soil Films. . . . . . . 25 Synthetic films for detergency studies . 25 Surface-film techniques utilized in other scientific fields . . . . . . . 28 Diphenylcarbazide Method for Measuring Chrouu. VI 0 O O O O O O O O O O O O O O O O 30 EXPERIMENTALPROCEDURE...........l.... 35 Measurement of Soil Retaining Capacity. . . . 55 Methods of Evaluating Cleaning. . . . . . . . 40 Methods of Evaluating Corrosion of Stainless Steeleeeeeeeeeeeeeeeeeeee 43 TABLE OF CONTENTS (Continued) EXPERIMENTAL RESULTS . . . . . . . . . . . . . Measurement of Soil Retaining Capacity. . . . Methods of measuring films . . . . Methods of evaluating cleaning . . . . . Methods of Evaluating Corrosion of Stainless Steel . . . . Conductivity . . . . . . . . . . . Emission spectroscopy. . . . Diphenylcarbazide for chromium VI. DISCUSSION . . . . . MI W CONCLUSIONS. . . O . . . O O O O . LITERATURE CITED . . vi 0 Page 51 51 51 53 58 58 59 62 74 TABLE 10 11 12 15 LIST OF TABLES Relative cleaning values of various detergent ingredientseeeeeeeeeeeeeeeee Extremes in the composition of milk stone . . Skimmilk film.deposition on glass microscOpe slides measured by interferometer . . . . . . Variation in weight of slides when weighed to the 4th.and to the 5th decimal place. . . . . Uniformity of skimmilk film weights on 25 x 75 mm. slide surfaces measured gravimetri- 0311’ O O O O O O O O O O O O O O O O O O O O Skimmilk film formation on 22 x 50 mm. micro cover glass slides when dipped intermittently and.measured gravimetrically. . . . . . . . . Skimmilk film formation on 22 x 50 mm. micro cover glass slides measured gravimetrically Variation in skimmilk film weight per rack of 22 x 50 mm. micro cover glass slides as af- fOCted by nuber of dips. e e e e e e e e e e Reproducibility of SpectrOphotelometer read- ings on 25 x 75 mm. glass microscope slides fihadWItthimlkeeeeeeeeee eee Reproducibility of SpectrOphotelometer read- ings on 25 x 75 mm. microscOpe slides filmed intermittently with skimmilk and a detergent n1xtnre................... Reproducibility of filming and cleaning pro- cedure within and between three racks of 25 x 75 mm. glass microscOpe slides as measured SpeCtrophOtelonetricallye e e e e e e e e e e Removal of skimmilk films from 25 x 75 mm. glass microscOpe slides by controlled washing as measured Spectrophotelometrically. . . . . Effect of heating on SpectrOphotelometer readings of skimmilk filmed 25 x 75 mm. glass microscope slides . . . . . . . . . . . . . . vii Page 86 87 88 89 89 9O 91 92 93 95 TABLE 14 15 16 17 18 19 2O 21 22 23 24 LIST OF TABLES (Continued) Spectrophotelometer absorbancy vs. number of dips of 25 x 75 mm. glass microscOpe slides into skimmilk . . . . . . . . . . . .'. . . . Sensitivity of gravimetric vs. SpectrOphote- lometer methods of measuring skimmilk film on 25 x 75 mm. glass microscope slides. . . . Recovery of l m . of nitrogen from standard samples of (N33 2804 as measured by the micro- kJeldahlnethO.e............. Effect of water hardness on detergency of 0.1% trisodium phosphate solutions in re- moving skimmilk films as measured by micro- kjelda-hl procedure. 0 O O I O O O O O O O O 0 Protein solubility of skimmilk-tap water soil film as influenced by chlorination of 0.1% trisodium phosphate solutions in tap water. . Effect of chlorinated buffer solutions at different pR's on protein solubility of skim- milk-tap water-sodium carbonate film soil . . Effect of trisodium phosphate and sodium hypochlorite concentrations on pH of tap 'at Gr 0 O O O O O O O O O O O O O O O O O O 0 Evolution of filming procedure to measure protein solubility by the micro-kjeldahl me thod O O O O O O O O O O 0 O O 0 O O O O O 0 Effect of chlorinating (156 p.p.m.) 015%ltri- sodium phosphate tap water solutions on pro- tein so ubility of soil films . . . . . . . . Effect of chlorinating (156 p.p.m.) 0.15% trisodium phosphate distilled water solu- tions on protein solubility of soil films . . Effect of exposure time on protein solubility of soil films washed with chlorinated (156 p.p.m.) 0.15%ltrisodium phosphate distilled water solutions . . . . . . . . . . . . . . . viii Page 95 96 96 97 97 98 98 99 101 102 103 TABLE 25 26 27 28 29 LIST OF TABLES (Continued) Page Evaluation of optimum levels of chlorination. on protein solubility of soil films washed with 0.15%ltrisodium phosphate distilled water3°1ution80eeeeeeeeeeeeee 103 Standard samples of chromium in chlorinated trisodium phosphate solutions measured by the diphenylcarbazide method. . . . . . . . . 104 Chromium losses from type 504 stainless steel exposed to chlorinated 0.5%ttrisodium phosphate distilled water solutions measured by the diphenylcarbazide method . . . . . . . 105 Standard samples of chromium in chlorinated preprietary detergent solutions measured by the diphenylcarbazide method. . . . . . . . . 106 Amount of chromium removed from 446 feet of 1.5 inch stainless steel pipeline by a chlorinated proprietary alkaline detergent as measured by the diphenylcarbazide method. . . 106 ix FIGURE 10 11 12 13 LIST OF FIGURES Mechanical fi and washing apparatus for gravimetric-m cro-KJeldahl analysis. . . Temperature controlled mechanical filming and washing apparatus for gravimetric-micro- KJeldahlanalysiso...e......... Electron micrograph, 8000 - 10,000 x, of smnfihreplicaeeeeeeeeeeee Deposition of skimmilk on 22 x 50 mm. micro cover glasses measured gravimetrically . . . Skimmilk film formation on 22 x 50 mm. micro cover glasses measured gravimetrically . . . Absorbancy of films measured by Spectro- photelometer vs. number of times the slide was dipped in skimmilk . . . . . . . . . . . Sensitivity of gravimetric vs. Spectrophote- lometer methods of measuring films . . . . . Effect of chlorinated buffer solutions at different pH's on protein solubility of soil films. 0 O O O O O O O O O O O O O O O O O 0 Effect of exposure time on protein solubil- ity of soil films washed with chlorinated (156 p.p.m.) 0.15%'trisodium phosphate dis- tilled water solutions . . . . . . . . . . . Evaluation of Optimum levels of chlorination on protein solubility of soil films. . . . . Standard curve showing recovery of chromium in a system containing chlorinated trisodium phosphate when measured by the diphenyl- carbazide method. (Average of two trials performed in duplicate). . . . . . . . . . - Effect of trisodium phosphate concentration on standard curve of chromium samples. . . . Mean of chromium removed from 1120 sq. cm. of type 504 stainless steel by 0.5%lchlori- nated trisodium phosphate in distilled water at 47° C. for 48 hours. I Page 107 108 109 110 111 112 115 114 115 116 117 ~ 118 FIGURE 14 15 LIST OF FIGURES (Continued) Page Removal of chromium, as influenced by tem- perature, from type 504 stainless steel by chlorinated (505 p.p.m.) 0.5% trisodium phosphate distilled water solutions. . . . . 120 Standard curve showing recovery of chromium in a.system containing a chlorinated pro- prietary detergent when measured by the di- phenylcarbazide method. (Average of two trials performed in duplicate) . . . . . . . 121 INTRODUCTION Marked progress has been.made in cleaning dairy equipment during the past quarter century. New cleaning agents have been.introduced and tinned-copper equipment has been replaced largely by stainless steel. Chlorinated detergents are one of the more recent introductions. A survey of detergent manufacturers and distributors in the United States revealed that 75 of them were producing or distributing chlorinated detergent com- pounds. This would indicate that chlorinated cleaners are being used widely and probably to a large extent in the dairy industry. In order of their frequency of use, the major chlorinated compounds were: a) chlorinated trisodium phosphate; b) trichloroisocyanuric acid; and c) dichloro- isocyanuric acid. Often problems and questions accompany the introduc- tion of new products. In the case of chlorinated cleaners the question arises as to the benefits gained through their use. _If beneficial, at what concentration do they yield the best results? What is the mechanism by which they ac- complish the purpose for which they were designed? Al- though some data were available answering these questions in part, further studies seemed necessary.' To this end the study described herein was undertaken. REVIEW OF LITERATURE Alkaline Detergents Definition of detergency. According to Niven (1954), detergency is "the removal of any objectionable material from anything and by any means." However, in the dairy industry he limited detergency to a three-component cleaning system involving: a) some liquid, b) some deter- gencybenhancing material dissolved in that liquid, and c) a mechanical device to facilitate the removal of the soils. Davis (1956) described a detergent as a substance used for cleaning or washing away offensive matter. _Lehn (1946), in-a closely related discussion, stated that cleaning is the release of contaminating matter from wet surface. The Milk Industry Foundation (1957) considered cleaning of dairy equipment to be the removal of soil from the surface of equipment by dissolving or by suspending the soil in a warm solution of suitable chemicals. Materials used. The chief detergent materials cur- rently used in the dairy industry are alkaline salts. Claybaugh (1950), Davis (1956), The Milk Industry Founda- tion (1957), Niven (1954), and many others have confirmed this statement. Also in use in conjunction with these alkaline materials are anionic, cationic, and nonionic Ivetting agents. These have been discussed by Scales and Itemp (1940) and Sisley and Wood (1952). - 2 - 'Properties of alkaline detergents. The Milk Indus- try Foundation (1957) summarizes the common dairy alkaline detergent ingredients and their major properties as shown in Table 1. The terms used to describe detergent preperties have been discussed by Harding and Trebler (1947), Lehn (1946), Little (1947). Mohr and Mohr (1954), Davis (1956), Shogren (1951), Trebler (1945), and others. These are as follows: a) b) e) d) e) f) s) h) 1) ¥pulsification is the mechanical action of break- ng up fats and oils into very small particles which are uniformly mixed with the water used. Sa onification is the chemical reaction between an aIkaII and an animal or vegetable fat result- ing in a soap. Wetting is the action of water in contacting all surfaces of soil or equipment. Dispersion implies the disintegration of the soil into sma er particles or draplets and the trans- port of the soil away from the cleaned surface. Suspension is the action which holds up insolu- b e part else in a solution. Pe tizi is the physical formation of colloidal squtIons from soils which may be only partially soluble. Water softening is the removal or inactivation of tHe hardness of water. Mineral deposit control is associated with a high sso ving power and a high neutralizing value of the deposits. Rinsabilit is the condition of a solution or suspensIon which enables it to be flushed from a surface easily and completely. - - Relative Cleaning Values of Various Detergent TABLE 1 . Ingredients. < n I 3.235.th meazeeezefimz§mz .2 a _ co m '< whsamozmhgiah .2 3 _ 0 GM X31dWOD SilVHdSOHd SONflOdWOJ wh '- Nouvwaonans U U U U U U U U U _.._m< m snmauosualm U U U it En >_k<¢..:.1 w:._<> w>_._. 30.. ....... U w:._<> I‘d—nu: ....... n 23¢) 20.: ....... < .555 9—. >3. J) Suds formation is related to foaming and the de- crease of surface tension.. k) Non-corrosiveness is the lack of ability to squbIIIze equipment with which it comes in contact. . Dairy soils. The requirements of a detergent solu- tion will vary with the extent and nature of the milk de- posits (Davis, 1956). According to the Milk Industry Foundation (1957). milk or milk product residues consist of: a) b) c) d) Milk sugar, which is readily soluble in water and does not appear to form insoluble products. Butterfat, which, in its normal, emulsified con- dition, is readily dispersed in water, but which, when the emulsion is broken, may form a continu- ous, insoluble film over soluble soil. Fats can be saponified with strong alkalies, particularly at high temperatures, but this method of removal is impractical except in bottle washing and cleaning by recirculation and spraying. Fats in stable emulsions, for example in raw milk, are readily removed by a cold water rinse. Otherwise they are most economically removed by the aid of emulsifying agents at temperatures above the melt- point of the fat (84° to 97° F.). Proteins, which when unchanged, as in fresh, raw milk films are readily dispersed or dissolved in water. Milk proteins are easily changed or de- natured by the action of heat or acid and then may no longer be soluble or dispersible in water alone. These denatured proteins may be dissolved readily in dilute alkaline solutions, but do not appear to be soluble in dilute acids. The addi- tion of dispersing agents speeds up the removal of denatured protein films. These agents include dispersive-type wetting agents for use in both acid and alkaline solutions as well as poly- phosphates for use in alkaline solutions. Mineral salts, which in fresh raw milk residues may be readily dissolved or dispersed in water. 0n the other hand, mineral deposits found in hot milk films or in film left from previous incom- plete cleanings or in deposits from hard water and alkaline materials are usually referred to milkstone. Mineral deposits are slowly soluble in acid solutions with a.pH of 5 or less. For rapid solution formation, the pH of the acid must be less than 3. At the lower pH level, the acid is very corrosive and should be kept away from skin and clothing. Dirt, dust, and many other foreign materials usually are removed by the same cleaning procedures which are adequate to remove milk residues, although occasionally special procedures may be required. More specific reference to dairy soils have been made by many investigators. Sommer (1958) noted that clarifier and separator slimes were high in milk proteins, fat, calcium phosphates, and other organic matter such as leucocytes. The composition of milkstone was discussed by Share (1942), Schwartz (1941), Johnson and Roland (1940), Leader (1955). Rupple (1960), and Sommer (1958). Sommer (1958) gave the composition.of milkstone as indicated in Table 2. The effects of salt concentration, pH, nature of solvent, added electrolyte, and ionic strength on the solubility10f proteins in general, were discussed by Mellanby (1906) and Cohn (1956). A brief summary of their discussion follows: a) The solubility of proteins is characteristically minimal at the isoelectric point. b) The water solubility of most proteins at their isoelectric points is increased by the addition of small amounts of salt. c) The solubility is found to increase with'inp creasing ionic strength, to pass through a maxi- mum, and then to decrease. TABLE 2. - Extremes in the Composition of Milk Stone° -Constituent Minimum Maximum (Percentage) Moisture 2.66 2.79 Fat 5.65 17.66 Protein 4.14 45.85 Ash 42.05 67.55 CaO 21.05 54.66 P20s 17.65 26.95 MgO 1.71 8.12 Fe203 0.0 0.29 Na20 1.40 7.35 ° Samples from various plants in Illinois, formed on different pieces of equipment under various conditions. d) Polyvalent electrolytes are more effective in increasing the ionic strength and are hence more efficient in producing the salting-in effect. a) Addition of organic solvents to aqueous solu- tions of typical proteins ordinarily leads to a reduction in solubility, and usually to pre- cipitation. McMeekin and Polis (1949) reported that the sedi- mentation constant of casein was lowered by small amounts of calcium, thus lowering the solubility of the casein. Little (1947) found that surface and interfacial tensions of detergent solutions were lowered by either ’ neutral or alkaline electrolytes with a resulting improve- ment in detergency. Clark and Hawley (1958) noted that the stability of hydrophobic sols was due to the presence of an electric double layer. Since the essential charge was frequently the result of adding small amounts of electrolytes, the latter became known as peptizing agents. Similar effects were observed with amino acids, peptides, and proteins. Physical factors iggluencipg efficiency of deter- n s on dai oi . Davis (1956) indicated that the most _1 difficult problem to be overcome by a detergent solution is the cleaning of equipment in which milk has been.heated, and particularly in the modern continuous process heat- treatment plants. The albumin and globulin of milk may be denatured by heat (Ramsdell and Whittier, 1953). Leader (1956) concluded milkstone formation was re- duced by a two-step procedure for heating dairy products. A holding time at low temperature resulted in coagulation of lactalbumin before the high-heating period. Leader and Martin (1952) reported that the use of a small electric current on the milk pasteurized by high temperature short- time heating reduced milkstone formation. The device is called the Hydrotron. During the processing of milk, two types of film may be produced on metal heat-transfer surfaces (Johnson and . Roland, 1940). One film was described as cheese-like in composition, and the other film was mineral-like. The two may be produced concurrently or independently. In removing milk films, Jennings (1959b) found that kinetics could be applied in establishing the effect of temperature. The reaction between solutions containing hydroxide ion and milk films exhibited a Q10 of 1.6. The effect of temperature on the reaction rate was linear, at least between the limits of 56° to 82° 0. Cleaning efficiency was increased by the scrubbing action of occluded air and bubbles under a condition of re- duced pressure (Jennings, 1959a). Even without the any trained air, cleaning was more effective under reduced pres- sure than under higher pressure. A turbulence—time-temperature-solution concentration relationship was studied by Jennings 23 31. (1957). Reynolds numbers were used to measure turbulence. In cleaning, turbulence was more significant than temperature.d Both temperature and turbulence became less important as~ the effectiveness of the detergent increased. Limitations of detergents. In addition to rating poorly on some of the desirable characteristics listed in Table 1, some additional ones have been reported below. Davis (1956) stated that a detergent solution in use may rapidly change in chemical composition, and physical preperties. Among the conditions of use listed were: a) addition of milk constituents; b) dilution by carry-over /-J 10 of rinse water; and c) absorption of carbon dioxide. The detergent is weakened in proportion to the length of time used. Thus, its efficiency is steadily decreased. Claybaugh (1950) detected the formation of‘a chloro- protein film when producers' cans were sanitized with ex- cessive amounts of chlorine Just prior to use. This film was extremely difficult to remove by ordinary detergents. In a study of sanitizers, Cox and Whitehead (l949)~' observed a similar circumstance in noting that hypochlor; ites may react with protein deposits on poorly cleaned equipment. Roland (1942) discussed another undesirable property of many of the alkaline detergent salts. Insoluble pre- cipitates of calcium and magnesium may be formed on equip- ment when these detergents are used in natural water cone taining calcium and magnesium ions. Chlorine Compgundg of chlorine used in detergents and sani- tizers. Reddish (1957) listed the sanitizing compounds as follows: a) gypochloriteg are available on the American market as powders containing calcium hypochlorite and sodium hypochlorite combined with trisodium phosphate and as liquids containing sodium hypochlorite. b) Chloramines. The simplest of the chloramines Has the formula Nfizcl. 11 c) Chloramine-T is p-toluene sulfondichloramide in which one Hydrogen of the amino group of p-toluene sulfonamide is replaced by chlorine. d) Dichloramine-T is p-toluene sulfondichloramide in which—BEEE—Eydrogens of the amino group of p- toluene sulfonamide are replaced by chlorine. e) Hal zone is the name given to p-sulfondichlor- aminobenzoic acid. 1 f) D chlorodimethyl hydantoin is an organic compound car ng ava a le c or ne. 9 g) Succinchlorimide is the chlorinated imide of succi c ac . h) Dichloroc uric acid is an organic compound carrying available chlorine. i) Trichloroc anuric cid is also an organic com- poun car ng av a la chlorine. Lewandowski (1959) reported on the chlorine materials currently in use as dairy sanitizers. Included were cal- cium and sodium hypochlorites, di- and trichlorocyanuric acid and dimethyl hydantoin. It is observed that some of the sanitizing chlorine compounds are used in detergent formulations. A survey of representatives of detergent manufactur- ers through personal correspondence (flaxcy, 1958; Baskin, 1958; Albrecht, 1958; Bothel, 1958; Than, 1958; Leighty, 1958; Ressler, 1958; Barrett, 1958; Wright, 1958; Young, 1958; Zaelke, 1958) indicated the following compounds are the major substances used currently in’chlorinated alka- line detergents: a) Sodium hypochlorite b) Trisodium phosphate 12 c) Sodium metasilicate f) Sodium chloride d) Tripolyphosphate g) Carbonate e) Trichloroisocyanuric acid h) Non-foaming wetting agents The survey showed that chlorinated trisodium phos- phate used alone was the most common detergent mixture of the chemical group. Chemistry of hypochlorite compounds. As most deter- gent operations in the dairy and food industries are in an aqueous system, reference is made here to the behavior of hypochlorite compounds in water. In aqueous solution both sodium hypochlorite and calcium hypochlorite yield hypochlorous acid (Fair, 1948). Hypochlorous acid in aqueous solution can decompose in two ways (Wilson and Bremner, 1948). Their account follows: a) 2 ROCl ;;h:0 + 2 H01 - a reaction slow at ordi- nary temperagures and much influenced by light and by such catalysts as cobalt salts and b) 3 8001 - ECO; + 2 301 It is this second reaction which is considered here. Actually, as hypochlorous acid is fairly readily attacked by hydrochloric acid, c) ROCl + 301 —.‘_-2-. H20 + 012 Reaction (b) does not occur, but hypochlorites behave similarly and we write d) 5 010' §#3 0103- + 2 CI. The reaction is very slow at room temperature but proceeds very rapidly in hot concentrated solutions. It is clear that a three-body collision between anions is most unlikely, but as in many other atom- transfer reactions, the exact mechanism is uncer- tain. Moderate amounts of free alkali stabilize hypochlorites; the solutions are least stable 15 at pH 5.7 and have maximum stability at pH 13.1. This supports an older theory that the reaction is between free hypochlorous acid and hypochlor- ite ion. e) 010‘ + 2 8010 - 0103; + 2 3* + 2 c1“ Owing to the small ssociation constant of hypo- chlorous acid, K - 5.8 x 10-3, some free acid will always be present. In the absence of free alkali, accumulation of hydrogen ions would cause reaction (c) to take place. Chlorites have been detected in the decomposition products, particularly from alkaline solutions. In.con~ sidering the reaction, the possibility of hypo- chlorous acid dissociating in yet another way should be remembered: _ + 8001 ;=§ (HO: ) + Cl The cationic chlorine will be a very powerful oxidizing agent and may well play an important part in the reaction. Sommer (1958) indicated an additional method for the decomposition of hypochlorous acid in water: 8001 g 301 + o. The importance of temperature and pH in the decompo- sition of hypochlorous acid has already been emphasized. Reaction with organic matter is another prOperty reported by many investigators. The remainder of the review on chlorine chemistry will be discussed under these three factors, namely, pH, temperature, and reaction with organic matter. , In regard to pH, Reddish (1957) gave the percentage of hypochlorite present in the form of hypochlorous acid for various pH values. At high pH the percentage of undis- sociated hypochlorous acid was very low. The data of Reddish (195?) are listed: 14 pH ' Hypochlorites present as undissociated H001 er cent 4.0 almost 100.0 5.0 9906 6.0 95.8 7.0 69.7 8.0 18.7 9.0 2.2 10.0 0.2 Afanasev (1948) concluded that the oxidizing action of chloramine was due in neutral solution to 010', and in acid solution to R010. This conclusion agreed with the data of Reddish (1957), and was supported by Johns (1951b). The influence of temperature on the bactericidal action of chlorine was studied by Ames and Smith (1944). To disinfect water containing Escherichia 321; with 0.25% organic nitrogen present, the time required at 8° 0., was more than nine times that required at 40° C. for a given chlorine level. Reddish (1957) reported data indicating that in the range of 20° to 50° C., the killing time of Bacillus metieng spores by calcium hypochlorite was reduced 46 to 66%ifor each 10° 0. rise. Increased rate of diffusion of the germicidal agent (undissociated hypochlorous acid) was believed to be responsible. The reaction of hypochlorites with organic matter has been studied by many investigators. Theoretically, the action of free halogens on.protein in aqueous solution can 15 take any of three paths: a) addition, b) substitution, c) oxidation (Herriott, 1947). wright (1926, 1936) concluded that hypochlorites normally react with amino acids and proteins to form (if only as unstable intermediate products) typical chloramino compounds. Cox and Whitehead (1949) corroborated the find- ings of wright. Faber (1947) suggested that a decrease in the oxidation-reduction potential may be used to measure the chemical activity when hypochlorous acid combines with organic matter. Lasmanus and Spencer (1955), Haller 23,51. (1941), and Wilkowske and Krienke (1955) found that milk dissipated“ the hypochlorous acid in chlorine sanitizing solutions. The presence of ammonia also decreased the effective-/ ness of sanitizing solutions containing hypochlorous acid (Van Hall, 1954; Weber gt a;., 1940). Cahn and POwell (1954) reported that nitrogen was liberated when ammonia and hypochlorite were allowed to react in an alkaline medium. Rerken and Siebersiepe (1955) reported that the pep-/~ tide bonds of several albumins and globulins were hydro- Mlyzed by'hypochlorites.. Materials other than proteins appearing in dairy,» soils may react with chlorine compounds. Lyness and Quackenbush (1956) found that methyl oleate, oleic acid and ethyl linoleate gave near quantitative yields of l6 addition products when treated with chlorine.{ Zoller (1925) concluded that other substances present in the milk were more easily attacked than the fat after observing that the presence of 3.8%»butterfat did not affect the decomposition rate of sodium hypochlorite in milk. Benefits of chlorine compounds as detergent aids. A comparatively recent development in cleaning has been the use of chlorinated detergents in the food industries. Hiscox and Briggs (1955) and Holland 3; 3;. (1955) reported beneficial cleaning effects from chlorinating alkaline de- tergents. Lewandowski (1959) stated the addition of sodium or calcium.hypochlorite, or organic compounds bearing chlo- rine, to a well-balanced alkaline cleaner gave brighter surfaces and reduced the tendency toward filming. Lewan- dowski emphasized that these chlorine compounds will not clean well when.used independently of alkaline detergents. - In a laboratory study, MacGregor g§_a;. (1954) recommended adding 25 to 100 p.p.m. sodium hypochlorite to certain alkaline detergents for improving the protein 9 cleanability of metal surfaces. Kaufmann and Tracy (1959) found chlorinated alkaline detergents of varying value in removing an iridescent dis- coloration from cleaned-inpplace pipelines. Shepard (1960) and Overman (1959) mentioned the — brightening effect noted by Lewandowski (1959). However, 17 both of these investigators and Rupple (1960) agreed that the major benefit of chlorinating the detergent was to im- prove peptization. Other benefits listed by Overman (1959) were : a) Changing the electro-static balance of the cleaning solution so that the soil which would normally be attracted by and adhere to the sur- faces becomes free rinsing. Utensil and machine odors were largely eliminated. b) Reduction in foaming of the washing solution. Corrosion by chloripe-bearipg compounds. Botham (1949), Foster (1949), Jacobsen (1954), Johns (1951a), Mitten (1954), and Myrick (1954) were a few of the many in- Vestigators who noted the corrosive properties of chlorine- bearing compounds under certain conditions of use. Of these authors, Botham (1949) and Johns (1951a) stated that corrosiveness was inversely proportional to alkalinity, and directly prOportional to temperature, time, and concentration of available chlorine. Jacobsen (1954) considered that corrosion by chlorine disinfectants was negligible when used in concentrations of 100 and 200 p.p.m. as is normally done in dairy processing plants. , Klenzade Products, Inc., (1960) stated that the cor- rosiveness of compounds containing chlorine may be modified by increasing the pH. Addition of sodium hydroxide or sodium silicate was recommended. 18 Evaluation of Corrosion of Food Processing Equipment Mitten (1954) has described the nature of corrosion. 11.;portion of his discussion, which follows, summarizes tezaiefly the principle of corrosion and some of the perti- nent terms. amount Metals and their alloys tend to revert to one or more forms in which they are found in their natural ores. Eventually they will do this. Man has discovered no way to prevent this earth-to- earth return. By skillful metallurgical processes and by proper care, man can prolong the process by slowing the rate of corrosion, but he cannot com- pletely prevent it. Corrosion is an electrochemical process which destroys the metal or alloy and (1) non-metallic solids such as sulfur, (2) liquids such as acids or water, or (5) gases such as chlorine and air. In the most common form of corrosion by the electrochemical process, there is generally an anode, a cathode, and an electrolyte. By an ex- change of electrons atoms of metal and ions in the solution (usually hydrogen ions) replace each other. Ions are atoms having an electrical charge. When the metal atoms and solution ions replace each other, hydrogen gas evolves or a second chemi- cal change takes place so that hydrogen and oxygen combine to form water. The metal particle then goes into solution. As this is repeated millions of times, the metal surface is corroded away. Some of the factors in corrosion are acidity of solution, aeration and oxidizing agents, tempera- ture, velocity, and effect of films. Uhlig (1948) listed the methods of determining the and influence of corrosion as: a) Visual observation c) Changes in electrical resistance b) Change in weight d) Oxygen evolved 19 e) Depth of pitting i) Electrochemical f) Hydrogen evolved J) Electrometric g) HicroscOpic k) Optical methods h) Changes in physical prOperties Use of specific methods for evaluating corrosion Iléis been reported by many investigators. Holland 95,51. (1953) have Judged corrosion visually in a long-term study c>x:.oleaned-in-place stainless steel pipelines. Botham (:JL949) combined visual evaluation with measurement of depth (>1E'pits resulting from corrosion. Electron diffraction, ::-—ray diffraction, and electron microscOpy have been used leay'Yearian 23,51. (1957) in determining the formation of (Datide films on 18-8 stainless steels. These steels were <>3cidized in air at 500 to 700° 0. By electron diffraction studies Kaufmann pp 1. (1955) found the composition of iridescent film on stainless steels to be metal oxides. (iarius (1956) followed the course of passivation of non- zmusting steels by recording changes of potential. The steels were exposed to solutions and the variation in po- tential observed. Changes in weight of test samples have ‘been the most common technique aside from visual observa- tion in detecting corrosion of dairy metals. The gravi- metric technique has been utilized by Barnum 22.51. (1937), Finley and Foter (1947), Haller pp 21. (1941), Mohr and Wortman (1955), Fluette (1960), and Klenzade Products, Inc., 2O ( 1960). Harper (1958) showed close agreement between weight changes and a radiometric method using P52 on type 302 stainless steel. Methods of Evaluating Cleaning Davis (1956) emphasized that the only completely satisfactory way of testing detergents and methods of cleaning is to try the method and the detergent under the existing conditions for the cleaning operation concerned. He acknowledged various laboratory procedures which have been suggested for testing the efficiency of detergents. He reported these can be very useful provided their limi- tations are always appreciated. Davis felt it was usually unwise to draw hard and fast conclusions from such results. The majority of methods to evaluate cleaning fall into two general areas. One is the assessment of viable bacteria present after the cleaning Operation. The other is the determination of the amount of soil retained on a surface after cleaning. Most investigators have used some combination of both of these principles. Bacon §_t_ a1. (1955) used a bacterial swab technique to show the cleanliness of beer glasses, Fleischman and Holland (1955) for glass pipelines used for milk, Hucker (1954) for surfaces of eating utensils, Jensen pp _a_1. (1959) for recirculation-spray versus manual cleaning of milk storage tanks, Kaufmann .e_t_ p1. (1955) for in—place-cleaning 21 emf stainless steel lines, and Kaufmann pp 51. (1960) for comparing cleanability of various stainless steel finishes. Ksufmann _e__t g. (1960) also used the bacteriological tech- niques of the Direct Agar Contact Plate, and the Direct E3tzrface Agar Plate Test for comparing the cleanability of xrsnrious stainless steel finishes. In testing the efficiency of detergents under factory <3c>nditions by microbiological methods, Meewes (1952) ex- aamnined the relative merits of plate counts of emulsions, impression plates, and thin layers of agar on the treated Surface. The capacity of surfaces to retain soil, a second Principle of evaluating cleaning, has been assessed in unsany ways. Calbert (1958) and Lewandowski (1954) reported re- sults obtained by gravimetric methods after investigating the cleanability of cleaned-in-place pipelines, and milk- stone formation, respectively. Measurement of light transmission through soiled glass surfaces was the technique used by: a) Jensen (1946) in determining the function of detergents employed in clean- ing dairy equipment as affected by various detergents and procedures, b) Claybaugh (1950) in his investigations of detergency applicable to mechanical can washing, c) Mead and Pascoe (1952) for assessing the cleaning efficiency of detergents, d) Leenerts 31; a1. (1956) in soil removal 22 tny dishwashing detergents, e) Gilcreas and O'Brien (1941) for evaluating detergents and methods of cleaning eating utensils, and f) Hughes and Berstein (1945) in studying compounds used in dishwashing machines. Hadioisotopic techniques now exist which are re- ported to avoid the pitfalls of the light transmission methods. The "light" disadvantages named, were low sensi- tivity at both high and low levels of soil residues (Masurovsky and Jordan, 1958). The same investigators (11:960) found that the procedure involving the determination C>13 Staphyloccus aureus labeled with isotopes was superior 1=<> macro-colony agar submersion counts, and Bacto-Strip Cleeterminations for measuring the cleanability of milk- <=<>ntact surfaces. Hays 22,21. (1958) and Hays (1960) used IEscherichia 2211 labeled with P32 in studying the cleana- bility of surfaces in contact with dairy-products. Ridenour and Armbruster (1955) utilized organisms labeled With P32 in an analysis of the cleanliness of eating sur- faces. Cucci (1954) labeled milk with P32 to measure the amount of milkstone deposited on rubber, Pyrex glass, and Tygon tubing. Seiberling and Harper (1956) found that P52 was unsatisfactory for labeling milk films in studying the cleanability of cleaned-in—place automatic valves. They reported that P52 reacted irreversibly with stainless steel and therefore preferred Cat"5 for evaluation of cleaning. 23 These same investigators found traces of 0a45 on surfaces shown to be bacteriologically clean. Thus, the isotopic Jpzmocedure was more sensitive in detecting soil than the bacteriological method. Extensive use of homogenized milk labeled with P52 liars.been.made by Jennings (1959a, 1959b), and Jennings 22 .2211. (1957). Criticism of the method involving the labeling of milk with P32 was made by Peters and Calbert (1960) who compared the relationship between the weight of soil re- moved from test films and the decrease in radioactive count. Peters and Calbert labeled milk by three methods. Onewas 1:37 direct incorporation of P32 into the milk used to make 1311c test soil. The second method involved the inoculation c>1r P32 into the blood stream of a goat. When these two Insethods were used to label milk there was an unsatisfactory Elssreement between loss in weight of the soil from the test films and the decrease in radioactive count. However, the third method of labeling milk, namely of incorporating bac- ‘berial cells labeled with P52 into the test soils, gave good agreement with the gravimetric method. Recently, Jennings (1961) reported a statistically controlled study in which 240 samples of milk labeled with P52 were used. Following a detergent treatment there was good correlation between methods involving determination of weight loss from films and decrease in radioactive count. According to Jennings, differences in the preparation of 24 test films were responsible for a lack of correlation be- tween losses of film measured by gravimetric analyses and by an isotopic method involving the direct labeling of milk with P32. He also reported a good correlation between the gravimetric method and a procedure in which labeled bacteria were incorporated into milk films, but indicated the latter required more effort in preparation. Many workers have made qualitative visual measure- ments of cleaning, either independent of, or in combination With, one of the above described quantitative techniques. Specifically, visual evaluation has been utilized by Jensen and Waterson (1950), Jensen 22 21. (1959), Kaufmann g 2.1.- (1955). Kaufmann and Tracy (1959), Wildbrett 22- a1. (1954), Fleischman and Holland (1953), Holland 2:; el- (1953). Blacker 2_t_ 21. (1951), Mohr and Junger (1955), Mohr, Junger, and Weinert (1955), and Mohr, Weinert, and Peters (1955). In determining the effects of several physical prOp- erties of detergents on cleanability, a few of the many in- teresting techniques should be noted: a) lohr and Mohr (1954) determined wettability of detergents by measuring the contact angle of the detergent solutions--no relation- ship was found between detergency and surface tension; b) Lindquist (1955) devised a method of eliminating obviously unsuited wetting agents by recording, photographically, the dimensions of created drops, dimensions of the drops were then used with a formula to calculate surface tension--by 25 this method, wetting agents which foamed excessively were Cietected by comparison with standard products showing de- sirable foaming characteristics; c) Bisio 21; 21. (1956) reported on the feasibility of recording the spreading rate of a drOp of oil on a metal surface and using this measure- ment as an indication of cleaning efficiency; d) Jennings (1959a) has successfully used Reynolds numbers to study the turbulence factor in cleaning; and e) Mohr 2p 21. (1952) suggested a special glass electrode containing lithium for determining pH of highly alkaline detergent solutions. Creation of Synthetic Soil Films Sypthepic films for detergency studieg. The results 01' studies concerned with the removal of films of soil have been discussed in a former section. Mention is now made of Various representative methods used to create synthetic films for detergency investigations. It will be noted that air drying, heating, and addition of various chemicals have been used widely to make the film materials tenacious. As previously mentioned, milk films have been made radioactive directly with P32 or indirectly with bacteria labeled with P52. Such films have been prepared by Cucci (1954), Jennings 22 21. (1957), Masurovsky and Jordan (1958). and Hays at. al- (1958, 1960). Cucci (1954) employed homogenized milk labeled with P32 to fill sections of rubber, Tygon, and Pyrex tubings. 26 The milk was removed after 24 hours and the tubings allowed to air dry before testing the detergent. Jennings e_t 2;. (1957) placed 0.5 to 1.0 m1. of labeled homogenized milk in the center of flat disks of stainless steel which were of 15/16 in. diameter. The disks were then steamed to dryness. Pasteurized homogenized whole milk or sterile 0.85% saline solution was tagged with radioactive cultures of E. 2211_or Micrococcu2 pyogenes var. a22eu2 by Masurovsky and Jordan (1958). The inoculated soil (0.05 ml.) was pipetted onto the center of l in. diameter disks of various materials used as contact surfaces. After mechanically spreading the soil, the disks were dried for 6 minutes at 60° 0. with an infra-red lamp and 10 minutes at 80° 0. in an oven. ~ Hayes 22 21. (1958, 1960) spread 0.4 m1. of Cream, buttermilk, homogenized milk, or chocolate milk inoculated with E. 2211 evenly over one side of plastic and stainless steel disks 2 inches in diameter. The films were then air dried. Beef and mutton fats, butter, skimmilk, egg, peanut butter, lard, and evaporated milk, alone or in mixtures, were manually spread over china, and plastic surfaces by Hucker 22 21. (1951) and Hucker (1954). MacGregor 22 21. (1954) manually distributed a slurry containing Ca003, CaHP04°2H20, spray-dried milk 27 milk powder, and distilled water on stainless steel strips. Drying of the films was accomplished by autoclaving for 25 minutes. . Jensen (1946) and Claybaugh (1950) manually immersed 5 in. glass squares, 1/8 in. thick, in raw milk and fol- lowed this with additional treatments. Air-dried raw milk films were prepared by dipping the glass squares twice into raw milk held at 40 to 50° F. After a l5-minute draining and drying period, the dipping was repeated several times according to the amount of film needed. Heat-treated raw milk films were made as above except that these films were heated at 180 to 185° F. for 15 minutes after the air dryh ing, but before the next dipping. Five complete sequences of dipping, drying, and heating were used to give the finished heat-treated film. In making "chlorine-protein complex" films, the glass squares were dipped twice in raw whole milk, drained, and air dried for 15 minutes. The ' glass was then immersed in a 250 p.p.m. NaOCl solution, and air dried for 15 minutes. Five complete sequences of this procedure completed the "chlorine-protein complex" film. Gilcreas and O'Brien (1941) prepared a synthetic wash- and rinse-water of known hardness, and deposited this on glass microscOpe-slides. Calcium chloride and magnesium chloride was added to a 0.5%rdetergent solution in distilled water. The ratio employed was six parts of calcium hardness 28 to four parts of magnesium hardness, each eXpressed as calcium carbonate. To simulate a milkstone film, the slides were dipped in this mixture, drained 5 to 5 minutes, and sometimes heated at 110° 0. for 5 minutes. By means of an Autotechnicon (Lewandowski, 1954), weighed stainless steel strips were dipped consecutively into a chlorine solution (200 p.p.m.), raw milk, water, detergent, and water. The water contained'500 p.p.m. hard- ness. This basic cycle was repeated 50 times. Various methods of heating were used to dry the slides. Surface-film techniqueg utilized in other scientific field2. Some surface-film techniques deve10ped in other' fields were adopted for use in this investigation. Rothen (1956) reviewed the results made possible by using surface-film techniques. Included were determina- tions of chemical and physical prOperties such as film formation, surface pressure, molecular weight, viscosity of surface films, film compressibility, and film thickness. Blodgett (1955) built films by transferring mono- molecular layers of various metallic soaps and fatty acids from a water surface to several solid surfaces. The thick- ness of "step" films was measured by means of the inter- ference of reflected monochromatic light. In similar work, Langmuir 22 21. (1957) suggested that variations in thick- 8 ness of much less than 10' cm. can be observed using opti- cal means. Blodgett (1957) reported that the process of 29 building films with substances such as proteins, required that the solid surface be lowered or raised through a water surface on which the film is spread. The importance of a mechanical slow-dipping device in producing uniform films was discussed by Dean 22 21. (1939). Joly (1959) stated that the surface viscosity of a _ protein film varies according to the care taken at the time of its spreading. It was believed by Langmuir (1958) that protein films were insoluble or denatured because hydrophilic groups were drawn into the liquid surface, distorting the molecule, and submerging hydrOphobic groups which normally ‘ occupy an internal position in the molecule. Langmuir sug- gested that methods of surface chemistry were of value in studying specific protein reactions. Absorbed films of proteins at air-water, and oil- water interfaces were investigated by Alexander (1951). Using the surface viscosity method, his studies showed that the thickness of absorbed films of proteins depends upon such factors as time, the nature of the interface, pH, salt concentration, and temperature. Braude and Nachod (1955) showed that multiple- reflection interferometer-methods made possible the direct determination of single monolayers deposited on solid sur- faces. 30 A comprehensive review of gravimetric, photometric,' interferometric, x-ray absorption, radioactive tracer, and electrical methods for measurement and control of the thick- ness of thin films was made by Greenland (1952). It was recommended by Gunn (1946), that in measur- ing films, the films be arranged to cover only a part of the plate. This made possible, at some position on the surface, an abrupt step equal to the thickness of the film. Lucy (1948) studied surface films by the reflection of polarized light. He noted that plane-polarized light is generally polarized elliptically by reflection. Since the molecules in a very thin layer are those chiefly concerned in the process of reflection, the ellipticity is very sensi- tive to the conditions at the interface, such as the pre- sence of films. . Rothen and Hanson (1948) used the ellipsometer to measure the thickness of transparent films, from one to many thousands of Angstrom units, deposited on polished metal slides. Diphenylcarbazide Method for Measuring Chromium VI Cazeneuve (1900) and Moulin (1904) were among the first to use diphenylcarbazide in estimating minute amounts of chromium colorimetrically. Sandell (1944) reported that diphenylcarbazide gave a clear red-violet color when 51 reacted with hexavalent chromium. Detergent solutions, after use in the dairy and food industries, may be regarded as biological systems because of contamination with the various, soils. In studies on biological materials, diphenylcarbazide has served to de- tect chromium in blood (Cahnman and Bisen, 1952); in water, sewage, and industrial wastes (American Public Health Asso- ciation, American Waterworks Association, and Federation of Sewage and Industrial Wastes Associations, 1955); in urine, tissues, and blood (Urone and Anders, 1950); and in urine (Saltzman, 1952). a Chemically, diphenylcarbazide is formed by the con- densation of urea and phenylhydrazine (Udy, 1956). AccOrd- ing to Welcher (1947), diphenylcarbazide has the formula c :- (NH - NH - 05H5)2, a molecular weight of 242.27, and is a. white crystalline solid. It is very slightly soluble in Water and slightly soluble in hot alcohol, acetone, and glacial acetic acid. In acid solution, the diphenylcarba- zide method is nearly specific for hexavalent chromium; molybdenum is the only element giving a similar, but less sensitive, violet color (Sandell, 1944). Morrison and Freiser (1957) reported that microgram quantities of hexavalent chromium can be evaluated by the diphenylcarbazide method. Saltzman (1952) found a recovery of 98% on 50 ml. portions of urine containing 0 to 15 pg. of chromium. The American Public Health Association, 32 American Waterworks Association, and Federation of Sewage and Industrial Wastes Associations (1955) recommended the method be performed on samples having 10 to 100,ug. of chromium. Many of the factors influencing the reliability of 'the diphenylcarbazide method have been studied and re- ported (Davis and Bacon, 1948; Feigl, 1946; Rowland, 1959; Sandell, 1944; Welcher, 1947; Saltzman, 1950; Cahnman and Bisen, 1952; Brookshier and Freund, 1951; Urone, 1955; Urone and Anders, 1950). Cahnman and Bison (1952) stated the main problem :in.applying the diphenylcarbazide method to biological ma- ‘terials, such as blood, was in eliminating the numerous .factors interfering with the precision of the method. The :factors noted were: a) incomplete oxidation of trivalent 'bo hexavalent chromium; b) presence of minute amounts of :roducing agents; c) loss of chromium through volatilization; and d) interference of heavy metals. Sandell (1944) reported the interferences of the VI III II Ineavy metals, Mo , Fe , and Vv, could be elimi- .Hs :nated by employing a green filter (Cenco No. 2) on the Spectrophotometer. Brookshier and Freund (1951) described a method for separating chromium from vanadium interference. If the ratio of molybdenum to chromium was less than 10, Rowland (1959) reported molybdenum interference could 33 be neglected. Urone and Anders (1950) noted manganeseas perman- ganate gave a faint yellow color with diphenylcarbazide. ‘Sodium azide was recommended to reduce the permanganate ‘before addition of the diphenylcarbazide. Feigl (1946) suggested heavy metal interference <3ould be prevented by the addition of suitable compounds wfihich reduced the ionic strength below that required for the diphenylcarbazide reaction. Development of color with diphenylcarbazide was shown by Davis and Bacon (1948) to be slower in phosphoric acid than in sulfuric acid. However, full color developed vuithin five minutes in acidities up to 2N. Welcher (1947) :reported the optimum acidity was 0.2N. At lower acidity 'tho color development was slow, and at acidities greater ‘than 0.2N the color was less stable. According to Saltzman (1950) ashing was generally Jsequired to destroy reducing materials such as organic Inatter. Saltzman also reported that polyphosphate com- plexed chromium making it unreactive to permanganate oxi- <1ation. Phosphate was converted to polyphosphate by strong heating and followed the reaction: 2 NaH2P04 g! N'2H2P2O 4» H20 This reaction was also found to occur to some extent when a wet ashing solution of nitric acid was taken to dryness following the ashing. In eliminating nitric acid after 54 ashing, Saltzman recommended low temperature evaporation on the steam bath. In this manner, the nitric acid was kept at 100° C. and the conversion of phosphate to poly- phosphate prevented. Urone (1955) found the sensitivity of the diphenyl- carbazide reagent varied inversely with the amount of dis- coloration developed. Therefore, Urone recommended dis- carding solutions which had discolored with age. EXPERIMENTAL PROCEDURE Measurement of Soil Retaining Capacity Measurement of soil retention was studied by means of the a) ellipsometer, b) interferometer, c) electron microscope, and d) gravimetrically. H. J. Trurnit of Rias, Inc., Baltimore, Maryland, and A. Rothen of the Rockefeller Institute in New Ibrk City performed the ellipsometer ins vestigations in this study. Trurnit (1959) used a Rudolph ellipsometer (Model 441) and followed a method of his own design. Stainless steel slides of No. 4 and No. 7 finishes were coated mechanically with 19 double layers of barium stearate followed by 10 single additional layers of the same substance. Each layer was approximately 50 A. thick and 1/8 in. wide. Rothen (1959), another cOOperator, also used a ARudolph ellipsometer (Model 200E) in measuring the thick- .ncss of films of pasteurized skimmilk soil deposited on .eimilar stainless steel slides as well as on glass micro- scope slides. Both sides of the glass and steel slides Imere coated with film by dipping the slides mechanically into skimmilk. All filming in this study was performed mechanically as indicated. The films covered the entire width of the slide and a length of approximately 40 mm. The interferometer measurements were made on the instrument in the Michigan State University Department of -35- 36 Physics and Astronomy. Dr. T. H. Edwards of that depart- ment supervised the work and cooperated in the study. The apparatus consisted, in part, of a light source, "light box," and an "Optical flat." A General Electric sodium vapor lamp served as the source of monochromatic light. This light passed into an adjacent light box through a dif- 'fusing glass. Here, the light struck a plain glass panel angled at 45°. The incident light was thus bent 90° and directed perpendicularly to the surface of an Optical flat. A nonreflecting background was obtained by placing black velvet under the Optical flat. The glass slides were dipped once into the skimmilk, then air dried and placed on top of the Optical flat. The slide was manipulated by hand to give the best visual Ob- servation of the displacement in the interference lines caused by the milk films. The electron micrographs of milk films were taken by Bass (1960) at a magnification of 8,000 to 10,000 using an R.C.A. electron micrOsOOpe (Model 20). The procedure was as follows: Clean, clear, glass, microscope slides were sprayed with very dilute, atomized drOplets of nitrocellu— lose, and allowed to air dry. Amyl acetate was used to dilute the nitrocellulose. One mechanical immersion of the slide in skimmilk served to apply the skimmilk film which was allowed to dry in air. The slide was then dipped once in a 1% solution of nitrocellulose in amyl acetate and air 37 dried. The last fiLm of nitrocellulose, when lifted away from the milk film surface, tore away some of the original atomized droplets of nitrocellulose. This produced a "re- plica" of the milk film surface, as well as showing its depth in the areas of the nitrocellulose drOplets. The replica was shadow cast with a Kinney (Type SC-5, 0.T.) high vacuum metal evaporator. Electron micrographs were then taken which revealed the surface t0pography of the film and its thickness. The gravimetric method consisted of weighing slides, filming these slides with skimmilk, and reweighing to Ob- tain the weight of the film. Clear, unetched, glass microscOpe slides were han- dled with forceps and cleaned chemically by submerging in a sodium dichromate cleaning solution for 24 hours in covered, glass, slide-staining dishes. All rinsing of the slides was done in the staining dish using running dis- tilled water. After rinsing, the slides were submerged for one hour in concentrated sulfuric acid, the initial tem- perature Of which was 60° 0. Following another thorough rinsing with distilled water, the slides were placed end up on paper towels in a 60 to 70° C. oven until used. Number 4 finish stainless steel sheets were made into slides having dimensions of 75 x 25 x 2 mm. These slides were cleaned by suspending them in a 4-1. beaker from clamps attached to a circular rack. The beakers 58 alternately contained hot solutions of prOprietary organic acid and alkaline cleaning materials. Agitation Of the solutions was performed by a magnetic stirrer. After thorough rinsing in running distilled water, the steel slides were handled and stored by procedures similar to those for the glass slides. The clean steel and glass slides were weighed be- fore and after filming on a four-place Voland balance (Model 750-D). Comparisons of accuracy were made between the Voland balance and a Rueprecht analytical-type balance allowing estimation of a fiveeplace weight. Before weigh- ing, slides and films were allowed to come to equilibrium with the atmosphere for one hour. While in storage, the filmed slides were protected from dust contamination by placing them in a large covered glass vessel. To minimize damage to the films, forceps and light metal storage racks were employed during the weighing and storage periods. The filming material consisted of skimmilk tempered as the experiment dictated. Various films were obtained by dipping the slides a variable number of times into the skimmilk. The filming apparatus used is shown in Figure l. The only variation made in this equipment throughout the study was the use of either a 4-1. Pyrex beaker or a one- liter Pyrex crystallizing dish to hold the dipping solu- tions. 39 Slide racks were constructed by welding 1/2 in. slices Of 4 in. steel pipe to ring-stand clamps. Six to 11 ball-Joint clamps were bolted around the periphery of the slice of pipe equidistant from one another. These clamps were designed to hold the glass and steel slides for the filming procedure. After placing the constructed slide racks on a ring stand, the racks were heated and ad- Justed to be in plane with a horizontal surface. The slides were aligned in the rack to allow equal filming depth. Alignment was accomplished by placing the ring stand with its attached rack and slides on a 1.25 ft. x 2 ft. x 1/2 in. clean "desk" glass. The slide rack was adjusted on the ring stand at a predetermined height. The slides were then manipulated in the clamps so that their .lower edges coincided fully with the flat surface of the ”desk" glass. A device designed to dip at a constant rate was nmade by modifying a Raytheon curd tension meter (Model 2- 505) to permit the attachment of the slide racks. Adjust- able base supports allowed leveling of the complete dip- ping assembly. A plumb bob suspended from the center of the slide rack acted as a gauge for dipping depth. Re- producible dipping depths were attained by manually switching the machine to reverse when the plumb bob reached an aocbitrary depth in the dipping medium. The filmed slides were heated in an air circulated oven for varying times and temperatures before reweighing. Methods of Evaluating Cleaning Two methods, the SpectrOphotelometer and the micro- kjeldahl, were used to determine the effectiveness of soil removal from the slides. The SpectrOphotelometer methods of Jensen (1946) and Claybaugh (1950) were used with some modification. Essentially, the method consisted of a) creating films on glass microscope slides, b) measuring the percent- age light transmission, c) subjecting the films to a con- trolled washing treatment, d) remeasuring the percentage light transmission, and c) then evaluating the change in percentage transmission resulting from the cleaning treat- ment. ' In addition tO the skimmilk used to film the slides ~as described previously, a 0.5%isolution of detergent mix- ture (w/v) in distilled water was used as a filming ma- terial. The detergent mixture was composed of 45%1Na5P04, 49% Na2005, and 2% Vel which is an anionic wetting agent. Six g. Of calcium chloride and.4 g. of magnesium chloride (Mg012-5H20) were added to each 4-1. portion of the dis- tilled water. The correctly aligned racks of slides were suc- cessively dipped once in skimmilk, air dried, and oven dried. Air drying was facilitated by shielding a round hot 41 plate with a paper device and suspending the slide racks inside this shield. The air drying was cOntinued until no ‘visual moisture was present. Oven drying during film mak- ing consisted Of suspending the slide racks in a 70 to 80° 0. air-circulated oven for 15 minutes. At the completion of the filming procedure oven drying was accomplished by heating the films for 15 to 16 hours at 70 to 80° C. Controlled washing consisted of suspending the films from their racks in magnetically stirred, 4-1. Pyrex beakers of detergent solution. A 24-1. capacity Blue M Electric (Model MW 1150) water bath furnished sufficient capacity for controlling the temperature of both the dis- tilled water and the 4-1. Pyrex beakers. . In preparation for the washing procedure, the beak- ers were removed from the water bath and filled to the 4-1. level with the heated distilled water. Sufficient détergent was added to give the desired concentration. Although the temperature of the detergent solution was not controlled, a maximum drOp of 1° 0. resulted during the washing pro- cedure which required three minutes. The percentage of light transmission through the filmed slides before and after washing was determined by a Cenco Sheard SpectrOphotelometer. The filmed slides were placed in the light path in a position normally occupied by the filter, and readings were taken on three different areas Of the slide. The three representative areas 42 selected for making readings on one slide were in the same positions as those used for all other slides. An arithme- tic average Of the three readings was recorded. The same clean microscope slide was used to adjust the percentage transmission to 100 before reading each slide. A one-hour= warm-up period before using the apparatus minimized the amount of adjustment necessary. The microkjeldahl technique for evaluating residues of film remaining after washing involved several specific procedures which are described below. The essential equip— ment for this method of forming film employed the device shown in Figure 1, combined with a microkjeldahl apparatus for nitrogen determination. When desirable, the tempera-_ ture of the washing solutions could be controlled as shown in Figure 2. Micro cover glasses (22 x 50 mm.) were used as the film supports. For the purpose of determining sam- ple size, the micro cover glasses were weighed on a Chris- tian Becker (Style AB-2) analytical balance before and after filming. Several materials in different combinations were used to create the films. After heating to various tem- peratures for extended periods, the films were given a con- trolled washing without agitation. Following the washing the two filmed micro cover glasses, comprising an individual sample, were "quantitatively broken" into a 50 ml. micro- kjeldahl flask with the aid of sharp pointed forceps. 45 Figure 2 shows the position of the unbroken slides in the neck of the flask. A nitrogen determination was performed = on the film residue remaining on the cover glass after the' washing period. Four milliliters of concentrated sulfuric acid were used to coat the film residues immediately after the washing period but prior to breaking them into the microkjeldahl flask. Each flask contained two glass beads and approximately 0.5 g. Of potassium sulfate (K2804). A clean glass rod was used to push the glass fragments down into the bulb of the kjeldahl flask and to crush the larger glass fragments to facilitate complete oxidation. One ml. of 10%icOpper sulfate (w/v, aqueous, CuSO4) in a 5 ml. hypodermic syringe was used to wash the glass rod be- fore its extraction from the flask. Nitrogen determinations were by the method of Luecke (1960). Results were ex- pressed as,pg. of nitrogen per mg. of film sample. Methods of Evaluating Corrosion of Stainless Steel The corrosion of stainless steel was measured by a) conductivity, b) emission spectrosOOpy, and c) diphenyl- carbazide method for chromium VI. Measurements were made comparing the difference in conductivity between hypochlorite solutions exposed and not exposed to stainless steel surfaces. An Industrial Instrument conductivity bridge (Model R0 16) with a 2 ml. cell, having a cell constant of 0.8500, was used in the 44 tests. Five 75 x 25 mm. stainless steel slides were washed collectively in four liters of distilled water. The wash- ing apparatus was identical with that previously described. Sufficient hypochlorite, Klenzade X1412, was added to the 4-1. portion of distilled water to give 44 p.p.m. avail- able chlorine. ,_ The available chlorine was determined by the starch iodide titration method (Milk Industry Foundation, 1959). The chlorinated solutions were held at 45° 0. during the respective 5-, lO-, and l5-minute exposures to the stain- less steel. Conductivity was determined by the method of Brunner (1960) who used the procedure to obtain identical conductivities of protein and buffer solutions prior to electrOphoretic studies. The emission spectroscopy analysis for iron was per- formed on chlorinated trisodium phosphate solutions after exposure to 1120 sq. cm. of the surface of type 504 stain- less steel beakers with a capacity Of 7-1. Three Vollrath 7-1. stainless steel beakers, bearing the factory identification number 7878, were used as the test surfaces. Four liters of triple distilled deionized water were added to each beaker. Addition of 12 g. of technical grade trisodium phosphate (Na5P04) gave a 0.5% solution (w/v). Sodium hypochlorite (NaOCl) was added to the phosphate solutions in varying amounts. One beaker contained no NaOCl, the second 42 to 49 p.p.m. available 45 chlorine, and the third 451 to 454 p.p.m. available chlorine. Each phosphate solution was exposed to the stainless steel surface at a temperature of 47° 0. for 48 hours. Temperature control was accomplished by placing the three 7-1. beakers in the 24-1. water bath previously described. Continuous and uniform agitation was furnished by three electric continuous stirring devices having pro- peller-type agitators. Evaporation losses were minimized by using heavy cardboard fiber circles as covers for the steel beakers. Evaporation losses from the original 4-1. volume were restored as necessary by adding triple dis- tilled de-ionized water. At the end of the 48-hour exposure period, the phos- phate solution in each beaker was quantitatively transferred to a clean 4-1. Pyrex beaker for evaporation. Without boiling, the 4-1. volume was reduced to 100 to 150 ml. over a low temperature hot plate. These evaporated solutions were, in turn, washed quantitatively into Berselius, tall- form 200 ml. Pyrex beakers. Further low-temperature evapo- ration reduced the volume to approximately 10 ml. A modification of the "8-Quinolinol" extraction pro- cedure for iron (Morrison and Freiser, 1957) was used to separate iron from the large amount of sodium present in the samples. The steps used were: a) Acidify the 100 ml. evaporated sample to approxi- mately pH 5 with 1:1 H01 (v/v, aqueous). 46 b) Filter the sample through Whatman filter paper (NO. 42) into a clean 400 ml. separatory funnel. c) Add 10 ml. of 1%18-quinolinol (w/v) in chloro- form. d) Shake 1 minute and let stand 10 minutes. e) Draw Off the bottom layer (chloroform) into a 200 ml., Berselius, tall-form Pyrex beaker. f) Repeat the extraction twice more using 5 ml. of 8-quinolinol for the last extraction. g) After final decanting, evaporate the solution to dryness on a steam bath. The samples were then examined spectrographically for iron according to the procedure of Bass (1960) which is briefly described in the following outline: a) Ash the previously evaporated samples at 490 to 500° C. for 50 minutes in a muffle furnace. b) Add an internal standard consisting Of 1 ml. of 2.4%» 0001206H20 (w/v, aqueous) to bring the ash into solution. c) Pipette 100‘>.of each solution onto a 5/16 in. high purity National Carbon Products carbon electrode. d) Dry the electrodes, containing samples, under a heat lamp O The emission spectrograph consisted of a National Spectrographic Laboratories power source and a large Hilger spectrograph with an interchangeable glass and quartz prism. The spectrograph was Operated on D.0. are for greater sensitivity. Duplicate standards containing 0, l, 5, 10, 50, and 100,ug. of iron, respectively, were used to evaluate the extraction and spectrographic analysis pro- cedures. 47 The corrosion of stainless steel was measured by using diphenylcarbazide to determine the amount of chro- :mium, the procedure being as follows: The procedures of Horrison and Freiser (1957), Urone and Anders (1950), and Sandell (1944) were modified in preparing the trisodium ' :phosphate samples necessary. The following steps were in- wo lved : a) ‘0) Clean all glassware by exposure to aqua regia for 15 minutes. Rinse the glassware seven times inutap water and three times in distilled water free from chromium contamination. Make chromium standards by placing 12 g. Na3P04. 12H and 25 ml. NaOCl in a 250 ml. Pyrex “be er containing approximately 150 ml. de- c) d) e) f) s) 11) ionized distilled water. Add varying amounts of chromium standard solution (5,pg. Cr./ml., K20r207, aqueous). Samples were Obtained in an identical manner to those previously described. However, instead of transferring the evaporated 4-1. samples to a Berselius, tall-form 200 ml. beaker, transfer the samples into 250-m1., low-form Pyrex beakers. Acidify the standard and steel samples to pH 2 with conc. H2604. Add 2 ml. 5% hydrazinedihydrochloride (w/v, aqueous), made daily, to each sample. Bring just to a boil. Bubble air, filtered through water, into the sample until a negative qualitative test for available chlorine is obtained. The test is done with starch iodide papers. Oxidize Cr to state VI by adding enough K2Mn04 crystals to give a residual pink upon bringing to a brief boil. Cool the samples to room temperature in a water bath containing tap water. i) J) k) 1) n) n) 48 Add a small amount of sodium azide (NaN3) cry- stals to reduce the residual K2Mn04 color. If necessary, filter out any precipitates with ashestos supported by a coarse sintered glass fi ter. Adjust pH to exactly 1.9 with a Beckman (Model G) pH meter and 1:10 82804 (v/v, aqueous). Add 1 ml. Of 0.25%»diphenylcarbazide solution (1:1 v/v, acetone and water) to each colorless sample. Allow 50 minutes for color develOpment « before taking the Optical density (0.D.) reading. Quantitatively transfer samples to 200-ml. volu- metric flasks and make to volume with deionized distilled water. Five hundred-ml. volumetric flasks may be substituted if color develOpment is intense. Invert three times to mix. Check 0.D. of all samples using a Beckman (Model B) spectrOphotometer employing wavelength of 545 mu. and sensitivity of 5. The machine is adjusted to zero 0.D. with the blank standard having no chromium. PrOprietary detergent samples were analyzed also for cdiromium. The procedures previously outlined for the tri- sodium phosphate samples were changed as described below: a) b) Make each chromium standard by placing 4 l. of tap water in a 4-1. Pyrex beaker. The tap water contains approximately 540 p.p.m. hardness ex- pressed as 0a003 by the EDTA titration (Milk In- dustry Foundation, 1959). Add sufficient chromium standard solution (Slug. 0r./ml., K CrgO , aqueous) volumetrically to give 0, 10, 58, an 100,ug. Cr per standard, respectively. Add 25 m1. Of NaOCl to each 4-1. standard. Acidify each solution by adding 5 m1. cone. H2804 and enough conc. HNO} to give pH 2 to 5. Evaporate to approximately 150 ml. without boil- ing. Four-1. samples of chlorinated prOprietary de- tergent were obtained from an automatic cleaning unit located in the Michigan State University dairy plant. Samples were taken after an elapsed c) d) e) f) e) h) 1) J) 49 time of 50 minutes in the washing cycle. In this automatic cleaning unit, approximately 446 ft. of 1.5 in. stainless steel pipe were cleaned at a temperature of 158 3 5° F. The available chlorine and detergent concentrations of the cleaning solu- tions varied widely. Available chlorine was fur- nished by NaOCl. The detergents were a mixture of Klenzade AC-l, and A0—6, used in approxi- mately 2:1 ratio, respectively. Acidify the 4-1. samples, taken in 4-1. Pyrex beakers, by adding 5 ml. conc. H 804, and enough conc. HNO to give a pH of 2 to . Evaporate to approx ately 150 ml. without boiling. Wash the evaporated samples quantitatively into 250-ml. Pyrex beakers with distilled deionized water. Adjust the pH to exactly 1.9 with 50%iNa0H (w/v, aqueous) and 1:10 H2S04 (v/v, aqueous). Add a few drO s Of 5%rhydrazinedihydrochloride (w/v, aqueous , made daily, to the warmed sample. Bubble air, filtered through water, into the sample until a negative qualitative test for available chlorine is obtained. The test is done with starch iodide papers. Add a large excess of KMn04 crystals tO the solu- tions and bring to a boil. Filter Off the pre- cipitate by filtering through asbestos supported by a coarse sintered lass filter. Repeat the KMnOh oxidation and f ltering twice more or until a residual KMn04 color remains upon boiling at least one minute. Sintered glass filters are cleaned by aqua regia and detergent solutions, bath of which are used in forward and reverse f ow. Add a small amount of crystalline NaN; to reduce the residual KMnOh color. Check, but do not adjust the pH of the sample solutions. Add 1 m1. of 0.25%ldiphenylcarbazide solution (1:1 v/v, acetone and water) to each colorless sample. Allow 50 minutes for color development before reading 0.D. - k) Quantitatively transfer samples to ZOO-ml. volumetric flasks and make to volume with de- ionized distilled water. Five hundred-m1. volumetric flasks may be needed if color de- valOpment is intense. Invert three times to m x. 1) Check 0.D. Of all samples using a Beckman A (Model B) spectrOphotometer employing wave- length of 545 mu. and sensitivity of 5. The machine is adjusted to zero 0.D. with the blank standard containing no chromium. All statistical analyses were performed under the direction of W. D. Baton, Statistician, Michigan Agricul- tural Experiment Station. The term "significant" in this thesis refers to significance at the 1%iprobability level as calculated by the "t" test. All reagents used were Reagent Grade unless other- wise noted. EXPERIMENTAL RESULTS Measurement of Soil Retaining Capacity Methods of measuring films. a) Ellipsometer. NO quantitative data for the method were 1)) tabulated. Films of barium stearate, 50 A. thick ap- plied on number 4 and 7-finishes of stainless steel slides, were measurable within an accuracy of plus or minus 5 A. However, the thickness of films produced by pasteurized skimmilk on number 4-finish stainless steel slides and glass microscOpic slides could not be meas- ured with reliability, because of the roughness Of the glass and stainless steel surfaces. Interferometer. The deflection Of interference lines, caused by increasing the thickness of skimmilk films, is presented in the data of Table 5. Difficulty was’ encountered in attempting to evaluate thickness of skimp milk film with the interferometer because it was impos- sible to determine the number of deflections caused by one film of skimmilk. Deflections were noted visually to be equal approximately to one-half of a wavelength of sodium light per individual film layer. When the apparent deflection of interference lines was one-half, the observer had no way of knowing whether the deflec- tion actually was one-half, one and one-half, or two and one-half, etc. For this reason, the interferometer - 51 - 52 method was discarded in this investigation. c) Electron.microsc0pe. The electron micrograph of a skim- milk film replica, taken at a magnification of 8,000 to 10,000, is shown in Figure 5. The large dark spots and their white shadows resulted from depositing droplets of dilute nitrocellulose on the glass surfaces prior to the skimmilk film. The droplet-shadows reflect film thickness. The variation in length of shadows of nitro- cellulose drOplets, having approximately the same di- ameter, supported the premise of film unevenness held by Rothen (1959). Shadows of equal length throughout the film would indicate uniform thickness. Perhaps the most significant point that might be noted from the micro- graph is the rough surface tOpography revealed in the other areas Of the micrograph not occupied by the nitro- cellulose drOplets and their shadows. <1) Gravimetric. Comparisons of variation between the four- ,place balance and another balance allowing estimation of the fifth place are illustrated in the data of Table 4. ‘Variation was equal or greater with the five-place balance than with the four-place balance. Consequently, the latter was used throughout the study. The uniformity of creating films of skimmilk on sur- fac es of glass and stainless steel, as measured gravimetri- cally, is illustrated in the data of Table 5. Evaluation '38 -made both within and between three individual racks 53 containing six slides each. Surfaces of stainless steel slides accepted weights Of skimmilk film with approximately equal uniformity. A similar result was noted with glass slides. The data Of Table 5 show a small variation in the weights of the film on the slides within and between racks relative to the filming procedure for one surface. The increase in weight of 22 x 50 mm. micro cover glass slides after each application of skimmilk is shown' in the data of Tables 6 and 7 and in Figures 4 and 5. After a slide was dipped once in skimmilk the weight of film deposited for each succeeding dip was linear, when the dipping was continued a maximum of 10 times. Within one rack of glass slides the comparative weights of skimmilk films deposited per slide was quite uniform as shown in the data of Table 8. However, more variation in weight Of film between the slides in one rack occurred when the slides were dipped 10 times, as compared to five. Methods of evaluating cleanipg. a) SpectrOphotelometer. The reproducibility of photelo- meter readings on 25 x 75 mm. glass microscOpe slides, dipped together in one rack, is given in the data of Table 9. These data show close agreement between re- peated readings. When every alternate layer Of film was made from a detergent mixture, instead Of each 54 layer being skimmilk, there was also close agreement between three repeated readings (Table 10). The reproducibility of photelometer values within and between three racks of microscOpe slides is again shown in the data of Table 11. Readings are given for clean slides, slides with films, and for slides with films after a controlled washing procedure. The read- ings for slides and films in racks l and 2 showed good agreement between and within racks. However, the read- ings made for slides in rack 5 showed wide variations. One possible reason was that the apparatus seemed sub- ject to voltage variations. The effect of washing, determined by the difference between the percentage of light transmitted through the washed slide and the filmed glass slide, is given in the data of Table 12. High percentage light transmission values for the washed slides resulted, indicating almost complete removal of ~the film by the washing procedure. Heating Of skimmilk films decreased the percentage transmission of light as shown in the data of Table 15. A change in the crystalline structure of the film by heating could account for the difference in light trans- mission. As has been noted, some difficulty was experienced in evaluating filming and washing procedures with the SpectrOphotelometer. Nonreliability of the photelometer 55 method for the determination of quantitative filming is shown by data in Table 14 and illustrated in Figure 6. The absorbancy did not increase in a linear manner with increasing weight Of film. That Beer's Law was not obeyed is confirmed by the data from Table 15 and Figure 7. The greatest departure from linearity is shown at both the higher and lower levels of film weight. b) Microkjeldahl. Data in Table 16 show results from pre- liminary trials using the microkjeldahl method. The percentage recovery of the standard samples containing 1 mg. nitrogen, ranged from 89.5 to 101.1%rwith the ' mean recovery at 95%. Wide differences in the comparative detergency values of hard and distilled water are illustrated in the data of Table 17. The soil film retained after washing, expressed inwug. of nitrogen per mg. of sample, showed a mean value of 15.1 for distilled water and 74.8 for the hard water. The protein solubility Of skimmilk-tap water films was increased significantly by chlorinating a 0.1%itri- sodium phosphate tap-water solution as shown in the data of Table 18. The protein remaining, expressed as pg. of nitrOgen per mg. sample, was decreased from 69.14 when no chlorine was used to 7.8 when 510 p.p.m. chlorine was em- ployed. The increase in protein solubility was accompanied by an increase in pH of the solution from 9.4 to 10.4. The rise of pH could have been at least partially responsible 56 for the increased protein removal. The pH of the trisodium phosphate tap-water solutions increased because sodium hypo- chlorite had been added for the purpose of chlorinating the solutions at different levels. " When buffer solutions at three different pH's were chlorinated, there was also a significant increase in pro- tein solubility (data from Table 19, illustrated in Figure 8). In these trials a greater increase in protein solu- bility resulted for the nonchlorinated buffer than for the chlorinated buffer, causing an apparent disagreement with results of Table 18. Later results indicated that the pH Of the buffers was not sufficiently high to show an increasei protein removal by chlorination. Figure 8 also shows close correlation between the curve for the amount of 2- Molar sodium carbonate used in the buffer and the curve in- dicating protein solubility by the chlorinated buffer. The effect of trisodium phosphate and sodium hypo- chlorite concentrations on the pH of tap water containing approximately 540 p.p.m. total hardness is shown in the data of Table 20. At a concentration of 0.5%itrisodium phosphate the solution was practically stabilized at a pH of 11.4. A concentration of 0.15% trisodium phosphate in distilled water gave the same pH of 11.4. The addition of 12 ml. of sodium hypochlorite to both tap and distilled- water solutions at pH 11.4 increased the pH only 0.2. 57 The data of Tables 22 and 25 show that significantly more protein was removed when 0.5%itrisodium phosphate tap- water solutions and/or 0.15%ltrisodium phosphate distilled- water solutions were chlorinated. Both studies were per- formed at pH 11.5 I 0.1. Numerous preliminary trials were performed to create a film sufficient in quantity and tenacity to permit meas- urements of cleaning for 10 minutes at 65° C. The informs! tion in Table 21 summarizes the procedures used in develop- ing the tenacious films. Highest resistance to washing was secured with Film N, which was relatively high in protein and withstood unagitated washing for 10 minutes in a 0.15% trisdodium phosphate distilleddwater solution. A variation of 56%»in the protein content of these films was observed. In preliminary studies time of reaction was impor- tant in Obtaining increased protein solubility resulting from chlorination. The data of Table 24, Figure 9, cone firmed the importance of the time factor. Protein solu- bility, due to chlorination, increased rapidly with time after one minute. At one-minute reaction time, no differ- ence was found between the protein solubility of chlorinated and nonchlorinated solutions containing 0.15% trisodium phosphate in distilled water at pH 11.5 1 0.1. A bit of evidence that chlorination was not a detergent aid when used for short periods Of time (less than 1 minute) was noted in making films. In preparing tenacious films, slides were dipped momentarily and intermittently in clarifier slime suspensions and solutions of trisodium phosphate, calcium carbonate, and sodium hypochlorite. The build-up of such films was much more rapid than when the sOdium.hypochlorite was not used. The more rapid build-up was easily Observed visually. One of the objectives of the study was to determine Optimum levels Of chlorination. Data in Table 25, shown graphically in Figure 10, give the relationship Of avail- able chlorine to protein solubility under the conditions of this study. Increases in protein solubility were noted at all levels of chlorination studied. However, a more rapid increase in protein removal was noted above the 75 p.p.m. available chlorine level. A lower rate of increase was generally observed when the chlorine content exceeded 226 p.p.m. The data also show (Figure 10) that at the comple- tion of washing, a smaller proportion of film remained on the slide above the 226 p.p.m. chlorine level than at the lower levels of chlorination. Therefore, probably insuffi- cient film was present to permit the maximum protein removal possible by the use of available chlorine in the solution. Methods of Evaluating Corrosion of Stainless Steel COpductivity. Attempts to measure removal of metal by determining differences in conductivity of chlorinated distilled water solutions after exposure to type 504 59 stainless steel did not show positive results.) No detect- able difference in conductivity was found when 1 liter of agitated chlorinated distilled water was used to wash, collectively, five 25 x 75 mm. stainless steel slides. One problem was the small proportion of metal present, if any, in relation to the much larger proportion of detergent salts present. Emission sp2ctroscOpy. Attempts to analyze used detergent samples for iron by the emission spectroscopy method were unsuccessful. Consequently, no quantitative data were collected. Diphepzlcarbazide for chromium VI. The diphenyl- carbazide method for determining chromium VI was utilized to evaluate residues removed from type 504 stainless steel when treated with chlorinated trisodium phosphate and anp other proprietary chlorinated alkaline detergent. Data for the analyses of standard chromium samples containing trisodium phosphate and two different amounts of sodium hypochlorite are given in Table 26, and illustrated in Figure 11. Increasing the amount of sodium hypochlorite did not affect the chromium recovery from the standard sample. The location of the curve of Figure 11 was esti- mated visually. The curve was linear for the standard samples containing 0 to l20,ug. chromium. 60 As shown in the data of Figure 12, increasing the concentration of trisodium phosphate, in the standard samples, reduced the lepe of the chromium recovery curve but did not affect its linear nature from 10 to 100,ug. chromium. However, the curve was not linear from 0 to 10 ,ug. chromium. In other words, the recovery of 0 to 10,ug. of chromium was not complete in the presence of 24 to 48 g. Of_trisodium phosphate. The curves were located visu- ally as previously described. When 1,120 sq. cm. of type 504 stainless steel were exposed to a chlorinated 0.5% trisodium phosphate distilled water solution, the amount of chromium removed is shown in the data of Table 27 and illustrated in Figure 15. Mean amounts of 1.5, 52.1, and 155,ug. chromium were removed from type 504 stainless steel when 0, 100, and 500 p.p.m. available chlorine, respectively, were used. Variation in chromium removed between individual samples was greater at the 100 p.p.m. chlorine level than at the 500 p.p.m. level. The mean rate of chromium removal increased with increas- ing chlorination but not necessarily in a linear manner (Figure 15). Results of a preliminary trial showing the increase _ in chromium removal with the increase in temperature is presented in Figure 14. The curve appears to be linear in nature. 61 The data in Table 28 (illustrated in Figure 15) show the recovery of chromium from standard samples con: taining a prOprietary chlorinated alkaline detergent of unknown composition. The data show that increasing the concentration Of the detergent two-fold did not affect the chromium recovery. The curve was linear from O to 100,ug. chromium. . Samples employing the proprietary chlorinated alka- line detergent taken from a commercial automatic cleaning unit (Table 29) were negative in chromium, with the excep- tion that a slightly positive test was secured from a de- tergent solution containing 254 p.p.m. available chlorine. The chromium content in the one positive sample was esti- mated to be 1 to 2,ug. in an original sample volume of 4 liters. DISCUSSION Perhaps the most significant result of this investi- gation_was the development of a cleaning evaluation test based upon the microkjeldahl procedure. The procedure gave quantitative information regarding the functions Of a detergent in removing protein at the soil-detergent- solution interface with results expressed in terms Of nitro- gen remaining on the soiled surface after the cleaning pro- cedure. Maxcy and Shahani (1960) found that a somewhat similar procedure possessed sensitivity for 2 p.p.m. of skimmilk solids in testing detergent solutions after use. There was a lack of uniformity in nitrogen recovery in preliminary trials Of this investigation on "used" de- tergent solutions of chlorinated trisodium phosphate COD! taining standard amounts of sample. An explanation for this phenomenon was Offered by Cahn and Powell (1954) who reported that nitrogen was liberated when ammonia and hypochlorite were allowed to react in an alkaline medium. Therefore, nitrogen can be assumed to be lost to a vari- able degree during the washing procedure before the micro- kjeldahl analysis. On the basis of the report by Cahn and Powell, the results of examining used alkaline detergent solutions containing available chlorine for nitrogen con- tent should be viewed with reservation. However, the value of the microkjeldahl method for determining nitrogen onfl .. -62- 65 soil films remaining after the washing procedure was well demonstrated in this investigation. Also, the absence of detergent chemicals in the kjeldahl flask facilitated the oxidation procedure by reducing the violence of the boil- ing. Another significant result was the develOpment of a special slime-type film high in protein and tenacity. Tenacious films were desirable in order to have sufficient film remain after the washing operation to furnish nitrogen for the relative comparisons of different conditions of cleaning. The films used by Roderig 22 21. (1956) were tenacious enough but too low in protein for effective com- parisons of cleaning by the microkjeldahl method. The films employed by MacGregor 22 21., Claybaugh (1950), Jensen (1946), and others were not tenacious enough to withstand the rather rigorous washing conditions demanded in the present experiment. Chlorination of trisodium phosphate, under condi- tions of this study, significantly increased protein solu- bility (removal) of soil films. Thus, one of the main Ob- jectives of the study, namely, the function of chlorina- tion in detergency of alkaline cleaning materials, was partially explained. The increased protein solubility of soil films due to chlorination of trisodium phosphate was dependent on time of exposure. After one minute the solubility of the 64 protein was much more rapid in the chlorinated than in the control solution. At one minute of exposure the protein solubility was identical for both the chlorinated and none chlorinated solutions. An explanation for the poor deter- gency value of chlorinated trisodium phosphate at one minute or less of exposure was provided in the literature by many investigators who have noted that chlorine sani- tizers combine with proteinaceous materials. Chlorine sanitizers are usually employed for short periods because of their fast action and corrosiveness to stainless steels. The results of this investigation did not indicate clearly whether the relationship of beneficial protein solu- bility due to chlorination was linear with time of exposure to detergent. Further study was indicated. Another unp clarified point was the importance of temperature on the beneficial effect of detergency due to chlorination. In these studies a beneficial cleaning effect was apparent at 65° C. but not at lower temperatures. There appeared to be almost a linear relationship between concentration Of sodium hypochlorite and improvement in protein solubility. More investigation may be required to establish the effect of higher levels of chlorination. However, the higher levels of chlorination may contribute to cOrrosion problems. A consideration of a combination of factors suggested that the colorimetric diphenylcarbazide method for measur- ing the amount Of chromium VI was a promising technique 65 for evaluating corrosion of stainless steels. The postu- lation was based on measuring the amount of chromium in detergent solutions which had been exposed to stainless steel surfaces. Chromium was selected as the element for corrosion evaluation because it was present in stainless steels and when found in the used detergent solutions, it would most likely have been removed from the stainless steel surfaces. Unlike iron, chromium was not commonly present in water supplies. Also, metal removal from stains less steel was indicated to be in the order of small magni- tude for any one exposure period to the detergent. The diphenylcarbazide method had been reported to be quantita- tively sensitive to microgram quantities of chromium (Morrison and Freiser, 1957; and Saltzman, 1952). The sur- face composition of type 504 stainless steel used in this investigation was known (Rhodin, 1955). In the studies reported herein, the diphenylcarba- zide method for measuring chromium VI was demonstrated to be a useful and sensitive means of determining metal re- moval from stainless steel. To retain the distinct advan- tages of the test and to eliminate problems of analysis, future study should be made of a distillation procedure to' effect a separation of chromium from the detergent samples. Chromyl chloride (Cr02012) has been reported to be vola- tilized at 116° C. from mixtures of chromium, sulfuric acid and hypochlorites (Udy, 1956). The removal of 66 chromium from detergent samples by the formation of vola- tile chromyl chloride seems possible. If the chromyl chloride distillation was efficient in removing chromium, a diphenylcarbazide determination for chromium could be made on the distillate. Extreme sensitivity would then be attained without the interferences of detergent salts, heavy metals, organic matter, and hypochlorites. Success in developing the distillation procedure would make avail- able a relatively simple, rapid method for evaluating re- moval of metal from stainless steels. Many problems were involved in applying the diphenyl- carbazide method for measuring chromium VI to the conditions of this investigation. The interferences have been men- tioned above. The presence of the detergent salts made the samples very difficult to boil and resulted in losses of samples from "pOpping" in.the later oxidation proced- ures. Another mechanical problem resulted from the method Of taking samples. Four-l. samples of detergent were evaporated to approximately 150 ml. The probability of encountering measurable amounts of chromium was enhanced by taking the large samples, but the problems of any inter- ferences present were magnified. Oxidation to destroy organic matter by wet or dry ashing methods also created difficulties. Wet ashing with sulfuric acid, nitric acid, and perchloric acid, separately or in combination, resulted in loss of chromium by volatili- zatione 67 Dry ashing of the samples sometimes resulted in an ash which was insoluble or difficult to get into solution. Loss of samples through "pOpping" in the ashing procedure was also encountered. Saltzman (1950) reported that phosphate was con- verted to polyphosphate by strong heating, either from wet ashing or dry ashing. Consequently, the chromium was com- plexed by the polyphosphate making the chromium nonreactive to diphenylcarbazide. Polyphosphates were undoubtedly pre- sent in this study and many of the negative results Ob- tained were attributed to their presence. Attempts to separate interferences with the chro- mium analysis by the troublesome extraction resulted in poor chromium recovery. As was true for the unsuccessful iron extractions, "sudsing" of the detergent and solvent was held responsible for the poor separations. Isoamyl alcohol, isoamyl alcohol and pyridine mixture, and chloro- form were among the solvents used without success tO ex- tract the diphenylcarbazide-chromium complex. Because of the difficulties encountered in separa- tion methods and destruction Of organic matter with wet and dry ashing, analysis for chromium was performed in aqueous solution. Potassium permanganate was employed to oxidize the organic matter present. This made possible the oxidation of chromium to the hexavalent state and permitted formation of the chromium-diphenylcarbazide complex. 68 In order that chlorites present did not interfere with the oxidation procedure, the hypochlorites were re- duced to chlorides. Several reducing substances were tested before one was found which would reduce the hypochlorites to chlorides without interfering in the permanganate oxida- tion of chromium from a valence of three to six. Hydrazine- dihydrochloride was the compound used successfully. Com- pounds used unsuccessfully were sodium azide, oxalic acid, hydroxylamine, stannous chloride, and hydrogen peroxide. Bubbling of air through the system assisted in eliminating any gaseous chlorine present. The interference of heavy metals, primarily iron, was minimized by reading the diphenylcarbazide-chromium complex in 200 or 500 ml. volumes. The principle employed was to dilute the heavy metal interference (other than chromium) below that required for the diphenylcarbazide reaction and was in agreement with the report of Feigl (1946). Dilution of the samples was possible because of the extreme sensitivity of the diphenylcarbazide reaction for chromium. The gravimetric procedure was superior to the other methods Of film measurement used because of the relative uniformity of the film deposition, the reproducibility of the results Obtained, and the flexibility Of the method. Comparison with the gravimetric method confirmed that the SpectrOphotelometer method did not obey Beer's 69 Law for the conditions of this investigation. This is in accordance with the statements of Masurvosky and Jordan (1958) that light transmission measurements are deficient at both high and low concentrations of film. The ellipsometer may have a very useful application in the measurement of stainless steel corrosion by deter- gents. The extreme sensitivity of the ellipsometer, as shown in these studies, supports this conclusion. Though in an area outside the immediate scape of this study, the mechanical filming technique may have an application in measuring the bacteriological quality of milk. An illustration of its application is in the Breed smear procedure for bacterial enumeration. Skimmilk films formed by the mechanical technique were shown by the elec- tron micrograph to be rough and uneven. 0n the other hand, milk films for the Breed smear are deposited and spread manually. Therefore, the Breed smears, presumably, would be less homogenous in bacterial content than the mechani- cally prepared film. If more attention was given to pre- paring the Breed smears, closer correlation between the Breed smear technique and other bacteriological enumera- tion methods might be expected. SUMMARY AND CONCLUSIONS Investigations were made of methods for measuring a the function of chlorinated trisodium phosphate detergents. Methods were develOped for evaluating a) the formation of milk films on glass and stainless steel surfaces, b) clean- ing, and c) corrosion of stainless steel. These methods ‘were applied under cleaning conditions of hard and dis- tilled water, varied levels of chlorination, varied pH, and 'variations in time of exposure to the detergent. ‘ The ellipsometer and other Optical methods were in- effective in measuring thickness Of skimmilk films because of the uneven nature of both the films and of the glass and stainless steel surfaces on which they were supported. Electron micrographs of skimmilk films supported this pre- mise. SpectrOphotelometer light transmission measurements of film deposition did not Obey Beer's Law, particularly at both low and high levels of film thickness. Consequently, the method was considered unreliable for evaluation of cleaning. Gravimetric measurements showed that the formation of skimmilk film on glass surfaces was a linear function when the slides were dipped from one to ten times. A combination gravimetric and microkjeldahl method was effective in measuring the function of chlorinated tri- sodium phosphate detergents. Useful visual observations of cleaning were also available during the washing of the afides. - 7o - 71 Films of skimmilk on glass surfaces were not suffi- ciently resistant to washing with chlorinated trisodium phosphate to permit study after the cleaning procedure. Methods were develOped for producing tenacious films from clarifier slime, calcium shloride, trisodium phosphate, and sodium hypochlorite. Distilled water was much superior to hard water in aiding trisodium phosphate to remove skimmilk films from glass surfaces. . Improved detergency resulted from adding sodium hypochlorite to hard water. However, increased protein solubility was accompanied by an increase in pH. When the pH was stabilized at 11.5 1 0.1, and thus was unaffected by addition of sodium hypochlorite, the improved protein solubility was attributed to chlorination. Distilled water solutions of trisodium phosphate also showed improvement in cleaning ability after chlorination at pH 11.5 i 0.1. At pH's lower than 11.5 I 0.1, chlorinated buffer solutions showed less ability to dissolve protein than did nonchlorinated buffers. However, ability of both chlori- nated and nonchlorinated buffers to dissolve protein in- creased with an increase in pH. Under the conditions of temperature and pH studied, the protein solubility due to chlorination of trisodium phosphate increased rapidly after one minute Of exposure ' to the detergent. 72 An optimum level of chlorination was not found. In- creased chlorination gave increased protein solubility at all levels Of available chlorine used, 0 to 512 p.p.m. The least increase in rate of protein solubility occurred at 75 and 512 p.p.m. of available chlorine. Chromium loss, of a small order of magnitude, oc- curred when type 504 stainless steel was exposed to chlorinated trisodium phosphate for 48 hours at 47° C. The chromium loss increased, as the available chlorine in- creased from 0 to 500 p.p.m., but not necessarily in a linear manner. Chromium losses from a commercial system of stain- less steel pipelines cleaned automatically with a prOpri- etary chlorinated alkaline detergent were almost non- existent. The highest available chlorine level of 254 p.p.m. yielded the only positive test and showed from 1 to 2,ug. of chromium present. All other tests were nega- tive. The following conclusions may be drawn from the . investigation: a) Chlorination, by the addition of sodium hypo- chlorite, increases the capacity of trisodium phosphate solutions to remove milk-protein soils. b) Conditions contributing favorably to increased protein solubility are: a temperature of 65° 0.; C) d) 73 a pH of 11.5 t 0.1; a time of exposure to the detergent Of 10 minutes; and a high, though noncorrosive, available chlorine content. 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Dairy Sci. é: 310-519 0 TABLE 3. - Skimmilk film deposition on glass microscOpe slides measured by interferometer Times dipped in 86 Slide skimmilk Deflection* (no.) (no.) A 1 7: B 2 O C 3 )6 D 4 O E 5 Y2 F 6 O ‘ Deflections in wave lengths of sodium light. 87 TABLE 4. - Variation in weight of slides when weighed to ‘ the 4th and to the 5th decimal place Variation Slide 4th decimal‘ 5th decimal‘ (meo) (ne.) 0 (>00ng OOOOOO OOOOOH H \O-PHWO‘G) \DVWONCD IIhO’UOZFS ENC-404K119 "dtljUOtfiP 000000 000000 000000 HHOI—‘H-P OOOOOO l-‘I-‘OOOH 000000 000000 000000 \I‘I'QO‘U'I Total 1.1 |-’ O N (D i 0.06 0.07 . * Represents mg. variation of three repetitive weigh- lngs o 88 TABLE 5. - Uniformity of skimmilk film weights on 25 x 75 mm. slide surfaces measured gravimetrically t Film weight of slides dipped in skimmilk Siifgze Rack 1 Back 2 Rack 3 Slide Film Slide Film Slide Film weight weight weight (ne.) (mso) (ms-) Stainless A 2.7 G 3.2 M 3.2 steel B 2.8 H 2.7 N 3.0 C 3.1 I 2.7 O 2.9 D 204 J 300 P 208 E 2.7 K 2.8 Q 2.8 F 3.2 L 2.6 R 3.0 i 2.8 2.8 2.9 Glass A 3.3 G 3.4 M 3.4 B 3.8 H 3.5 N 3.4 C 3.6 I 3.3 O 3.4 D 3.5 J 3.5 P 3.7 E 505 K 507 Q 3.5 F 3.4 L 3.7 R 3.5 MI W U1 \N \n \N 4: TABLE 6. - Skimmilk film formation on 22 x 50 mm. micro cover glass slides when dipped intermittently and measured gravimetrically Film Times dipped Fi1m weight‘ (no.) (ms-) m¢m coco ONH N!“ \O' #W fl- hu- 0 HHmQW muowb m I .Hm mnmonm H0 .02 Ammflc smospon N N N N N N N N N N N N opMHm pom nobov mmmwmw Haw z E H M h H m 0 m m Q 0 SHHH .vouoonnsm ones msHHH noHns on pamspwons msowpmnogo msHaHHH Ho ooqoSdom coanpnoo I .Hm mmmds 101 TABIIJ22. - Effect of chlorinating (156 p.p.m.) 0.5% tri- sodium phosphate tap water solutions on protein solubility of soil films _: I Residual protein film after washing in Trial Unchlorinated - Chlorinated trisodium trisodium phosphate‘ hosphate‘ (pg. N/mg. g. N/mg. sample) sample) A 18.68 10.31 B 14.74 10.51 C 16.17 . 10.74 D 19.25 15.00 E 23.18 17.02 F 30.31 15.71 x 20.39 13.22 0' 5.71 3.01 ‘ Represents‘pg. nitrogen remaining after washing per mg. film sample (10 min. unagitated wash, 65 to 54° 0., pH 11.5 - 0.1 . - 102 TABLE 23 - Effect of chlorinating (156 p.p.m.) 0.15% tri- sodium phosphate distilled water solutions on protein solubility of soil films Residual protein film after washing in Trial Unchlorinated Chlorinated trisodium trisodium phosphate‘ phosphate‘ (pg. N/mg. (pg. N/mg. sample) sample) A 13.17 15.19 B 16.52 16.11 C 15.86 9.62 D 24.46 16.11 E 29.53 14.96 F 22.19 8.88 G 27.22 10.33 H 24.26 '9.71 I 6.59 6.36 J 15.22 3.08 K 20.00 3.19 L 7.47 9.17 M 9.01 8.38 N 31.96 3.59 0 10.31 4.32 x 18.45 9.27 6' 7.65 4.65 ‘ Represents pg. nitrogen remaining after washing per mg. film sample (10 min. unagitated wash, 65 to 54° 0., pH 11.5 i 0.1). 103 TABLE 24. - Effect of exposure time on protein solubility of soil films washed with chlorinated (156 p.p.m.) 0.15%Itrisodium phosphate distilled water solutions M Protein film removed by washing in Time (minutes) Unchlorinated Chlorinated trisodium. trisodium hosphate‘ phosphate‘ pg. N/mg. (pg. N/mg. sample) sample) 1 15 15 4 1? 33 6 20 46 8 15 67 ' Represents mean of three trials,,pg. nitrogen removed by'gashing per mg. £11m sample (10 min. unagitated wash, 65 - 1° Ce, pH 11.5 " 001). TABLE 25. - Evaluation of optimum levels of chlorination.on protein solubility of soil films washed with 0.15% trisodium phosphate distilled water solu- tions Residual protein film after washing Sodium Available . _ hypochlorite chlorine i 0’ (31¢) (p.p.m.) (’18. N/ . sample 0 0 68.92 9.8 2 78 55.31 9.9 4 156 24.42 14.3 6 226 9.31 6.4 8 312 7.30 4.5 f ' Represents mean of 8 trials,,pg. nitrogen remaining after washing per mg. 11m sample (10 min. unagitated wash, 65 to 540 Ce, pH 11.5 " 001). 104 TABLE 26. - Standard samples of chromium in chlorinated trisodium phosphate solutions‘ measured by the diphenylcarbazide method Absorbancy of standard samples Chromium Trial 1 Trial 2 0180) 25 ml. 50 ml. 25 ml. 50 ml. BaOCl NhOCl NaOCl NaOCl (AB) (1,) (Is) (As) 0 0.000 0.000 0.000 0.000 15 0.036 0.050 0.051 0.040 30 0.075 0.085 0.088 0.090 50 0.152 0.126 0.132 ..... 70 0.200 0.187 0.184 0.165 90 0.241 0.230 0.245 0.239 120 0.322 0.302 0.289 ..... ‘ Each standard sample contained 12 g. Na P041123 150 ml. distilled water. or equated volume of 200 ml. Absorbancy was read in 8.1180 105 TABLE 27. - Chromium losses from type 304 stainless steel exposed to chlorinated 0.3% trisodium phosphate distilled water solutions measured by the di- phenylcarbazide method _r Chromium removed‘ Trial No 100 p.p.m. 500 p.p.m. chlorine chlorine chlorine (pg. Cr) (pg. Cr) (pg. Or) A 4.0 17.0 107.0 C ... 26.0 103.0 D 4.0 26.0 152.0 E 0.0 48.0 136.0 F 0.0 47.0 174.0 G 0.0 35.0 168.0 H 2.0 37.0 111.0 I 4.0 31.0 107.0 Total 14.0 289.0 1194.0 i 1.5 32.1 133.0 6' 107 1005 2705 ‘ Represents,pg. Cr. removed from.1120 sq. on. steel by 48 hour exposure at 47° C. TABLE 28. - Standard samples of chromium in chlorinated proprietary detergent solutions measured by the diphenylcarbazide method 106 Chromium Absorbancy of standard samples 048-) Trial 1 Trial 2 IX-conc.‘ 2X-conc.‘ 1X-conc.‘ 2X-conc.’ (1,) (1,) (1,) (1,) 0 0.000 0.000 0.000 0.000 10 0.045 0.040 0.032 0.036 50 0.150 0.155 0.118 0.160 100 0.310 0.315 0.298 0.295 ‘ Represents 2:1 ratio of 2 proprietary detergents roprietary detergent, (ll-concentration equals 4 ml. of lst and 2 m1. of 2nd proprietary detergent water evaporated to 150 ml. in 4-1. of tap TABLE 29. - Amount of chromium removed from 446 feet of 1.5 inch stainless steel pipeline by a chlorinated proprietary alkaline detergent as measured by the diphenylcarbazide method Trial Available chlorine (1)-pm.) Chromium remove HNQH IEIUOUIP ....I 0000 00.500 ‘ Represents chromium in a 4-1. sample elapsed time of 30 minutes in.the washing cyc taken at an , 135 1 5° F. Fig. 1. - Mechanical filming and washing apparatus for gravimetric-micro-Kjeldahl analysis. Fig. 2. - Temperature controlled mechanical filming and washing apparatus for gravimetric-micro-Kjeldahl analysis. 109 Fig. 3. - Electron micrograph, 8000 - 10,000 x, of skimmilk film replica. 110 0 l8 “ e 3. E. E, . i’ . E L: e 6 6* e ./ ./ O l 2 3 4 5 6 7 8 9 l0 Dips (no.) Fig. 4. - Deposition of skimmilk on 22 x 50 mm. micro cover glasses measured gravimetrically. 111 F I'Im weight ( mg.) Dips (no.) Fig. 5. - Skimmilk film formation on 22 x 50 mm. micro cover glasses measured gravimetrically. F— ,__.__+H (120 * 0J5 r' Absorban cy P 6 0.05 P— +--il,-ll_-,_, _llii_kl ._______,,,, l (,,,._ ._.___1 l I TRIAL 1 ' 1 \ ,X: ; TRIAL 2 [MpsIno) P10. 6. - Atsorbancy of f llms measlrei bv photelometer vs. number of times the slide was dipped in skimmilk 112 we t1" 0- 113 A 8 -— ”W 0.20 A 7 +— 6 IL‘ A C —-1 0J5 3, ! GRAVIMETRIC \ 1 . \E- s L ' z; E; i -'___—_’ S '6 I 4——— E g 4 L A 3 g !‘ A --i 0J0 .& l: . 3 ~— A /' SPECTROPHOTELOMETER 0”. 2 »- 1 0.05 I I- o i l 1 l J L 0 I 2 3 4 5 6 Dips(n0) Fig. 7. - )ensitivity 0f uzaxlmetrlc vs Spevtoehete- lometer methods of measuring films. Film Sample Remaining (pg. N / mg. 5 ) . No CO IN BUFFER 80 L. 2 3 . / 60 -— CHLORINATED BUFFER ___, . 40 r— UNCHLORINATED BUFFER <— +— e‘ 20 r 0 O 0 l l *__. L 9.4 I00 I07 pH 114 50 l00 I50 2M Na2 CO‘3 Used In Buffer(ml.) 200 Fig. 8. - Effect of chlorinated buffer solutions at different pH's on protein solubility of soil films. 115 70 0 60 — CHLORINATED u; ‘E” 50 i— ‘\ 2 3' e 'b 0) > E 40 ’- 8 E I: .E . 3: o L Q 30 __ UNCHLORINATEO 20 F— /. g... \ 9—7 e .0 1 1 l l l l I 0 l 2 3 4 5 r“ 7 8 Time (minutes) Fig. 9. - Effect of exposure time on protein solu- bility of soil films washed with chlorinated (156 p.p.m.) 0.15% trisodium phosphate distilled water solutions. 80 03 O A O "Pg—T Residual Film Sample Remaining (pg. N/mg. s.) N O O f‘ "— ‘" J 200 Chlorine (p.p.m.) 116 Fig. 10. - Evaluation of Optimum levels of chlori- nation on protein solubility of soil films. 117 0.40 ‘ e 0.30 *‘ i | >. i U C O E Q 0.20 In D ‘1 OJO 1 Q o L i l 1 l _w J ' I20 vlf) 3C 50 70 3C Chromium (pg) «m4 *n Fig. 11. - Standard curve showing recovery of chreilum in a system containing chlorinated trisodium phosphate when measured by the dlphenylcarbaZlde method. (Average of two trials performed in duplicate) .Absorbancy 1 0.5 0.4 9 OI on 118 I0 50 100 Chromium (9.9.) Fig. 12. - Effect of trisodium phosphate concentration standard curve of chromium samples. Li 7 77—7 3 9 ~“—7, ‘ l I 250 *— L l i 200 ‘~~ l —~ L 3:50 ~— L e I 3 , E 3 . l e + ‘ C L U l i l00 r- L l g L 50 L L L O o ' - __ ,l, k, l, _Vll_ __»llnw1 0 l00 500 Chlorine (p.p.m.) Fig. 1f. - Mean of Chromium removed from 1120 sq. or. of type 304 stainless steel by 0.3% chlorinated trisodium phosphate in distilled water at 47° C. for 48 hours. 120 0£5*“ 04 *- Absorbancy 0.3 i— i L i 45 55 65 Temperature (°C.) Fig. 14. - Removal of chromium, as influenced by temperature from type 304 stainless steel by chlorinated (503 p.p.m.) 0.3% trisodium phosphate distilled water solutions. 0.5 ' L l l 0.4 L— i l x | 0 a g 0.3 ~— O k i 3 l «O F <1 e 0.2 L— OJ 4 l o mui_im-_m_l.l,l_ _ ill, 0 IO 50 Chromium (p9,) 121 fig. 15. - standard curve snowing recovery of chromium in a system containing a chlorinated prOprietary detergent when measured by the diphenylcarbazide method. two trials performed in duplicate) (Average of