THE. FLUOROMETRIC DETERMINATION OF CITRATE Thesis for the Degree of M. St MICHIGAN STATE COLLEGE Sidney Katz 1946 w“... —.._-7 - “43:133. ‘. This is to certify that the thesis entitled Thefluorometric Determination of Citrate presented by has been accepted towards fulfillment of the requirements for ' Master of Science degree in Chemistry (Analytical) v Major proffir Date December 9, 1946 mm a . , 1‘ I ’ :51- ~_>a .‘W P.— .' A“ V; $§.ow. .‘ ..| :97. A! :3”. (—1 It " ...\." " k a 0‘ .(o I" O- P ‘1 Cl 'l‘ ‘- I a '5 I‘,\ ,‘. .34 l~" wit" I‘ I 1 1. Aw, : . ._ (E39. ”is; ' Q.‘ .,Q'A‘ I4: t 4 ' I ”II-1:33,"; ‘53“ 9? .“ v I I . 51?? 1 A - I}? ‘3’ I: 1-: ’- I-I f1 . n,‘ ‘ ' v ' . p . ., ‘ d. I fit ‘3' "55.. ' " (‘1'! 31*. I '. u‘fii‘lhj“ I . ‘3“, . .‘ _ _. . “fli‘ _' .317” "‘~ .' “01.7 .,' ,- n 1'. -,‘- J! {1' ' r . .. 7. 4 . C .u .'.,' I .| ‘-u‘, - “ v . nit-fla‘ . _,‘.| 'xtt,V’ '4 I ' fl . (45-; . V ‘ f4 ‘2'1’7'§.l ._ " .3.» "\ -_ “2‘ ‘ V I no'. ..‘_ l‘ , ". "4'3 ‘ . ' THE FLUOROJLTRIC DETERMINATION OF CITRATn By Sidney Katz A THESIS Submitted to the School of Graduate Studies of Michigan State College of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Chemistry 1946 The author wishes to acknowledge his deep appreciation and gratitude for the guidance and timely suggestions offered by Dr. Elmer Leininger in this investigation and in the preparation of this manuscript. 187837 FLUOROMETRIC DETERMINATION OF CITRIC ACID HISTORICAL STATEMENT OF PROBLEM EXPERIMENTAL 1. 2. 3. 4. 5. 9. Selection of primary and secondary Filters Selection of fluorescence standards Dehydration of citric acid Reaction of thionyl chloride and citric acid Preparation of aconitamide Formation of citrazinic acid Preparation of measured volume of solution of ammonium citrazinate Variation of fluorescence intensity of ammonium citrazinate with concen- tration, temperature and pH Preparation of the calibration curve METHOD SUMMARY 11 15 16 19 21 24 26 31 HISTORICAL sabanin and.Lasknowsky (1) in 1878 prepared citra- zinic acid as a derivative of citric acid by heating ammoniacal solutions of citric acid in a sealed tube at 110-1200 C for several hours with.subsequent exposure to air. The blue fluorescence of the alkaline solutions was noted as characteristic of citrazinic acid. A de- tailed study of citrazinic acid was made by Behrman and Hofman (2) in 1884. They prepared citrazinic acid as a derivative of citramide by heating with hydrochloric acid. Easterfield and'Sell (e) heated di-ammonium citrate for three hours in an open vessel at 150° C to obtain the same highly fluorescent material. The intense blue fluorescence of ammonium citra- zinate in ultra violet light led F. Feigl (4) into the development of a micro spot test for citric acid based upon the derivation of citrazinic acid from.citric acid. The procedure for the test as outlined by F. Feigl (5) is as follows: "A dr0p of the test solution is evapo- rated to dryness in.a.micro-cruoible and the residue treated with four drOps of thionyl chloride and fumed. About eight drops of concentrated aqueous ammonia are then added and.the mixture boiled over a micro burner until about two drOps of liquid remain in the crucible. On cooling, six drops of concentrated sulfuric acid are added and heating is continued to dense fumes. tents of the crucible are poured into a test tube and rendered,ammoniacal. An intense blue fluorescence ap- pears in ultra violet light if the sample contained citric acid." Feigl presented the series of reactions in the test as follows: CITRIC ACID 03C-OH /C:0H Hg'c 0’32 ‘ I SOClg 0' 0:0 ‘ on 63 AMMONIUM CITRAZINATE 0= 0NH4 H-C 0-H II I NH HO-C\ ,IC-OH<' 2' N ACONITYL CHLORIDE (TRICHLORIDE) 0=C-Cl 0 H2- 0 ’ \\C-H l I NH o-q q=d““5" Cl 01 CITRAZINIC ACID o-q-OH I t H- C 0-3 H ' H SO HO- C 40-0HVLL \ N ACONITAMIDE (TRIAMIDE) 0:3-NH2 / e 3230 C-H I I ozq c-o NH2 NH2 II Czq-NHQ H-c’ *c-H II I HO-C c-oa N32 N H The cone STATEMENT OF PROBLEM At present the pentabromoacetone method of Hart- mann and Hillig (6) for determination of citric acid is prescribed for general use by the Association of Official Agricultural Chemists ('7). The greatest disadvantage of the pentabromoacetone method is that it requires at least one overnight period of standing. A rapid method for determining citric acid would have wide application. The research problem hereby investigated is that of determining the applicability of Feigl's qualita- tive test to a rapid quantitative method for the deter- mination of citric acid. EXPERIMENTAL 1. Selection of primary and secondary filters for the Lumetron fluorescence meter, model 402 EF. The wave lengths of the band of fluorescent light emitted by the fluorescent solution of ammonium citra- zinate were determined by taking a spectrogram. A Raman spectra tube, a Gaertner spectrograph and a mercury vapor lamp were used in the investigation. The fluorescent band was found to extend from the wavelength of 4000 Angstrom units to the wavelength of 5200 Angstrom units with the most intense area in the wavelengths from 4300 to 4500 Angstrom units. The spectrograms were taken without use of a filter with the mercury vapor lamp for a two-hour exposure and with a filter transmitting the 3650 Angstrom units wavelength for an eight-hour exposure. Inasmuch as the spectrograms were identical except for the presence of visible mercury lines in the first spectr- ogram, the 3650 Angstrom units wavelength is the acti- vating wavelength. The secondary filter selected, consisting of a combination of the Corning lantern blue filter number 5543 and the yellow filter furnished by the Photovolt <{ Corporation for use in Vitamin Bl determinations, reaches a peak of transmission at a wavelength of 4400 Angstrom units with.a sharp drOp after the wavelength of 4600 Angstrom units. The yellow filter absorbs light of the wavelength of 3650 Angstrom.units preventing scat- tered primary light from activating the photocells. Weak fluorescence in the range of wavelength of 5200 Angstrom units, emitted by the sulfuric acid reagent used in this work, is absorbed by the secondary filter. The primary filter selected for this investigation possesses maximum.transmission.at the wavelength of 3650 Angstrom.units and is furnished by the Photovolt Corpor- ation for Vitamin Bl determinations. The transmission curves of the primary and secondary filters are drawn on the accompanying graph (graph A, page 6). 2. Selection of fluorescence standards for the Lumetron fluorescence meter, model 402 EF. The fluorescence of solutions of ammonium.citrazinr ate prepared by Feigl's series of reactions becomes less intense on standing at the rate of approximately two percent daily. This apparent slow decomposition pre- cludes the use of this solution as a standard and.makes necessary the use of an outside standard. Consideration of known fluorescent materials (8) and their properties led to specific investigation of sodium salicylate. Preliminary investigation of other possible substances eliminated them on the basis of their being weakly fluorescent in the wavelengths desired, highly sensitive to changes in temperature or pH, subject 100 90., Yellow Vitamin _.= I x 80;L 70" g .I liYitamin 81 a 60 Primary I g - ;_ {IV-311“? ,I earning 5543 s50 . Filter E4 540* W. q ; E 30. . ,. O Corning a 204-..- - 5643 plus N yellow ‘10 Vitamin.Bl 0' , I I 5000 4000 at000 WAVELENGTH - ANGSTROM UNITS Gra nh A Transmission curves for primary and secondary filters. to decomposition on standing or under ultra violet light, or absorbent of the activating light to such a high degree as to cause erratic instrument response. Absorption must be so weak that the fluorescence intensity is practically uniform throughout the cell. as the absorption increases the fluorescence intensity diminishes along the beam of exciting light. when the absorption has increased to the point where a large share of the fluorescent light eminates from a small section of the cell the instrument response becomes highly sensitive. Sodium salicylate does not conform in every respect to the Optimum characteristics for a standard fluorescent material. It is subject to mold growth and the fluores- cence intensity is not uniform throughout the cell. It was found however that a few drops of toluene shaken with the sodium salicylate solution inhibited mold growth. The absorption of activating light by the solu- tion can be decreased sufficiently to cause the error so incurred to be negligible by lowering the concentration of sodium salicylate. This, in effect, places an upper limit on the concentration of sodium salicylate to be used in a fluorescence standard. Investigation of the range of fluorescence intensi- ties of sodium salicylate solutions (see graph B page 9) indicated that the upper limit of concentration of sodium salicylate for use as a fluorescence standard is approximately a two tenths percent solution, 1.6., two grams of sodium.salicy1ate dissolved in enough water to make one liter of solution. The characteristics of such a solution with respect to temperature and pH were inves- tigated. The accompanying graphs (graphs C and D, pages 9 and 10) illustrate that these factors are favorable in that the change of fluorescence intensity with.tanperature is very small and the fluorescence intensity is constant at a pH of five to ten. Blank runs were made to provide solutions for the zero setting of the fluorescence meter. The blank solu- tions gave readings of the order of one half unit when the fluorescence meter was set with distilled water as zero standard and two tenths percent sodium salicylate solution as "100" standard. Distilled water was selected as zero standard. 5. Dehydration of citric acid. Thionyl chloride reacts violently with water giving hydrochloric and sulfurous acids. In order to avoid this reaction, the sample must be made as nearly anhydrous as possible. Citric acid normally has one water molecule attached. This water molecule can be removed by heating at 70-750 C (9) or by standing over concentrated sulfuric acid. In order to dehydrate the citric acid rapidly, high temperature and the use of the vacuum oven were investigated. Aliquot portions of water solutions of citric acid were used in this investigation. These were delivered by O O (D O O O .13. O FLUORESCENCE SCALE UNIT;J as O O 4 6 8 ODIUM SALI YLATE IN SOLUTION. Graph 3 Instrument setting: 2% solution of sodium TE: lb 0‘ w 0 O O O O [\‘J O FLUORESCENCE SCALE URI 0 salicylate equals 100. 6 8 10 ‘ Um SALICYLAIL SOLUTION. Graph C Instrument setting: .2% solution of sodium salicylate equals 100. .au. ..u'.'~| o ”as. - . —f“ .o.‘ mama» -¢”i y I rLuoasserncs SCALE orxrs -L rtuoasscrncs SCALE UNITS 10 100 1:— : t as -=:_ 99 a: 98 97 96 95 l_ 20 221 234 26 28 30 TEMPERATURE CENTIGRADE Graph D Effect of temperature on 0.2% solution of sodium salicylate. Instrument setting: I 0.2% solution of sodium salicylate at 24° C equals 100. 100 95 9O 85 ‘80 75 20" 22 24 26 28 30 ~11.""):‘T‘n H 7:“ "Mrs-r rvw 'I' ‘MIJL“A“;LL7.0L CLAJ inLkIALLA‘ a Graph 13 Effect of temperature on 2% solution of sodium salicylate. Instrument setting: 2% solution of sodium salicylate at 24° c Sequel to 90. 11 a calibrated pipette from solutions prepared from.anhy- drous citric acid. Baker 0. P. citric acid monohydrate was heated in a 70-750 C oven to constant weight to pre- pare the anhydrous citric acid. Temperatures of 100-1100 C for 15 hours caused as much as sixty percent decrease of fluorescent intensity. Decomposition was indicated by browning of the residue. A 65-750 C oven with a pressure of approximately thirty five millimeters of mercury was found to dehydrate the citric acid without decomposition after'one and one half hours if a milliliter or less of solution were used in the aliquot portion. Exposure to these same conditions for periods up to four hours did not change the results. Preliminary investigation indicated the suitability of a twenty-five milliliter flask for this and the subse- Quent reactions. The requirement for a ground glass standard taper mouth is brought out in the next section. Standard conditions selected for this step are: one milliliter or less of solution of the sample is placed in a twenty-five milliliter flask which has a ground glass standard taper mouth. The sample is dried and dehydrated in a 65-700 vacuum oven, with the pressure approximately thirty five millimeters of mercury, for two hours. 4. Reaction of thionyl chloride and citric acid. The direct action of thionyl chloride on citric acid produces varying amounts of a tarry material in 12 addition to the product desired. Benzene (10) and pyridine (10,11) have been used to increase the yield of reactions of organic acids with thionyl chloride to produce the chlorides, and anhydrous sodium carbonate (12) has been used to decrease formation of tarry residues and increase yields. Investigation of these reagents indicated that anhydrous pyridine increased the formation of tarry deposits which interfere with.suc- ceeding reactions. Anhydrous benzene increased the fluor- escence intensity about two fold whereas anhydrous sodiwm carbonate increased the fluorescence intensity about ten fold and prevented formation of tarry residues. Benzene in addition to sodium.carbonate gives no better results than sodium carbonate alone. Anhydrous calcium carbonate and.sodium bicarbonate were investigated as substitutes for sodium.carbonate but the results obtained indicated that they were not superior although giving good fluorescence intensities. The volume of thionyl chloride used was varied from one half to seven.milliliters and the maximum fluorescence was obtained in all volumes above one and one half milli- liters. Redistilled Eastman practical thionyl chloride gave reproducible fluorescence readings. However the best grade Eastman thionyl chloride gave better reproducibility with the fluorescence intensity approximately ten percent greater. The amount of sodium carbonate used was varied and it was found that less than ten milligrams produced erractic and low results whereas ten to fifty milligrams gave maxi- mum fluorescence intensities. 15 Refluxing increased the fluorescence intensity'but exposure to atmospheric water vapor or water during or after refluxing lowered the intensity. Refluxing over a loo-110° 0 oil bath.equipped with a mechanical stirrer for twenty minutes gave maximum.f1uorescence intensity. Refluxing for longer periods gave no increase in the in- tensity of the fluorescence. In order to reflux the thionyl chloride solution and prevent access of‘water vapor the twenty five milliliter flask and a reflux condenser constructed of fifteen.millimeter pyrex glass tubing were fitted with standard taper ground glass Joints. A constriction was made fifteen centimeters from the ground glass Joint on the condenser. A drying tube was prepared above the constriction for another fifteen centimeters, plugged with glass wool and filled with anhydrous magnesium.per- chlorate (Dehydrite). (For drawing, see figure 1, page 30). The reflux condensers so prepared were used for over fifty determinations without replacement of the magnesium.perchlorate being necessitated. The Joints of all the twenty five milliliter flasks and the Joints of the reflux condensers were ground with fine grinding compound in rotation in order to insure interchangeability and nonvleaking Joints. The excess thionyl chloride is removed by evacuation through a system consisting of a water pump connected through a safety bottle to as many outlets as there are 14 samples being run simultaneously. The reflux condenser is connected to each outlet through pressure hose and a three-way stopcock. The three-way stopcock is arranged so as to allow evacuation for a period of time and a flushing of air back into the condenser flask system. To prevent a rapid reentrance of air and subsequent blowing of particles from the flask a short piece of glass tubing with.a capillary and is connected to the stopcock through which the air enters. (For drawing see figure 1, page 30). When the vacuum is first applied the flask is rotated gently so as to prevent splattering. As the flask cools the palm of the hand is applied to the bottom of the flask to warm.it and the rotation continued until the thionyl chloride is driven off. The evacuation is continued for four minutes after which air is flushed in by turning the three-way stepcock. The system is reevacuated for one minute and reflushed. The evacuations and flushings are continued for a total of one four-minute and three one- minute evacuations. . Standard conditions selected are as follows: Approximately fifteen.milligrams of anhydrous sodium.car- bonate and two milliliters of thionyl chloride are added to the anhydrous sample. The solution.is refluxed over a loo-110° 0 oil bath for twenty minutes using a drying tube. The excess thionyl chloride is evacuated and the system.flushed for a total of one four-minute and three one-minute evacuations. 15 5. Preparation of aconitamide. The use of concentrated ammonium hydroxide with reduction of volume by boiling presents difficulties in application to a quantitative method in that the succeed- ing reaction requires an exact volume of residual solution remaining. Evaporating rapidly to dryness gives lowered fluorescence intensity. Investigation of the possibility of transforming the chloride to the amide through the use of gaseous ammonia gave fluorescence intensities comparable to those using concentrated amonium hydroxide and circumvented the requirement of a definite volume of residual solution. Results at room temperature do not differ from those obtained at 100° c. The ammonia chamber (see figure 1, page 30) built of cardboard or other suitable material, is L-shaped with the long part horizontal. The dimensions are: horizontal section: five centimeters wide, eight centimeters high and thirty centimeters long; vertical section: five centimeters in both horizontal directions and fifteen centimeters high. Ammonia enters from a cylinder connected to the long hori- zontal chamber and flows through the long chamber, up through the vertical chamber and out through a gate. The thirty- centimeter horizontal chamber holds four twenty five milliliter flasks. A block of wood (see figure 1, page 31) approximately four by four by twenty centimeters, through which a hole 16 has been drilled down the long axis to fit over the condenser, is used to detach the twenty-five milliliter flasks from the condensers after the flasks are at the bottom of the short section of the ammonia chamber. The flasks can then be moved into the long section by tilting and gently tapping. Investigation of the length of time necessary to obtain maximum fluorescence intensity with gaseous ammonia revealed that in some cases thirty seconds is enough and in no case does it exceed two minutes. The excess ammonia can easily be removed by placing the vessel under the hood for two minutes. Gaseous ammonia as compared to concentrated ammonium hydroxide for this reaction while giving comparable re- sults is superior in that it requires fewer and simpler Operations, the end product is not in solution, and the excess is easy to remove. The final method chosen for this step is as follows: the flask with condenser attached is introduced into a chamber through which gently flows gaseous ammonia. The condenser is detached from the flask in the presence of gaseous ammonia and removed from the chamber. The flask is then removed after ten minutes and allowed to stand under the hood for two minutes. 6. Formation of citrazinic acid. Preliminary investigations indicated that four variable factors are critical to this reaction. These 1? factors are the concentration of sulfuric acid, the volume of sulfuric acid solution, the temperature of the oil bath used to control the temperature of the solution during the reaction and the length of time that the reaction flask is kept in the oil bath. De- tailed inquiry into each.variable indicated that these conditions can be controlled. Graphs for each variable were compiled by varying one of the factors at a time and keeping the other three constant at the value finally selected for standard conditions. There is a gentle increase in fluorescence inten- sity as the concentration of sulfuric acid rises to 75%, a plateau from.75-79% and a rapid drOp after 79%. (see graph F, page 18). The term concentration of sulfuric acid as used herein refers to percentage sulfuric acid by weight in an aqueous solution. Concentration of sulfuric acid was computed from density using the Inter- national Critical Tables. The intensity of fluorescence rose rapidly to a maximum.at a volume of one milliliter’of sulfuric acid solution and remained at this maximum.for larger volumes. (see graph G, page 18) The fluorescence intensity increased rapidly as the oil bath temperature rose to 155° C, increased slowly as the bath temperature rose from.155-165° C, decreased slowly as the bath.temperature rose from 165- amousc‘mcn sour UNITS rmonnscmcn scm mums 18 7O 71 7B 7B 74 75 76 77 78 79 80 81 80 78 76 74 72 _7O 58 -ECLTTA“; ”VZFUTIU .813 I] ACQULOUS SOLUTION. Graph F ample size and other variables held constant. O .o 0.6 1.2: 1,8 2.4 vetunn ow entwuxlc ACID 3 LUII 3 IR nILLILIEnzn. Graph G Sample size and other variables held constant. 7"» .. '0 -< '..“’.‘*q ‘ Mr. 19 200° C and- decreased rapidly with the bath temperature , -over 200° C. (see graph H, page 20) The intensity of fluorescence increased rapidly to a maximum as the time in the oil bath reached four minutes, and maintained the maximum until after eight minutes. (see Graph I, page 20) Standard conditions selected for this step are: two milliliters of 76-77% sulfuric acid solution are introduced into the flask and the contents heated for six minutes in a mechanically stirred oil bath whose temperature is 165° 0 plus or minus 5°. 7. Preparation of a measured volume of solution of ammonium citrazinate. Dilution of the hot residual sulfuric acid solution with water and neutralization with dilute ammonium hydrox- ide accomplished the same results as those obtained by cooling and adding concentrated ammonium hydroxide.‘ One hundred milliliter glass steppered volumetric flasks were found well suited for the preparation of the final fixed volume of fluorescent solution of am- monium citrazinate. The cell of the Lumetron fluorescence meter used in this investigation requires twenty-five ' milliliter portions for each filling. Therefore, several additional readings can be made if necessary. Final conditions selected for this step are: the sulfuric acid solution is carefully diluted with about five milliliters of cold water. The diluted solution is FLUORESCENCE SCALE UNITS FLUORESCEHCE SCALE UNITS -74} 150 180 170 180 190 £00 ELITEXATVRh OF OIL BATH,SUL?UHIC ACID Kin T173. Graph H Sample size and otner variables held constant. 80 78 t 76 72 7O as h i o 2 4 e s 10 73"~V\-~r F x" (\T‘ 'r‘T‘ - ~ T-T r\' -p T .. '11? T.T .FT‘TI‘IF‘. | u...._‘)-l kl} ---a4.4 .bai bfi—J o‘..;; 4‘! “JpLIU.—.L4U. (}rs:p11 I Sample size and other variables held constant. ill.- 21 transferred to a glass stoppered one-hundred milliliter volumetric flask. The reaction flask is washed several times with small volumes of water and the wash is added to the solution in the one-hundred milliliter flask. Dilute ammonium.hydroxide is introduced until litmus paper indicator changes to blue. The solution is brought to volume with water. 8. Variation of fluorescence intensity of ammonium citrazinate with concentration, temperature and pH. The accompanying graphs (see graphs J and K, page 22) indicate the amount of citric acid that may be de- termined under the standard conditions selected. The fluorescence meter was reset for the group of readings plotted on each graph so that 100 on the meter corres- ponded to the largest sample of each group plotted. As the sample size reaches two milligrams the sensitivity decreases, 1.6., for each increase in sample size there is a smaller and smaller increment in instrument reading. The effect of temperature of the solution in the cell was investigated over the range of concentration, 0-80 micrograms of anhydrous citric acid, and illustrated on the accompanying graph (see graph L, page 25). The effect of increased temperature is to lower the fluores- cence intensity in a linear manner. Raising the pH beyond neutralization with litmus does not change the degree of fluorescence. 100 80‘, 60 4o 20 FLUORESCENCE SCALE UNITS 0 200 400 600 800 H CiOGRHHS SAXPLL LHZYLROUS CITEIC ACID. Graph J Instrument setting; '800 micrograms citric acid equals 100. 100 80 60 4O 20 FLUORESCENCE SCALE UHITS O 0 2 4 6 8 IILLIGIAJS SAXPLL AHHYDSOUS CITRIG ACID. Graph K Instrument setting; 8 milligrams citric acid equals 100. 22 I o n .. ' u v . 1 ‘a. o x 1 u s " - ' I ‘ - . n as... - . a .- -, . " - I o ‘ . A _ ‘r . a. » - . a4 . a n I I e . k \ . a o . ‘ \ . o ' - ‘ .---....0.-.-.uo. rwouscmrcs SCALE owns 160 90 so 70 so to 4,0 30 so . 10 23W * 70 microgram sample of citric acid (anhydrous) 50 microgram sample of itric acid (anhydrous) * 50 microgram sample of t_______ citric acid (anhydrous) . * i * ‘—fir- 20 22 24 26 28 30 TEMPER.’.TURE - CEIITIGRADE Graph L Temperature effect on fluorescence of ammonium citrazinate solution. Instrument setting: 0.2% sodium salicylate equals 100. 24 The range of concentration for use with the two- tenths percent sodium salicylate solution standard is determined as 0-80 micrograms of anhydrous citric acid. The temperature selected as standard for solutions in the fluorescence meter cells is 24° C. 9. Preparation of the calibration curve. A calibration curve is plotted on the accompanying graph (graph M, page 25) for anhydrous citric acid sam- ples of’0-80 micrograms. Several samples were run at each sample size and the average taken for each plot. Following is a compilation of samples taken and devia- tions from.the average: Anhydrous cit- ric acid sample in micrograms: 10 20 50 40 50 60 70 Number of same ples taken: 3 6 3 6 2 29 2 Average Reading: 9.9 22.6 55.9 47.8 61.9 77.0 91.6 Average Deviation: Meter units: 0.4 0.9 0.6 1.4 0.1 1.4 2.0 Percentage: 4.0% 4.1% 1.7% 3.0% 0.2% 1.8% 2.2% Greatest Deviation: Meter units: 0.9 1.4 0.9 2.7 0.1 2.9 2.0 Percentage: 9.1% 6.2% 2.8% 5.6% 0.2% 8.8% 2.2% rmoasscrucs 8cm: UNITS 100 90 80 7O '6. 5O 4O 30 20 10 O O 20 40 60 80 AHHYDROUS CITRIC ACID IJ MICEOGRAAS Gmmhm Instrument setting: 0.278 sodium salicylate solution equals 101. 26 METHOD Equipment peculiar to this method is sketched on page 30 and described in detail in the section on ex- perimental. The following reagents are used: Anhydrous citric acid. Baker 0. P. Citric acid monohy- drate is heated to constant weight in a 70-75° C oven. Eastman best grade thionyl chloride. Anhydrous sodium carbonatg. Anhydrous sodium carbonate is heated twenty four hours at 110° 8. 133111319313. A cylinder of ammonia is the source. 76-77% sulfuric acid. Seventy-three milliliters of concentrated sulfuric acid are added to thirty-five milliliters of water with cooling. The percentage sulfuric acid by weight is determined by density measurement and reference to density tables, page 59, Volume III, International Critical Tables. AdJustments are made until the desired solution is obtained. 0.2% sodium salicylate. Two grams sodium salicylate is diluted to one liter. Several drOps of toluene are added and the solution shaken. An aliquot portion of solution containing 10-80 micrograms of anhydrous citric acid, preferably one . milliliter or less, is placed in a twenty-five milli- liter Erlenmeyer flask equipped with a standard taper ground-glass mouth. The flask is placed in a 65-700 C 27 vacuum.oven.with a pressure of approximately thirty- five millimeters of'mercury for two hours. The sample thus dehydrated may be stored if necessary in a desic- cator over anhydrous magnesium perchlorate. Approximately fifteen milligrams of anhydrous sodium carbonate and approximately two milliliters of thionyl chloride are added to the sample in that order. A reflux condenser with a standard taper ground-glass Joint and a built-in drying tube is connected to the flask and the flask is immersed in a 100-1100 C mechanically-stirred oil bath so that the level of liquid inside the flask is even with.the oil in the bath. After twenty minutes the flask with the reflux condenser attached is removed from the bath and evacu- ated through the top of the reflux condenser. In order to avoid splattering in the initial evacuation the flask is rotated gently. As the flask cools,the palm of the hand is applied to the bottom.of'the flask and the rotation continued until the thionyl chloride is removed. The evacuation is continued for a total of four minutes after which air is flushed in by turning the three-way stopcock. The system is evacuated for one minute followed by another flushing with air. The evacuations are continued for a total of one four-minute and three one-minute evacuations. 28 The flask with reflux condenser attached is intro- duced into a chamber through which gently flows gaseous ammonia. The top half of the reflux condenser will proJect from.the ammonia chamber. Using the special wooden block, the reflux condenser is disengaged from the flask while the flask is in.the ammonia.atmosphere, leaving the flask within.the chamber. After ten minutes the flask is removed from.the ammonia chamber and placed under a hood for two minutes to allow escape of excess ammonia. Two milliliters of 76-77% sulfuric acid solution are added and the flask tipped and rotated so as to dissolve all the sodium carbonate. The flask is then.immersed in a 165° C plus or minus 5° mechanically stirred oil bath to a slightly greater depth than that of the liquid inside the flask. At the end of six minutes the flask is removed from the bath.and the liquid inside carefully diluted with five milliliters of water. The liquid is then transferred and washed with.about twenty five milliliters of water into a one hundred.milliliter glass steppered volumetric flask. The solution is made alkaline to litmus with dilute ammonia and brought to volume with water. The solution is thoroughly mixed and.brought to 24° C. The fluores- cence reading is taken.and the weight of anhydrous citric acid is determined from.the calibration curve. 29 The calibration curve is prepared by running known portions of anhydrous citric acid in the range 0-80 mi- crograms through the entire procedure and preparing a graph; fluorescence scale readings as ordinate and citric acid portions as abscissa, such as that on page 25. . The Lumetron fluorescence meter, model 402 EF, equipped with.the Vitamin B1 filter transmitting a mmximum.wave length of 5650 Angstrom units as a primary filter and with the Corning lantern blue filter number 5543 and yellow Vitamin B1 filter as secondary filters is set with the following as standard solutions for the preceding readings: two tenths percent solution of sodium salicylate as the "100" standard, distilled water as the zero standard; all solutions at 24° C. 30 ‘_ S Three way stepcock leading to water pump and safety bottle. aha-’5‘? '1 ' mun. ‘ “'25-. a Reflzx condenser with built in drying tube. my: ‘L Wm Twentg7 Sive milliliter flask with ground glass mouth. a Q Wooden block for (1017201.:1’18 reflux eondcnnunf from flsufli. juinonitl Chzrfller. 4. Equipment for preoaraeion of fluorescent solution. (7 .igure 1) 51 SUMMARY A fluorometric method for the determination of citric acid is presented which is accurate to less than forty parts per thousandfor quantities ranging in size from.fifty to eighty micrograms of anhydrous citric acid. In the range of ten to fifty micrograms the error is somewhat greater. 2. 5. 4. 7. 8. 9. 10. 11. 12. 32 LITERATURE CITED sabanin, A., and Lasknowsky, N., Zeit. Anal. Chem., ‘11, 74, (1878) Behrman, A., and Hofman, A., Ber., 17, 2687 (1884) Easterfield. T., and Sell, W., J. Chem. 800., pg 28, (1894) Feigl, F., Anger, V., and Frehden, 0., Microchemie, ..11, 55 (1955) Feigl, F., "Laboratory Manual of S 0t Tests", p.196, New York, Academic Press. (1945 Hartmann, B., and Hillig, F., J. Assoc. Official Agr. Chem., ls,lgg (1950) Assoc. Official Agr. Chem., "Official and Tentative Methods of Analysis", 6th ed., p. 595 (1945) Dement, J., "Fluorochemistry", p. 146, New York, Chemical Publishing 00., (1945) Witter, H., Ber., gs, 1160 (1892) Hill! G“: and Kelley, L., "Organic Chemistry": p.126, Philadelphia, The Blakiston Co. (1943) Brooks, L., and Snyder, H., Org. Syntheses, 25 84, (1945) Coleman, G., Nichols, G., McCloskey, G., and Anspon, H., Org. Syntheses, 25, 88 (1945) 7i ,(l 1") y ) em 19 '53‘ 001 2 3 1993 7, gu.'.....-..'.JI".LL 1.3.2.1. L‘. ‘ J'T545 - 187887 K19 Katz -_.‘_l_f_..._,. 7 -_ a a ,, -. , , llllllillll HIll!liIHWIIMIIIHIII HIHIIII Illll‘Hll 31293 02446 7585