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RJ Reynolds

Explanation of Time Estimation Grid.

Date: 1988
Length: 132 pages
510690089-510690220
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R&D
01-04-01-11
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Minnesota
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19961216
Referenced Document
Official Methods of Analysis, by Association of Agricultural Chemists, 600000. A Modified Single Solution Method for the Determination of Phosphate in Natural Waters, by Murphy J, Riley J, Anal. Chem. Acta., 620000. Method for Quantitative Determination o
Date Loaded
07 Jan 1999
Named Person
Gf Smith Chemical
Mohr
Eh Sargent & Co
Seaman, W.
Allen, E.
Cundiff, R.H.
American Optical
K&K Laboratories
Natl Bureau, O.F. Standards Grade
Eastman
Matheson Coleman Bell
Harrell, T.G.
Somogyi, M.
Johns Manville
Nelson, R.A.
Sensabaugh, A.J.
Rush, K.L.
Scientific Glass Apparatus
Mi Capsule
Hewlett Packard
Applied Science Lab
Labline
Will Scientific
Carl Schleicher & Schuell
Mallinckrodt
Sigma Chemical
Eastman Organic Chemcials
Fisher Scientific
Hege, R.B.
Precision Scientific
Mitchell, J.
Smith, D.M.
Interscience Publishers
Laurene, A.H.
Sullivan, B.N. Jr
Hamilton
Erlenmeyer
Sloan, C.H.
Sublett, B.J.
Corning Fiberglas
Markunas, P.C.
Oc Rudolfph & Sons
Westphal
Reimann
Brookfield
Bausch & Lomb
Lewis Benedict
Ace Glass
Author
Rjr
Box
Rjrc4023
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Marginalia
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psx53d00

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EXF'L.AINIATIUw OF TI14E LS"! IMATIUN GRID A time estimation grid of the type shown below has been placed at the bottom of the last page of each method. This article offers a bri ef descri pti on of ;Lhe gri d and an expl anati on of i ts use. TIiiE ESTIMATIU;d GRID S"~ 1~?13 ~~ !i 6 7 8~9 ~~ ~ O~U' ~~U 5t~ u0 10 8Q ~J I UU 200 300 ~ RT 1 134 (5; 6 I~3 9 I i) 20 3ii ~tJ 5U 6U ~U) LC~ ~?~ I UU ~10~)i 3UU~^ _ _ ~__ _. __ . . ___, . _. L _ I1H ._! 2 3~+ 5~6 7 ~i 9 10 2U 3U 40 50 6U /U 80 90 100 200 300 - .___-_ This grid was included to provide aa rapid reference concerning the amount of time requi red to perform the analysi s, and the man-hours required for a set of samples. It enables anyone interested to obtain this information easily without being thoroughly familiar with the procedure in question. This will assist in estimating how 1 ong i t wi l l take to obtain resul ts `after the sampl es have been received and about how much it should cost the customer. The gri d contai ns three separate data 1 i r•es . They are ex; l a i ned as fol l olws: Line S •- The sum of the numbers circled shows the number of samples that can be run most economically in a single set. Usually, this means that this number of samples can be run in the same time as onee can be due to grouping. Line RT .'The sum of the numbers circled shows the real elapsed timc in tenths of an hour required to perform the analyses on the sample set from the time the samples are received by the analyst until the results are available. Li ne MH - The sum of the numbers ci rcl ed shows the amount of man-hours i n tenths of an hour requi red to do the work involved in the procedure that requires the presencc.~ of `the analyst. It does not i nci ude such items as oven dryinca time, conditioning time, soxhlet extraction tir+,:,`and the like. The use of the grid can best be described by the use of an exampl e. Usi ng the exampl e gri d, the fol l owi ng i nformat.i on can be obtained: ~
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-2- l. The samples will, be run in sets of 12 (10 + 2) because it takes about the same time to run a group of 12 as it does to run one alone. 2. It requires 27.5 hours [(200 + 70 + 5) X 0.1] total elapsed time to obtain the results on a set of 12 samples. 3. The analyst is actively engaged in performing various steps of the procedure for 3.5 hours [(30 + 5) X 0.11 out of the total elapsed time of 27.5 hours for a set of 12 samples.
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NATIONAL INSESTABLE-2AB INDEXES EIQABLE YOU TO MASE YOUR OWN SVSlECT ARBANGERIM1?, USING PLAIN IIVSERTS ON WHICH TO WRITE YOUR OWN CAPTIONB. The Beaded edye on tab makes it eaay to insert. captions MadelnU.S.A. T600 690TS t, I SQOH.tSK NOIZWIQ2i6'QTIVZS
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t 1. STANDARDIZATION METHODS (ST) t ST NO. PREPARATION AND STANDARDIZATION OF SODIUM HYDI:OXID.~.~.', SOLU'TIONS 2 PI:1?PARA1'7 ON AND S:CANI)ARhI7.ATION OF IIYDROC:xLORIC ACID SoLUTJ:ONS 3 PREPARATION AND S:CANDARDIZATIOIN, 01' SULti'URIC ACID SOLUIION'S •4 PREPARATION AND STAN D.',RD I ZA'_ I O\' OF TENTH NOI'u'%1AL SODIUM THIOSULFATE 5 PREI'ARATION AND S?'ANDAF.DIZATION OI' TENTH NO10IaL IODINE SOLUTION PRI)PAiMI0.N AND STA N DA RDI Z AT ]:,O N OF 'SENTH r,OPUAhh POTASSIUM PERAIANGAX'1TE 7 1'REI'AI'.ATIO'v AND STANDARDIZATION OF SILVE,R NITRATE SOLUTIONS 8 PREPARATION AND STANDARDIZATION OF PI;RCIIL,ORIC ACID IN GLACIAL ACETIC ACI.I) SOLUTIONS PREPARATION AND STANDARDIZATION SOLUTIONS OF TE f 1;A15U 1'YLA' uf0'~TL'~I IIXDROXIDT: : , i
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R. J. REYNOLDS 7.'013ACC0 COMPANY 2,E SI:A1ZCll 1)UAI:TMi;NT Analytical Method No. ST-1 PREPAI:ATION ANl) S7'A*;DARnI7.ATI0:1 OF SODIUM HYDROXIDE SOLUTIONS PRINCIPLE . Potassium acid phtfialate is dissolved in water and titrated to the phenolphthalei.n end point with sodi.um hydroxide. APl'Al'ATUS AND RF.AGENTS BURF.TTEy Normax or equivalent grade. When using automatic burettes, protect from air with ascarite or soda°lf.me tubes. STORAGE BOTTLE, preferably fabricated of alkali-resistant glass. WATER, carbonate-free. (1) Boil distilled water for 20 minutes and cool with soda-l.ime orr ascarite protection, or (2) bubble air, freed from carbon dioxide by passing`thr.ough a soda-lime tower, through water for 12 hours. SODIUM HYDROXIDE SOLUTION (1: 1). Add one part water to one part sodium hydroxide, Analytical Reagent grade, swirl until solution is complete and close with a rubber stopper. Allow to stand until the sodium carbonate has settled, leaving a perfectly clear liquid (about 10 days). This solution should be approxi_mately 1.8.5 N. Prepare the sodium hydroxide titrants by diluting the 18.5 N solution with water to the desired strength according to the following table: . TABLE I i DILUTION OF 18.5 N SODIUM HYDROXIDE TO PREPARE TITRANTS Approximate Norma Volume of 18.5 N lity NaOH to Prepare Desi.red 1 Liter 17 Liters 0.01 0.54 ml. 9. 2 ml. 0.05 2.7-- "- 46. 0.10 5.4 92. 0.50 27 460. Ln ~ 1.00 54 920. m m kD m m
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POTASSIUM ACID EHTIiALATE, Primary standard grade. Dry for two hours at 120° C., cool, and store i.n a desiccator. PHENOLPHTHALEIN INDICATOR SOLUTION. Dissolve 1.0 g. of the indicator •i.n 100 ml. of S.I). 3A ethanol. PROCEDURE Accurately weigh the amount of potassium acid phthalate indicated in . Table 71 into a 250 ml. Erl.enmeyer flask that has been swept free of. carbon ~ dioxide with nitrogen. Add 50 ml. of carbon dioxide-free water, stopper ~ flask and swirl gently until sample is dissolved. Add three drops of i indicatorr solution and titrate with the solution being standardized until ~ production of a slight pink color, which persists for 15 seconds. # TAL'LT: II ' AMOUNT OF_ P_0 TASSIU_M FCIp_ PHTiIALATE_ TO WEIGH _ ~ FOR STAN])AP.])I.'1.ING VAR10US STREW-TH SOLUTlO:IS I Appr.oxinate Nor.mality Amount of Potassium Acid Pht:hal.ate to be Wei. hed 0.01 0.07 - 0.08 g. 0.05 0.35 - 0.40 0.10 0.7 - 0.8 0.50 3.5 - 4.0 1.00 7.0 - 8.0 CALCULATIONS Normality _ Sample wt. x 4.89668 Volume of Base QW m m ~P
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R. T. REYNOLDS TORACCO COMPANY R}',SI.ARCli Dk:PARTMENT Analytical Method No. ST-2 PREPAPW7'1'.0N ANI) STAT:DA,I:DI`I,ATT.ON OF HY))ROCHLOR7,C ACID SOLUTIONS PI:'1NC 7 PLE '1'ri.s(hydiotiymethyl)aminomethane is dissolved in water and titrated to a visual. end point with hydrochloric acid. REAGF:NTS 1IYDROCII).,0i;7:C ACIl) SOLUTION, reagent grade, approximately 12 normal, dilu,te-d to desired strength with water according to the following table: ' TABLt: Z DILUT70N OF 12 N HYDROCHLORIC ACID TO PREPARE TITRANTS Approximate Volume of 12 N HCJ- Volume of 12 N HC1 Nor-ma J-i.t Desired' to Prepare 17 Liters to-Pr-epare 1 Liter 0.01 14 ml. 0.84 }. 0.05 71 4.2 0.10 1.42 8.4 0.50 ' 710 42. 1.00 7.420 84. 9.'RIS(HYDROXYMI,TilYL)AMI:NOMETIIANE, (CI3 OH) CNH?), (T13AM), G. F. Smith Chemical Company.. Dry at 105° C. ~or ~wo hours in a forced draft oven and store in a desiccator. INDICATOR SOLUTION. Triturate 0.1 g. bromcresol green in a mortar with 1.5 ml. 0.1 N NaOH, and dilute to 1.00 ml.-with distilled water. Dissolve 0.1. g. sodium alizarin sulfonate (alizarin Red S) in 100 ml. of water. Mix the two solutions.
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~. PROCE01)1:3: l:ccurar_cly weigh the amount of tri.s(hydr.oxymet.hyl)arninomethane indicated in Table. II into a 125 ml. Erlencneyer flask. Add 30 ml. of distilled water and three drops of the indicator solUt.ion standarcla.-r.ed. The solution changes color to green t.o yellow. The end point is the p1l at tho end point: is 4.70. TABLE 7I and titrate with the solut:ion being progressively from violet to blue change from Sreen to yellow. * The. ATfOL'.NN-7 OF TlWi TO 'W_EIC-H FOR ST_ANDART)I7.ING VARIOUS S`J'f, T„~Gi ii~ SOLiiTl:O\S~__--- -- ---- CALCULATIONS Normality = Sam~~le_Wt. x 8.25G9 Volume of Acid~ Amount of L1iAT1 to be Wei_ghed m ko ~ m
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of S. D. 3A ethanol. SULFURIC ACID SOLUTIONS. Prepare the sulfuric acid solutions by diluting concentrated sulfuric acid to the desired strength with water according to the following table: DILUTION OF 36 N SULFURT.C AC]I) TO PREPARE TITI:ANTS R: J. REYNOLDS TOBACCO COMPANY . -. *-RESEARCH DEPARTMENT Analytical Method No. ST•-3 PREPARA'IIO.~ AIID SfiANDARD7LATI0N OF StTI,I'UR7C ACID SOLUTI'ONS PRXNCTPLE -Sulfuric acid solution is titrated to the phenolphthalei.n end point with standard sodium hydroxide solution. 0.05 t SODT.UM HYDROh]:DE SOLUTIONS. Prepared and standardized as described in Analytical Method No. ST-1. 1'HENOI}.'HTHAI:L'1N I.NDICATOR. Dissolve 1.0 g. of the indicator in 100 ml. . 0.1 0.5 1.0 Volume of Concentrated Sulfuric Acid to Prepare 1 Liter 17 Li.ter. s 0.28 4.8 0.57 9.7 1.42 24.1 2.84 48.3 14.2 241.4 28 4 482.8 u, ~ . m rn ~ m m
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PROCI:DURh Pipet 25.0 ml. of the sulfuric acid solution into a 250 ml. Erlenmeyer •£lask. Add three drops of phenolphthalein indicator and titrate with the ,appr.opriate standard sodium hydroxide solution until production of a slight pink color which persists for 1.5 seconds. CALCUh.A`tTON Normality NaOH x Volume NaOH 1 SO l I horma Ity It, 1. - 25
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PREPAI:AT,IC)N ANI) STA'~llART)IZATIOti. 0I' ONE-Tl?N1'ti NORt•:AL SODIUM T1iTOSULFATE I PRINCI-.PLE Iodine. is liberated from potzr;si.um end point with sodium tlliosul.fat-e. APPARATUS BUfiETT):; Normax or equivalent: iodat:e , and titrated to the -starch REAGENTS POTASSIUM IODATE, Primary Standard Grade. Dry torr two hours at 120-125° C., and store in a desiccator. .`~ , POTA ;SIUM IOD7])E, ACS grade. I-il'DItOCI1L0RIC ACID, 6N. Add 500 ml. concentrated hydrochloric acid to " water and dilute to one liter with water. _ STARCH INI)ICATOR. Grind 5 g. soluble starch with 20 mg. mercuric iodide to a thin smooth past:e with about 25 ml. water. Pour this suspension into 500 ml. of boiling water, while stirring, and boil. for 5 minutes. Allow to stand overnight and decant the clear supernatant iiquid. SODIUM Tf1I0SULFATE SOLUTION, 0.1 N. Dissolve 25 g. of sodium thio,sulfate (Na S 20•5 H?0) i.n•one liter of water. Boil gently five minutes and transler whiIe hot to an amber storage bottle. Standardize weekly. Never return unused solution to the storage bottle. PROCEDURE . Accurately weigh 0.1-0.12 g. potassium iodate into a 250 ml. Erl.enmeyer flask. Add 35 ml. of water and 6 g. of potassium iodide. When both solids have dissolved, add 3 ml. of 6 N hydrochloric acid. Titrate slowly with the sodium thiosulfate solution until the yel.low color nearly disappears. Add 2 ml. of starch indicator and titrate until the blue color just disappears. CALCULATIOy Normality Sodium Thiosulfate _ Wt:. R103 x 28. 0348 Vol. of. Thiosulfate
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.. R. J. REYNOLDS TOBACCO C014I'ANY ItESEARCH DEPARTMENT PREPARATION AND STANDARI)IZATION OF TENTH NOP.IIA7:. IOI)INR SOLUT.ION t PRINCIPLE Iodi.ne solution is titrated *to the starch end point with standard sodium thiosulfate solution. APPARATUS $URETT.h', Normax or equivalent. REAGENTS IODINE SOLUTION, 0.1 N. Dissolve 13 g. of iodine and 20 g. of potassium iodide in 50 ml. of water. Wtien the iodine is completely dissolved, dilute to 1. liter with water. Store in amber colored glass-stoppered bottle. POTASSIUM IODIDE, ACS grade. SODIUM T1110SULFATE SOLUTION, 0.1 N. Prepared and standardized against potassium iodate as described in Analytical Method No. ST-4. STARCH INDICATOR. Grind 5 g. -soluble starch with 20 mg. mercuric i.odidee to a thin, smooth paste with 25 ml. water. Pour this suspension into 500 ml. of boiling water, while stirring, and boil for 5 minutes. Allow to stand overnight and decant the clear supernatant liquid. HYDROCHLORIC ACID, 6 N. Add 500 ml. of concentrated hydrochloric acid to water and dilute to 1 liter with water. ~ . PROCEDURE Pipet 25.0 ml. of the iodine solution into a 250 ml. Erlenmeyer flask. Add 1 drop of 6 N hydrochloric acid then titrate with standard 0.1 N'sodium thiosulf.ate until the iodine color almost idsappears. Add'2 ml. of starch indicator and continue the titration until the blue color just disappears. CALCULATION Normality Iodine Solution ~ N~ormal. 7hiosul.fatee x Vol.. 7"ni_osulfate ~ ~ ~_ 2 5
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R. J. Rf:Y.NOLDS TOILACCO COMPANY RESEARCH DEPARTMENT Analytical Method No. ST-6 AND STA_NDAKDIZATI_0_N OF TFN1'H NORMAL PRk:PARUjION _ P07'ASSIUt.i PI.RMANGANATE PRINCIPLE The reaction of potassi.um'permanganate and sodium ox.alate in acid solution is quantitative and can be represented by the following equation: 5 C2o4 + 2 Mno4 -r 16 x+ ---> 10 CO2 + 2 rtn'H' + 8 1120 .APPARATUS •' BURETTE, Normax or equivalent grade. SOU]:Uri OXALATE primary standard grade. Dry for one hour at ].05-110° C. . and store in a desiccator. - SULFURIC ACIZ, 18 N. Add concentrated sulfuric acid, ACS grade, to an equal volume of distilled water. POTASSlUri PERMANGANATE SOLUTION, 0.1 N. Dissolve 3.3 g. of potassium perrnanganate, ACS grade, in 300 ml. of hot water, dilute to one liter and es ~ntly boil for 10 minutes. Let the solution cool to room temper- ature and pour into a glass-stoppered bott:le. Allow the solution to stand in the dark for at least one week. Carefully siphon the solution into an amber-colored bottle, discarding the first 25 ml. and allowing the last inch of liquid to remain in the bottle. Do no t allow perman- ganate solutions to touch rubber, filter paper or other organic matter. REAGENTS PROCI:DUi:E Accurately weigh 0.25-0.30 g. of sodium oxalate into a 500 ml. Erlenmeyer flask. Add 200 ml. of distilled water to the flask, heat nearly to boil.ino and add 10 nil. of 18 N sulfuric acid. When all off the oxalate is dissolved, titrate inunediately with the permanganate solution to the first faint, permanent: pink tinge. Swirl the solution constantly throughout the titration, and main- tain the temperature above 70° C. Ln ~ m m W m ~ m ~ ~ .n~.!.~...~..L'I ~...«.. «~.~.. .
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CA~ 70N Nor mality Wt. of Sodium O::alate x_1.4.92.4 T'Y ~ of. Fermanganate Vol.wne .' 'Ph
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RESEARCH DEPARTMENT Analytical Method No. ST-7 PRk:PAItATION AND STANDARDIZATION OF SILVER NITRATE SOLUT:[:O\S Diohr's method is based on *the quantitative fracti_onal precipitation of the chloride ion as silver chloride in the presence off a second indicator ion, the chromate ion, capable of producing a highly colored insoluble silver salt. The end point in the instrumental method is realized from the second der• i•vati_ve fUncti.on of the potentiometric inflection at quantitative precipi- tation of the silver chloride. SOLUTION BOTTLES.~ Fabricated of _~a_mber glass, preferably painted black if large volume$ of silver nitrate are prepared. BURETTE, Normax or equivalent grade. If Machlett type burettes are ' employed, they should be fabricated from amber glass. .~ AUTOMATIC TITRATOR, Sargent-Malmstadt, E. H. •Sargent and Co., Catalog No. S-29690. ELECTRODES: Indicator •- platinum or platinum-rhodium. Reference •- calomel. POTASSIUM CHLORIDE, ACS grade. Dry 2 hours at 120° C. and store in a desiccator. BIANK SOLUTION. Dissolve 20 g. of potassium chl.oride in l liter of water. SODIUi•i CHLORIDE SOLUTION, 0.1000 N. Dry sodium chloride for 2 hours at 120° C. and store in a desiccator. Accurately weigh 5.854 g., dissolve in water, and dilute to exactly 1 liter with water. • POTASSIUM CHRO%,fATE SOLUTION. Dissolve 20 g. of potassium chromate, K2CrO~, in 100 ml. of water. DJ:STII~LLD WATER. Must not give any turbidity when tested as follows: Fill a test tube three-fourths full with the water, add 1 drop of dilute, nitric acid and 2 drops of silver nitrate splution and shake. NITRIC ACID, dilute. Add one volume of concentrated nitric acid to nine volumes of water. SILVER NITRATE SOLUTION. Weigh the respective amount of silver nitrate, as listed in Table I, and dilute to the des~red volume with distilled water. I R. J. REYNOLDS TOBACCO COMPANY
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TABLE I W}:IGt1T. OF SILVER NITRATI's TO PREPARE TITRANTS Approximate Amount- of Silver Amount: of Silver Normal.i.ty nesired Nitrate t-o l:'repare _1 Li ter Ni.t:rate to Pi.e:par.e ].7 Liters 0.01. g. 0.025 , 4.3--4.4 0.05 g.6-8.7 0.1 17 . 1-17 . 5 PROCEDURE Mohr Method `:146 . 0 -1.48:. :.291. -298; Accurately weigh the amount of potassium chloride indicated in Table II into a 250 ml. Erlenmeyer flask and dissolve in 50 ml.-of water. Pipet 10 ml. of blank solution into another flask"and add 40 ml. of water. Add 5 drops of- potassium chromate to each of the flasks. Titrate the blank with silver nitrate solution until a definite red color persists. Add 1 ml. of blank solution to -. remove the red color and use this flask as the color blank. Titrate the potassiv: chloride sample witn silver nitrate solution until one drop pr.oduces a permanent color slightly darker than the coriiparison. Potentiomet.r. i.c Method blank. Shake the color blank before each. Accurately weigh the amount of potassium chloride indicated in Table II into a 250 ml. electrolytic beaker. Add 100 ml. of water and 7.5 ml. of dilute ni.tric; acid. Adjust the flow rate of the automatic titrator so that 9-10 ml. of silver nitrate per minute are delivered from the burette. Position .the beaker under the electrodes, close the burette stopcock, press the automatic button and when an audible click is heard, open the burette stopcock. In some instances the instrument will cut off with the addition of only a few drops of ti:tr.ant. Mien this happens, close the burett:e stopcock, press the automatic switch, and when the click is heard again, reopen the stopcock. The stirrer and valve shut off automatically at the end point. Read the burette, record the volume of titrant, then press the manual button to allow 2-3 drops of titrant to flow through the tip. Rinse the el.cctrodes and tip, then proceed with the next tit:ration.
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TABLE II Ar;OUNT_ OF I'OrASSY_U''f CHLOI:_TDF: TO I4TT.CH FOR S7ANDAkI~I'LING ~'~~RICJS STIa?NGTH SOLU`t'7.0\S .. Approxinat:e NormaliU 0.05-0.07 g. 0. 10-0. 13 S. .0.20-0.25 Standardization A~,,ai.nst 0.1 N._Sodi-um Chloride Solution Standai-d 0.1000 N sodium chloride solution may be substituted for the potassium chloride in either of the above listed methods. Pipet the appropriate amount of sodium chloride solution into the f:l.ask or beaker, dilute to the prescribed volume withjwater, then proceed as described in the above methods. I . CALCULATIONS ].. Using potassium chloride: F Wt. of KC1 x 13, 41 N ~ Volume of Silver Nitrate 2. Using 0.1000 N Sodium Chloride: N ` 0.1_x Volume of_NaCl sol.ution ~ Vol.ume of Silver Nitrate ~~ Amount of Potassium Chloride to be j-lei.ghed ~ ~ m m ~ m ~ m u, -•w.....,• . .....e^a-^..s.rr•.r. .:...-x•<v.,v.n.•...~~...~r...~,•.+-wp-..Y . .... . .,-.,'..y .......... ...,. ...~.......-..
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R. J. RI:YNOLUS TOBACCO COMPANY R1:SEA1'.CH DEPARTMENT Analytical Method No. ST-8 }'iZrPARATICN AND_ SIANDAI:D_IZATION OF P1;RML0RLC AC7.D IN GLhCIAL ACETIC ACID SOLUTIONS Potassium acid phthalat::e i.s dissolved in glacial acetic acid and titrated to the crystal violet end point with perchloric acid solution. APPARATUS.. AND -1:EAGENTS following table. DILUTION OF 11.6 N PERCHLORIC ACID TO PR1:I'AR.E TITRANTS Volune of 70-72% Volume of 70-72% . Norma] i.ty 1)esired 11C104 to Prepare 1 Liter HClOI} to Pr. epare 17 Liter. s . automatic type burettes are used, protect from atmosphere with drying tubes. ACETIC ACID, glacial, ACS grade. PERCHLORIC ACID, 70-727a, ACS grade. POTASSIUM ACID 1'HTHALA:Cr, primary standard grade. Dry for 1 hour at 120° C:, and store in a desiccator. CRYSTAL VIOLET INDICATOR. Dissolve l g, of- crystal violet in 100 nil. of glacial acetic acid. 1'ERCILLORIC ACID SOLUTIONS. Dilute 70-727o pE-rchloric acid to desired FURETTF., Normax or equivalent grade. If Machlett strength with glacial acetic acid according to the 0.01 0.9 0.025 2.1 0.050 4.2 0.100 8.4 0.200 16.8 0.500 42.0 PROCEDURE 15.3 35.7 71./+ 142.8 285.6 " 714.0 Accurately weigh the amount of potassium acid phthalate indicated in Table II into each of three 250 ml, l:rlenmeyer flasks, add 30 ml, of glacial acetic acid and heat gently to effect solution. Cool, add one drop of a-.a 51069 0106
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. crystal violet indicator and titrate to a bluc-green end point with the perchloric acid solution. The solution to blue-green. changes color f:roTn violet to blue APiOUNT OF POTASSIUM ACID P}IT}U LhT:: TO t•TJ;XGl-T _ ~ FOR SJ'ANI)AF`t)ILI\G VAKIOUS STRI:NG'Ci1 SOLUTIONS A_ pproximatc NormaLi.ty CALCULATION Anount ~ Sam 1e Wt x 4 8967 Seaman, W., and Allen, E., AN.AL, CHL•M., 23, 592 (1951). 0.06 - 0.07 g. 0.15 - 0.18 g. 0. 3 - 0.35 g. 0. G - 0. 7 1.2 ~ 1.4 g. 3.0 - 3.5 g. Normal ity Volume of Titrantw-- REFF,RENCE of Potassium Acid Phthalate to be T%Ie9.ghed.~y .
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It. J. REYNOLDS TOBACCO CC):,IPANY . REST:ARCH I)ErARTMEN'P I PREPARATION AND STANDARDIZATION OF TETIZAI3UTYI.AMDfONIImi HYDROXIDE SOLUTIONS PP.INCIPLE Benzoic acid is dissolved in the desired solvent and titrated either to a visual or potentiometric end- point with tet:rabutylamnonium hydroxide. APPARATUS ammonium bromide in methanol.'-,Change this electrolyte daily. }3URET7'E, 10 ml,, Normax or equivalent, equipped with Teflon stopcock. pH METER, Precision••Shell Dual. AC Titrometer, Leeds and Northrup, or equivalent, Precision-Dow Recordomatic Titrator may also be used. CI.ASS I:I,F.CTRODE, General Purpose, Beckman No. 41262. CALOi•4EL I.LECTR01)E, Fiber, Beckman No. 39170. Replace the saturated aqueous potassium chloride in the out:er jacket with 0.01 M tetrabutyl- REAGENTS with nitrogen, restopper, shake vigorously and replace in the sa].t-ice bath for 1.5 minutes. Filter under a nitrogen atmosphere through a fine porosity si.nter.•ed glass funnel. Transfer the filtrate to a l.-lit:er volumetric flask, allow to warm to room temperature and dilute to volume with benzene. Allow to stand for. 24 hours, and if any silver oxide separates, ref:i.lter. Store the titrant in a reservoir protected from carbon dioxide and moisture. 1'ETRABUTYIArM0NIUM HYDROXIDE, 0,02 N. Dilute 200 ml, of 0.1 N tetrabutyl- ammonium hydroxide and 30 ml. of methanol to 1 liter with benzene. Prepare other dilute titrants using proportionate amounts of 0.1 N tetrabutylammonium hydroxide, methanol and benzene as for the 0.02 N titrant. moist,silver oxide. Flush the flask with nitrogen, tightly stopper, and replace in the salt-ice bath. Allow the reaction to proceed for one hour with frequent shaking, replacing the flask in the salt-ice bath after each shaking. Add 900 ml. benzene to the flask, reflush 95 ml. methanol in a. 1 liter flask. Chill the solution in a salt-ice bath (-5 to -10° C.), then add.the freshly precipitated, methanol- si.lver nitrate in 50 ml, of carbonate-free water, add 55 ml. of 4 N. carbonate-free sodium hydr.oxide, shake vigorously, then filter under a nitrogen atmosphere through a medium porosity sintered glass funnel. Wash the precipitated silve.r oxide with 750 nil. boiling water, then with 300 ml. methanol. Dissolve 40 g. tetrabutylammonium iodide in TETRAhUTYIAMONIUM HYDROXIDE, 0.1 N. ni ssol.ve 30 g. of reagent grade ~_.....^. ..1~„1.Y'V+.(i.X"S1..n ry nN:^Mr.MO.'.^rv, n r,..nv~v~•~,•ir`+TT'.~. . . ~. ^.Y.y.:TM/^ER.~. .r. , ~.. w . n. . -. ^.
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PXRInII\E, Flask-distill technical grade pyridine from barium oxide using an upright condenser, discarding the first and last 10% of the distillate. Alternatively, add 80 g. reagent aluminun oxide to 2 liters of pyridine, shake, and allow to stand overnight. Filter through a medium porosity sintered glass funnel containing 20 g. of Celite. Sparge with nitrogen . for 5-10 minutes before use. ACETONE, drum grade. Sparge with nitrogen for 5-1.0 minutes before use. ACETONITRILE, drum grade. Sparge with nitrogen for 5-1.0 minutes before use. tert-BUTYL ALCOIIOL, Eastman White Label Grade. 5-1.0 minutes before use. BENZOIC ACII), NBS grade. Dry for 1 hour desiccator. AZO VIOLET INDICATOR SOLUTION. Dissolve azoresorci nol in 1.00 ml. of pyridine. TI11TlOL BLUE INDICATOR SOLUTION, Dissolve in 100 ml.. of acetone. NITP.OGI:N~ oil-pumped. PROCEDURE : : Sparge with niL• rogen for at 105° C. and store in a 0.5 gram off p-nitrobenzene- 0.3 gram thymolsulfonephthalein Vi.sualyStandardi.zation, _ PyridineySolvent` Accurately weigh the amount of benzoic acid indicated in Table I into a.1 25 nil. Erlenmeyer flask. Add 40 ml. pyridine and one drop of azo-violet indicator solution. Titrate under nitrogen to a violet end point. The color change at the end point is from red to violet. Perform a blank titr.ation and correct the vol:zrce of titrant accordingly. .. Visual Standardization, Acetone, Acetonitri]e, tert-Butyl Alcohol Solvent Accurately weigh the amount of benzoic acid indicated in Table I into a 125 ml. Erlenmeyer flask. Add 40 ml. of the desired solvent and 3 drops of thymol blue indicator solution. Titrate, preferably under nitrogen, to a blue end point. The color change is from yellow to green to blue. 1'erform a blank titration and correct the volume of titrant accordingly. Potentiometri.c Standardization., All Solvents Accurately weigh the amount of benzoic acid indicated in Table I into a 250 ml. electrolytic beaker. Add 40 ml. of the desired solvent and place beneath the electrodes. Titrate potentiometrically, under nitrogen, using either the automatic or a manual. titrator, recording volume of titrant and milli.voltage. Plot the volume of titrant vs. millivoltage and determine the end point from this curve. Perform a blank titration and correct the volume of titrant accor.dingly.
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- ~ .~AI3LN 1 AMOUNT_ OF YEN?.OIC ACID TO WEIGH FO_R_ STAND:IIZI)I ZING I'At:IOUS~ STRENGTH SO7.UTIONS CAI,CUT-.ATI ON Normality . NOTES Amount of Benzoic Acid to be Weighed 0.01 - 0.011 g. 0.02 - 0.022 g. 0.05 - 0.055 g. 0.1 - 0.11 g. Sample Wt. x 8.1.8867 Volume of Titrant For most accurate results it is,.preferable to standardize the titrant using the solvent to hee employed in subsequent analyses. It is also preferable to standardi-ze the titrant for each mode of titration rather than use the value obtained visually for potentiometric work, or vice versa. Pyridine is the most useful solvent for this work and its use is recormnended wherever feasible. - REFEF.}:NCE G`undiff, R. H. , RDR, 1-961, No. 46 (November 7). \
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ANALYTICAL METHODS SUiylMARY SHEET DETERHINIATIOid P-IEDIA* TECHWIQUE** RANGE 6 A.I1. NO. Acid Detergent Fiber TUB GRAV 2033 Acids TOB TITR 2064-1 Acid Value TOB TITR 2064 Additives, Total PAP & FIL GRA`J 5-15% .677 3002 Al cohol Sol ubl es TOB GRAV 20-40% 2.33 2020 Alkalinity of Ash TOB TITR 2061 Alkaloids, Total TOB TITR 2012 Alkaloids, Total 'f 0B TITR 2035 Aluminum TOf3, 0TH A.A. 2045 Al Umi nUm HO A.A. 5045 Ammoni a T6B TITR .1-2% .06 2008 Aimnoni a H20 0TH .01-17 mg/L 1.59 5002 Ant i niony TOl;, OTH A.A. 2045 Antimony H20 A.A. 5045 Arseni c TOB, 0TH A.A. 2045 Arsenic 11 A A 5045 Ash 6~ T . . GRAV 8-25% 1.58 2017 Ash 0TH GRAV 4015 Ash, Water Insoluble TOB GRAV 2061 Ash, 4:ater Soluble TOB GRAV 2061 8arium TOB, 0TH A.A. 2045 E3arium H20 A.A. 5045 Beryl 1 i um TUfi, 0TH A.A. 2045 l3eryl l i um H20 A.A. 5045 Bismuth TOE3, OTH A.A. 2045 Bisrnuth H20 A.A. 5045 Boron TO$, OTH A.A. 2045 Boron A.A. 5045 Burning Rate T6B 0TH 40-70 mg/min 2.59 2030 Burning Rate TOB 0TH 8-13 min/cig .54 2030 Cadmium TOB, 0TH A.A. 2045 Cadmium H0 2 A.A. 5045 Calcium TOf3, 0TH A.A. 1-4% .059 2045 Calcium HO A.A. 5045 Calcium Carbonate PAP K FIL TITR .2-35% 3007 .Cellulose TOB GRAV 2033 Cesium TOB, 0TH A. A. 2045 Cesium B A.A. 5045 Chloride T6 TITR 0.1-3% .028 2014 Chromium TOfi, 0TH A.A. 2045 Chromium H,0 A.A. 5045 Citric Acid T~B G.C. 2025 Cobalt TOB, 0TH A.A. 2045 Cobalt H20 A.A. 5045 Dyshrosium TOEI, OTH A.A. 2045 Dysprosium HO 2 A.A. 5045 Fats ft1 0 GRAV 4002 . 51069 0112
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2 DETERIMITlATIOld MET)IA* TECHNIQUE** RANGE cr A.11. 140. Freon 11 (H-20) TOB G.C. 2022 Glycerine TOB G.C. 2026 ycyrrhizic Acid Gl 0T I-I COL 4019 , Gold TOl3, 0TH A.ti. 2045 Gold_ H 0 A.A. 5045 Iron 2 TO[3, OTH A.A. 2045 Iron H 0 A.A. 5045 Lead 2 TOG, OTH A.A. 2045 Lead H, 0 A.A. 5045 Lignin T~13 GRAV 2033 Lithium T0[3, 0TH A.A. 2045 Lithium H20 A.A. 5045 f-1agnesium TOB, 0TH A.A. .3-.7% .015 2045 Magnesium A.A, 5045 Malic Acid T6U G.C. 2025 Manganese TOB, 0TH A.A. 2045 Manganese A.A. 5045 Menthol T6E3 COL, AUTO 0.2-0.6% .026 2002 Mercury TO[3, 0TH A.A. 2045 Mercury A.A. 5045 F1oi sture T6B GRAV 2011 Moisture TOB G.C. 2051-1 Moisture TOB 0TH 2051 Moisture 0TH GRAV 4001 Molybdenum TOB, O,TH A.A. 2045 Molybdenum H 0`' A.A. 5045 Nickel 2 TOB, 0TH A.A. 2045 tJi c6:el A.A. 5045 Nicotine TOS TITR 1-5% .202 2012 fJi coti ne TOB COL, AUTO 1-6% .082 2001 Witrate TOB 0TH 0.1-3% .097 2004 tJi trate OTH 0.1-100 mg/L .876 2004 Hitrogen,-Insoluble T0~ TITR 0.5-3% .396 2009 Nitrogen, Soluble TOB TITR 0.5-3% .270 2009 Nitrogen, Total T06 TITP. 1-5% .148 2003 Ni trogen, Total TITR 0.5-130 mg/L 2.39 5006 Norni coti ne T6B TITR 2012 Oxalic Acid TOB G.C. 2025 Pal l adi urn TOB, 0TH A.A. 2045 Pal1adium A.A. 5045 Pectin T6B COL 2036 pH TOB OTH 4-7 .095 2010 pH 0TH 0TH 4014 Phosphorus TOB COL 2072 Phosphorus COL 0.1-30 mg/L .166 2072 Phosphates T~l~ COL 2072 Piiosphates H20 COL 0.3-90 rng/L .48 2072 Platinum TOE3, OTH A.A. 2045 a Platinum H20 A.A. 5045 51069 0113
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3 • DETERMINATION ME U T A* TECHNIQUE** R ANGE v A.M. 110. Potassium TOE3, 0TH A.A. 1- 5% .063 2045 Potassium 11 A A 5045 Potash T 6~ . . A.A. 2045 Propylene Glycol TOB G.C. 2026 Protein TOB TITR 2009 Refractive Index 0T}{ 0TH 4009 Rubidium TOE3, 0TH A.A. 2045 Rubidium ~ A.A. 5045 Sand T6 GRAV 0- 90% .728 2013 Sel eni um TOQ, 0TH A.A. 2045 .Selenium H?0 A.A. 5045 Silicon TOB, OTH A.A. 2045 Silicon H 0 A.A. 5045 Si l ver 2 TOB, OTH A.A. 2045 Silver H?0 A.A. 5045 Sodium TOR, 0TH A.A. 2045 Sodium HO A.A. 5045 Sodium Chloride T68 TITR 2014 Solids, Total OTH GRAV 5003 Solids Total W0 GRAV 10-5 000 mg/L 807.7 5003 Solids, Volatile RH GRAV 5003 Solids, Volatile H'0 2 GRAV 14 000 mg/L 370.1 5003 Specific Gravity 0 fH 0TH 4003-1 Specific Rotation, Menthol 0TH 0TH 4003 Starch TOl3 COL 2016 Strontium TOB, 0TH A.A. 2045 Strontium 11 O A.A. 5045 Sugar, Reducing H R TITR 4005 Sugar, Reducing ToB, 0TH TITR 4- 20% .862 2005 Sugar, Reducing T08 COL, AUTO 2- 25°0 .308 2001 Sugar, Solids OTH 0TH 4009-1 Sugar, Total 0TH TITR 4005 Sugar, Total TOB, 0TH TITR 2- 20% .374 2005 Sugar, Total TOB COL, AUTO 5- 20% .318 2001 Sulfate TOB GRAV 2015 Tellurium TOf3, 0TH A.A. 2045 Tellurium H20 A.A. 5045 Ti n TOE3, 0TH A.A. 2045 Ti n H?0 A.A. 5045 Titanium. TOB, OTH A.A. 2045 Titanium H0 A.A. 5045 Triacetin PAP ~ FIL COL, AUTO 20-' 60 mg/rod 2.437 3003 Tria.cetin PAP & FIL G.C. I 3003-1 Triacetin PAP & FIL G.C. ~ 3001 Triethylene Glycol TOl3 G.C. i 2026
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• DETERMINATION h1EDIA* 4 TECHJdI(~UE** RANGE Q - A.M. 110. Triethylene Glycol Diacetate PAP & FIL G.C. 3001 Tungsten . TOB, 0TH A.A. 2045 Tungsten }120 A.A. 5045 VanGdium T0t3, OTH A.A. 2045 Vanadium t.{ 0 A.A. 5045 Viscosity ON 0TH 4007 Water Soluble TOB GRAV 5-50% .481 2021 Water Soluble TOB GRAV 2021-8 Water Soluble, Cold 0TH GRAV 4014 Water Soluble, Hot 0TH GRAV 4014 Ytterbium TOD, 0TH A.A. 2045 Ytterbium H,O. A.A. 5045 Zinc TO[3,20TH A.A. 2045 Zinc H20 A.A. 5045 Zi rconi urn TOQ, 0TH A.A. 2045 Zirconium H20 A.A. 5045 LEGEPdD , iIED I A* SMO - Smoke TOB - Tobacco PAP & FIL - Cigarette Paper and Filters 0TH - Others H20 - Waste Water TECHNIQUE** GRAV - Gravimetric TITR - Titrametric COL - Colorimetric G.C. - Gas Chromatography A.A. -- Atomic Absorption AUTO - Automated 0TH - Other
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. ANALYTICAL MST$4DS - _ 5i069 0116 N
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R. J. REYNOLDS TOBACCO COMPANY RESEARCH DEPARTMENT Analytical Method No. 2001 AUTOMATED METHOD FOR [)ETERMINATION_ OF NICOTINE ALKALOIDS, ~ REGUGING SIJGARS, A(~D TUf/~LTSUGARS L3Y ROBOT CHEMIST PRINCIP[.E Nicotine alkaloids and sugars are extracted from a tobacco sample and analyzed by automated colorimetric procedures; nicotine by color of schiff base formed by action of CNBr and sulfanilic acid, and reducing sugars by formation of ferrocyanide with. a diminution in color of the ferricyanide originally present. Total sugars are determined by inverting non-reducing sugars with invertase solution, followed by reducing sugars end determina- tion. APPARATUS AND REAGENTS ~ ROBOT CHEMIST, American Optical Corp. SHAKER, [3urre1l Wrist-Action. FILTER PAPER, S & S 560 EXT[ZACTION SOLUTION, 10 ml Glacial Acetic Acid'and 20 ml of methanol, diluted to one liter with distilled water. SULFANILIC ACID-AMMONIUM ACETATE SOLUTION, 10 g sulfanilic acid and 50 g ammonium acetate in one liter aqueous solution. CYANOGEN BROMIDE SOLUTION, 40 g of CNBr (reagent grade) in one liter aqueous solution. CAUTION: CNBr vapor is toxic. All weighings and handling should be done in a hood. ~~ MONOPOTASSIUM PHOSPHATE (KH2P04) SOLUTION, 0.1 M. Dissolve 13.61 g KH?PO4 in;one liter aqueous solution. SO]DUM HYDROXIDE SOLUTION, 0.1 N. Dissolve 4.0 g NaOH in one liter aqueous solution. PHOSPHATE BUFFER SOLUTION. Mix 500 ml of 0.1 M KH2PO4 and 296.3 ml 0.1 N NaOH. Dilute to one liter. SODIUM HYDROXIDE SOLUTION, 0.5 N. Dissolve 20 g NaOH to one liter aqueous solution. FERRICYANIDE SOLUTION. Dissolve 0.90 potassium ferricyanide and 20 g sodium carbonate in water. Dilute to one liter. INVERTASE SOLUTION, 1%. Dissolve 1.0 g invertase (calbiochem) in 100 ml distilled water. Add 0.5 ml toluene as preservative. Keep refrigerated when not in use. CHARCOAL, Darco G-60 NICOTINE SALICYLATE, U.S.P. grade, K & K laboratories. GLUCOSE, National Bureau of Standards Grade. i.
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2 Analytical Method No. 2001 NICOTINE STANDARD, STOCK SOLUTION. Dissolve 1.8513 g nicotine salicylate in extraction solution to yield one liter of stock solution containing 1.0 mg/ml. GLUCOSE STANDARD, STOCK SOLUTION. Dissolve 1.0000 g of NaS glucose (dried in vacuum oven) in extraction solution to yield one liter of stock solution containg 1.0 mg/ml. Calibration Curves NICOTINE STANDARD, blORKING SOLUTIONS, 1.0, 1.5, 2.0, 2.5, 3.0, 4.0, and 5.0 nil of the stock nicotine solution containing 1 mg/ml are diluted with extraction solution to 100 nil in volumetric flasks. These solutions contain 10, 15, 20, 25, 30, 40 and 50 ug/ml, respectively. GLUCOSE STANDARD, WORKING SOLUTIONS. Glucose solutions containing 50, 100, and 150 jjg/ml are prepared by diluting 5.0, 10.0, and 15.0 ml, respectiv,ely of 1 mg/ml glucose solution to 100 nil with extraction solution. ~ i Calibration curves ~Ire prepared by substituting quantities of the above prepared solutionsfor samples and performing the appropriate analyses. The resulting absorbances and concentrations are treated by least squares calculation to determine the absorbance/concentratidn factors that may be used to calculate the results desired from the analyses of samples. PROCEDURE Accurately weigh a catchweight of 0.1 g of dried, ground tobacco and transfer to a 125-m1 glass--stoppered Erlenmeyer flask. Pipette 100 ml extraction solution into the flask, stopper and extract for 15 minutes using a Burrell wrist-action shaker. Filter half of this extract through S&S 588 folded filter paper. Collect this filtrate in a stoppered container for nicotine alkaloid analysis. Analyze using the automated nicotine alkaloids procedure. Add 0.10 g Darco G-60 to the flask~ containing the balance of the original extract, stopper, and shake vigorously by hand for 10 seconds. Immediately filter through the S&S 588 folded filter paper used above. Collect this filtrate in stoppered containers. Take approximately 5 ml of this filtrate for the reducing sugars determination on the Robot Chemist. 1 Add 1 ml of 1% invertase solution to the remainder of the above filtrate. Let this solution stand for at least 10 minutes. Take a portion of this filtrate for the total sugars determination on the Robot Chemist. ~
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Analytical Method No. 2001 The Robot Chemist program for nicotine is as follows: Set water bath temperature at 40° C -Robot Turntable Station Number 0 1 4 7 9 11 15 18 . Operation Pipet 0.5 ml sample. Pipet 2.25 nil sulfanilic acid solution. Stir. .Pipet 0.5 nil CNBr Solution Sti r. Pipet 4.2 nil phosphate buffer Stir.~ Withdraw sample and read at 466 nm. Operation of the Robot Chemist is contained in the Robot Chemist Operation Handbook. Absorbance values are printed out digitally and/or fed to on-line computer for calculation, Manual calculation can be done as follows: . - Concentration Factor = ~ F (Concentrations).2 E ~(Absorbances J~' % Ni coti ne (Absorbance)(Concentration Factor 10) ~ Weight of Sample~(~g) r The Robot Chemist program for both reducing and as follows; Robot Turntable Station Number 0 2 3 5 53 total sugars is Operation _ _ Pipet a 0.50 nil aliquot of either sample or standard solution. Add 2.50 ml of extraction solution. Add 1 nil sodium hydroxide solution. Add 2 ml ferricyanide solution. Stir, heat att a bath temperature of 80° C. Read at 420 nm. Treat the absorbance values in the same manner as was done for nicotine. T47-6-"T- BS~)`0,T20304 ~5U ~(0 80 90~~b~~00 SUO _I Fl l~~ 2 3~l i?~~6 0 f3 9 f U) 20 30 F Ei0 Gii /0 &0 90 10200 300 I =1fill` ~'I-03 ~4 ~ j ~~~ ~ ~~_~01_~'_ZO 30~0 ~U 60 70 80 ~O~1~U ~~G 300 ~
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R, J. REYNOLDS TOBACCO COMPANY RESEARCH DEPARTMENT. Analytical Method No. 2002 AUTOMATED DETERI=1INATION OF MENTHOL I-N_ yMENTHOLATED TOBACCO BY ROE30~f CHE~•1IST~ ~ PRINCIPLE The menthol is separated from tobacco by distillation and determined colorimetrically after reaction with p-dimethylaminobenzaldehyde. APPARATUS AND REAGENTS DISTILLATION EQUIPMENT. See Figure 1. A is a 500 nil round-bottom flask, B a curved glass tube of 20 mm O.D. tubing, C an adapter made with 8 mm glass tubing, and D a 100 ml volumetric flask. Standard taper 24/40 glass joints are used throughout. ROBOT CHF:MIST, American Optical Co rp. ETI-IANOL, 95% ETHANOI,-•WATER MIXTURE. Mix equal parts of water and 95% ethanol. SULFURIC ACID, concentrated, ACS grade. p-DIMETHYL.Af,iINOBENZALDEHI'DE, Eastman, white lab'el. COLOR REAGENT. Prepare a dilute sulfuric acid solution of 1.6 volumes acid and 1.0 volume water. Dissolve 0.5 g p-dimethylaminobenzaldehyde in 100 ml of this dilute acid. MENTHOL, Eastman, white label. MENTHOL STANDARD. Accurately weigh 0.1 g menthol into a 100 ml volumetric flask, add ethanol to dissolve, and dilute to the mark with ethanol. PROCEDURE A. Preparation of Calibration Curve From the standard menthol solution prepare dilute standards by pipetting quantities containing 3, 4, 6, 8, and 10 mg of menthol into 100 nil volumetric flasks, and diluting to the mark with the ethanol-water mixture. - Analyze by same procedure used for samples after distillation on Robot Chemist. Compute an absorbance/concentration factor using least squares system, if needed. . m N B
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3 .r
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Analytical Method No. 2002 B. Analvsis If sample is loose tobacco, accurately weigh 2.00-2.15 g and tran5fer to distillation flask (A). If sample is cigarettes., remove paper and accurately weigh the tobacco from 2--3 cigarettes. After weighing the tobacco of filter cigarettes and transferring it to the distillation flask, remove the wrapper from the filters and add the filter material to the flask. Thus, menthol which may have migrated from the tobacco to the filter will be accounted for in the results. Add 80 nil of water and a few boiling stones, connect flask A to the condenser with tube B, attach the adapter C to the condenser and immerse the tip in 20 ml of ethanol contained in the receiving flask. Gently heat the sample flask with a Bunsen burner flame until the distillate begins to come over, at which time the heat may be increased and the receiving flask lowered so that the tip of the adapter is no longer immersed. Distill until 20 ml of distillate has been collected. Disconnect condenser from tube B and wash down the-undenser with ethanol, remove the receiving flask, dilute the distillate to approximately 70 nil with ethanol and add water almost to the mark. Mix, add ethanol to the mark and mix again. The end-determination is done using the Robot Chemist. The Robot Chemist program for menthol is as follows: Robot Turntable Stati on Number 0perati on 0 Pipet 1.0 mi sample or standard. 2 Pipet 4.5 ml color reagent 5 Stir. 15 Stir. 20 Withdraw sample and read at 550 nm. The Robot Chemist is usually run on-line with computer. Calculations can be done manually, if it is desired to do so, by proper treatment of the absorbance values obtained by digital print-out.
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Analytical Method No. 2002 CALCULATIONS Concentration Factor Jz (Concentrations)2 _ L7Absorbances Concentration Factor 10 (Absorbance Value 1 th ~ M l ' ~ ~ '~~ _ en o REFERENCE eight of Sample ( cJ } L. Harrell, T. G., RDR 1956, No. 6 (JULY 31) --4-0 59-60 7T- c90- TO-M7~3-or- s -FT~ 03 0 , - - ------- --, ~~j ~1 2345678 ~Ur2~030~44050607080a0,100200300
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R; J. REYNOLDS TOBACCO COMPANY RESEARCH DEPARTMENT Analytical Method No. 2003 DETERMINATION OF TOTAL NITROG[:N IN TOBACCO AN[)_ FERTILIZER ~ BY Y.JE:LDAHL KROCkQURE PRINCIPLE In the presence of a catalyst, the organic matter of nitrogeneous substances is destroyed by digestion with concentrated sulfuric acid and the nitrogen is changed to ammonia. The digestion mixture is made strongly alkaline with sodium hydroxide, the ammonia is distilled quantitatively into a two percent boric acid solution, and titrated with standard 0.1 N hydrochloric acid. APPARATUS AND REAGENTS FLASKS, KJELDAHL, 800-m1 capacity. flasks, erlenmeyer, 500-ml capacity. DIGESTION APPARATUS, electrically heated. DISTILLATION ASSEMBLY, electrically heated. SULFURIC acid, concentrated, low nitrogen, ACS grade. SALICYCLIC ACID, reagent grade. SODIUM THIOSULFATE, anhydrous, ACS grade. ZINC METAL, granular, 30 mesh, ACS grade. SODIUM HYDROXIDE SOLUTION. Dissolve 500 g of sodium hydroxide in 500 nil of water. SULFURIC-SALICYLIC ACID MIXTURE. Dissolve 152 g of salicylic acid in 4 Kg of sul furi c aci d. HYDROCHLORIC ACID SOLUTION, 0.1 N. Prepared and standardized as described in Analytical Method No. ST-2. BORIC ACID SOLUTION, 2 percent. Dissolve 20 g of boric acid in a liter of water. HIGH BOILING MIXTURE, prepacka ed, individual packages. INDICATOR, N-Point (trade nameg}, manufactured by Matheson, Coleman & Bell. PROCEDURE Accurately weigh 1-2 g of ground tobacco or 0.25 g fertilizer sample into a Y~jeldahl flask, add 40 nil of the sulfuric-salic,ylic acid mixture, swirl until thoroughly mixed, and allow to stand at least two hours. (For the analysis of tobacco stems, allow to stand'30 minutes.) In analysis of fertilizer add approximately 5 g sodium thiosulfate, shake and allow to stand for 5-10 minutes. Add one package of high boiling mixture, place on digestion rack, turn on electric heaters to heat and digest until all danger of frothing has passed. Increase the heat until the acid boils briskly and continue the digestion until the mixture is colorless. ~
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2 r If the contents of the flask are likely to become solid before this point is reached, add 10 ml more of sulfuric acid and continue heating. Allow the solution to cool for 20 to 30 minutes, add approximately 200 nil of water, and allow to cool again. Pour 50 nil of 50 percent sodium hydroxide solution down the side of the flask so that it does not mix at once with the acid solution, add several pieces of granulated zinc and connect the flask to a condenser on the distillation rack. Pipet 50 ml of boric acid solution into a 500 nil Erlenmeyer flask and place beneath the condenser so that tip of the condenser extends below the surface of the solution. Mix contents in the Kjeldahl flask and distill until 150 ml of distillate is received. Wash down the condenser tubes.and titrate.the distillate with 0.1 N hydrochloric acid using 0.5 ml N-Point indicator. Titrate through gray warning color to red end point. CALCULATION Percent Total Nitrogen = V x N x 0.014 x 100~ Wt. of Sample~l~1-F f~asisT where: . . I V = Volume of 0.1 N hydrochlor,ic acid required N = Normality I Pf hydrochloric acid REFERENCE 1. Methods of Analysis, A.O.A.C., 7th ed., p. 13 (1950). S 2_3 4 5 6 7 8 9 1~ 7 30 40 50 60 70 80 90 100 2_00 300 ! RT 1~_3 4 5 6 7_8 9 100130 40 50 60 70 80 90 1Q0 ?_00~300 . 9H 1 2 3 4 5 E 7 8 9+8~~30 ~+0 010 6U 70 f30 9(i ~ 00 200 300 ~
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R. J. REYNOLDS TOBACCO COMPANY RESEARCH DEPARTMENT Analytical Method No. 2004 DETERMINATION OF NITRATE CONTENT WITH A SELECTIVE ION ELECTRODE PRINCIPLE The nitrate content of a sample is determined by immersing the selective ion electrode into its aqueous solution and relating the resulting meter reading to a known concentration response. APPARATUS AND REAGENTS SPECIFIC ION METER, Orion Model 407 NITRATE ION ELECTRODE, Orion Model 92-07 REFERENCE ELECTRODE,; Fiber Junction Calomel, Beckman No. 39170 tfiAGNETIC STIRRER I NITRATE STANDARD, concentrated. Accurately weigh 0.3261 g of potassium nitrate ~which has been dried overnight at 102° C. Dissolve in water and dilute to 100 ml for solution containing 2 mg nitrate per ml (2000 ppm). Prepare fresh every two weeks. PROCEDURE FOR TOBACCO Procedure "A" (Direct Measurement Method) Pipet 1 ml and 3 nil of the concentrated nitrate standard into separate 200-m1 volumetric flasks and dilute each to volume. These standards contain respectively 1 mg and 3 mg nitrate per 100 nil. Transfer 100 ml of the 1 mg per 100 ml standard into a 150-ml beaker containing a stirring bar. Place on si;irrer and stir at a fixed rate. Insert electrodes and adjust meter with calibration knob to read 100 on red scale. Repeat operation, up to adjustment of meter, with 3 mg per 100 nil standard and, if necessary, adjust slope to attain a reading of 300 on red scale. Then set temperature knob to proper temperature. For best results, sample readings should fall between these points. Dry sample and grind to small particle size. Accurately weigh approximately 0.05 g burley tobacco or 0.1 g of most other types of tobacco or blends. Transfer to a 150-ml beaker and accurately add 100 ml water. Add a few ml of the water initially to wet the tobacco before adding the remainder of the 100 nil. Allow mixture to stand foril5 minutes with occasional stirring. With a stirring bar in mixture, place on magnetic stirrer and stir at a fixed rate. Inrnerse electrodes and read uppermost (red) scale of meter. ~ (6 ~
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Analytical Method No. 2004 % Nitrates (NO3-) ~ Meter readinq : 1000 Sample~Wt. Procedure "Q" (Known Addition Method) Follow the directions set forth in the second paragraph under Procedure "A" up to the last sentence. Insert electrodes and adjustt meter with calibration knob to obtain center scale reading (->) on green scale. Without removing sample, pipet,l ml concentrated standard into sample and read green scale. °d Nitrates (N03-) = Meter Reading X 0.2* Sampl e : llt. *Based on addition of exactly 2 mg nitrate ( in 1 ml) to sample. Different strength standards would require proportionately different factors. PROCEDURE FOR WATER SAMPLES ~ Pour 100 nil of sample into 150-m1 beaker and analyze as in procedure "A" for tobacco samples. % N03- = Meter Readi ng 1000 NOTES For best results the following conditions should be observed: 1. Always use same size beaker for measurements and immerse electrodes to approximately the same depth. 2. Use approximately same stirring rate for all measurements. 3. Recharge electrode each month or earlier if unsteadiness, drift or slow response is observed. For best response and stability, soak freshly charged electrode in potassium nitrate solution (2 rng nitrate per 100-m1 concentration) for one hour before use and store immersed in this solution between uses. 4. For determinations on low nitrate tobaccos (flue-cured and Turkish), extract a 2-g sample in 200 nil water, filter and make measurement on 100 rnl of the filtrate. 5. In analyses of untreated stems (high in nitrates), extract 0.1 g in 100 ml water• and dilute 10 nil to 100 nil for measurement. ~
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Analytical'Method No. 2004 When using the Known Addition method, the 1 ml of nitrate standard added should contain one to four times the quantity of nitrate present in the 100 nil of sample extract. 7. When using the Direct Measurement procedure, calibrate instrument with standards daily and make one standard mea.surenent: after each 25-30 sample measurements, adjusting meter if necessary. The dilute standards should be prepared daily. REFERENCES 1. Harrell, T. Gibson, RDM, 1971, No. 55 (NOV. 1971}. T- ~ 1 2 3 ~ 4 5~6 `"1^8;- 9 'r0y20 '3 Z 40 50 'I I '2 34;,i 6 7~~i)'9F 10 20 30 4 0 5 0 60 I(0 80 90 100 200 300 ~~IifT'~~2~3~ 5J 6 7 8 9 10 20 30 40 50 60 !0 80 90'100 200 30U ~
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R: J. REYNOLDS TOBACCO CC?f,SPANY RESEARCH DEPARTh1(:(dT Analytical Method No. 2005 DETERMINATION OF SUGARS ):N TOCACCO ~~ f1ND ~l.N LI(UID SIJGI~f~!^T~^ _-- --_ -~ .-- ---- PRINCIPLE The sugars present are extracted from ground tobacco with water and determined by the Somogyi (1) method. Similar techniques are used to determine the total sugar, the invert sugar, and sucrose content of liquid sugar. APPARATUS AND REAGENTS c. DISTILLED WATER INVERTASE SOLUTION. Dissolve 1 g of invertase, GBI analytical grade, in 100 ml of water. Add 0.5 ml of toluene as a preservative. Keep refrigerated when not in use. SODIUM PHOSPHATE, dibasic, Na2HP04, anhydrous, ACS grade. SODIUM POTASSIUM TARTRATE (ROCHELLE SALT), NaKC,6Hti0c•4H?0, ACS grade. SODIUM SULFATE, anhydrous, ACS grade. SODIUM HYDROXIDE SOLUTION, 1 N. Dissolve 40 g sodium hydroxide pellets in water and dilute to 1 liter with water. POTASSIUM IODATE SOLUTION, 1 N. Dissolve 35.67 g potassium iodate in 1 liter of water, SODIUM THIOSULFATE SOLUTION, 0.1 N. Dissolve 25 g of sodium thiosulfate crystals (Na2S?03-5H20) in 1 liter of water. Add 1.0 ml of 1 N sodium hydroxide as a preservative: SODIUM THIOSULFATE SOLUTION, 0.005 N. Dilute 110 nil of 0.1 N sodium thiosulfate to 2 1 with water. POTASSIUM IODIDE SOLUTION, 2.5%. Dissolve 25 g of potassium iodide in 1 liter of water. Add approximately 0.1 g sodium carbonate as a preservative. COPPER SULFATE SOLUTION, 10%. Dissolve 100 g copper sulfate (CuSO4•5H20) in 1 liter of water. STARCH INDICATOR SOLUTION. Prepare a thin paste of 3 g soluble starch with a small amount of water. Add to 500 ml of boiling water and boil 3-4 minutes. Add about 10 mg mercuric iodide as a preservative. SULFURIC ACID, 2 N. Dilute 55 ml concentrated sulfuric acid to 1 liter with water. ACETIC ACID, 10%. Dissolve 100 ml glacial acetic acid in water and dilute to 1 liter with water. CELITE ANALYTICAL FILTER AID, or equivalent. m ~ N ~ t0
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Analytical Method No. 2005 SOMOGYI REAGENT SOLUTION. Dissolve 56 g anhydrous dibasic sodium phosphate and 80 g sodium potassium tartrate in 1400 ml of water, heating to affect solution. Acid 200 nil of 1 N sodium hydroxide, and while stirring add 160 ml of 10% sulfate solution. Add 360 g anhydrous sodium sulfate and stir until dissolved. Add 50 ml of 1 N potassium iodate. Transfer to a 2-liter volumetric flask, allow to cool, then dilute to volume with water. Mix thoroughly, allow to stand overnight, and filter befo re use. STANDARD SUCROSE SOLUTION, 0.2 mg/ml. Dissolve 0.0500 g NBS sucrose in water and dilute to 250 ml. Store in refrigerator when not in use. STANDARD GLUCOSE SOLUTION, 0.2 mg/ml. Dissolve 0.0500 g NE3S glucose in water and dilute to 250 nil. Prepare fresh weekly, and store in refrigerator when not in use. STANDARD INVERT SUGAR SOLUTION, 1%. Dissolve 0.5 g NBS sucrose in water in a 1 liter volumetric flask. Add 5 ml concentrated HC1 and dilute to about 100 ml. Store at room tempe rature for 3-4 days then dilute to volume with water. This solution is stable for several months. ! ~. INVERT SUGAR STAf4DARp SOLUTION, dilute. Pipette 2.0 ml. of 1% invert sugar solution into a 100 ml volumetric flask. Add a few milliliters of water, 2 drops of phenolphthalein, and neutralize with sodium hydroxide solution, then dilute to volume. Five milliliters of this solution contains 1.0 mg invert sugar. Prepare this dilute solution immediately before use.v PROCEDURE A. Determination of Total Reducing Sugars in Tobacco Accurately weigh 0.07-0.08 g tobacco dust and transfer to a 125 ml Erlenmeyer flask.: Pipette 50 nil water into the flask, stopper, and extract 15 minutes, using the shaking machine. Add approximately 1 g Celite to the flask, restopper and shake vigorously, then filter through Whatman No. 5, or equivalent, filter paper. Pipette 5 nil of the filtrate into a 25 x 200 mm test tube, 5 nil of water into a second tube, 5 ml of standard glucose solution into a third tube, and 5 rnl of standard sucrose solution into a fourth tube. Add exactly 1 drop of 10% acetic acid and 2 drops of invertase solution into each tube. Mix, and place in a 55-60° C water bath f,or 15 minutes. Cool, add 5.0 nil of the Somogyi reagent to each jtube, and rni x. P1 ace the tubes in a boiling water bath for 15 minutes. Cool, add 2 nil of potassium iodide solution and 1.5 nil of 2 N sulfuric acid, mix vigorously and titrate the solution with 0.005 N sodium thiosuli'ate until the yellow color almost disappears. Add '.i drops starch indicator solution and titrate until the deep blue color just disappears. ~
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Analytical Method No. 2005 CALCULATIONS Glucose factor (G) _ Wl = Milligrams of glucose per 5 ml standard solution. VQ = Volume of thiosulfate for blank determination. VS = Volume of thios.ulfa.te for sample determination. , % Total Reducing Sugars as Glucose NOTES (Va - VS) (G) Grams of Sample ~ The reducing sugars, before inversion, may be determined and calculated by the above procedure, omitting the inversion step. It is necessary to prepare separate blank and standard glucose solutions for the non-inverted samples. It is not necessary to calculate the standard sucrose sample when analyzing for sugars in tobacco. One such sample should be inverted daily, however, to test the efficiency of the invertase. On titration the volume difference between the blank and the sample should be at least 7.0 ml. If, less than.7.0 ml, the invert.ase is not sufficiently active. The glucose factor should be determined with each series of analyses. This factor is expressed as the milligrams of glucose per milliliter of thiosulfate, and since it is determined with each run, it is not necessary to determine the exact normality of the 0.005 N thiosulfat:e. This frequent determination also compensates for any changes in strength of the Somogyi reagent. 6. Determination of the Sugar_.Content of l.i,guid Sugar c. PROCEDURE Accurately weigh 0.2-0.3 g(4-5 drops) of liquid sugar into a tared 100 ml volumetric flask, and dilute to volume with distilled, water. Mix, then dilute 10 ml of this solution to 100 ml with water. A. Pipette 5.0 ml of the dilution, 5.0 ml water, and 5.0 nil of the dilute standard invert sugar solution into each of three 25 x 200 mm test tubes. Add 1 drop of 10% acetic acid and 2 drops of invertase to each tube.
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4:. Analytical Method No. 2005 Mix and place in a 55-G0° C water bath for 15 minutes. Cool, add 5.0 ml of Somogyi reagent, mix and place in a boiling water bath for 15 minutes. Cool, add 2 ml of KI solution and 1.5 ml of 2N H2S01+, then titrate with 0.005 N thiosulfate to the starch end point. B. Pipette a second 5.0 nil aliquot of the dilution, 5.0 nil of water and 5.0 nil of the dilute standatd invert sugar solution into each of three 25 x 200 mm test tubes. Add 5.0 ml of Somogyi reagent, mix,-and place in a boiling water bath for 15 minutes. 'Cool, add 2 nil of KI solution and 1.5 nil of 2N H?SO,,, then titrate with 0.005 N thiosulfate to the starch end point. CALCULATIONS I = mLinvert suqar/5 nil ~ ~ (From A.) II = V B V S mg invert sugar/5 nil_ ~ (From B ) 0 VB-. VS . A. % Total Sugar = (V -` VS)(I)(20) (F om A ) (wet basis) G~arns af Sample r . B. % Invert Sugar = (VB •• VS)(II)(20) i b ) (From B.) s (wet as Grarns of Sample C. % Sucrose = A - B (Wet basis) = Milliliters 0.005 N thiosulfate required for blank. VS = Milliliters 0.005 N thiosulfate required for sample. 100 To convert to dry basis, multiply by factor: T Solias C. Analysis of Glucose Solut.ions , PROCEDURE Dilute the solutions as received as follows: If the expected amount of glucose is less than 0.05 percent, pipet a c.f aliquot of the dilution and analyze. 5 ml aliquot of the solution into a 25 x 200 mm test tube, add 5 nil Somogyi reagent solution and proceed as in the regular procedure, omitting the inversion step. If the expected amount of glucose is more than 0.05 percent, but less than 0.5 percent, dilute 10 ml of the solution to 100 ml, take a 5 nil,
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. Analytical Method No. 2005 'If more than 0.5% but less than 1.0%, dilute 5 ml of the solution to 100 ml, take a 5 ml aliquot and analyze. If more than 1.0% but less than 3.0%, dilute 10 nil of the solution to ~00 ml, take a 5 nil aliquot and analyze. If more than 3.0% but less than 6.0%, dilute 5 ml of the solution to 500 nil , take a 5 nil al iquot and analyze. If more than 6.0% but less than 12%, dilute 5 nil of the solution to 1000 ml, take a 5 nil aliquot and analyze. CALC ULATION D = (VB -VS)(0.0?_)(G) x Dilution factor D = Grams of glucose/100 mi solution VB = Volume of 0.005 N thio \ fai:e for blank V = Volume of 0.005 N thiosulfate for sample S G = Glucose factor Dilu tion factor: If run as is = 1 If diluted 10/500 = 50 If diluted 10/100 ml = 10 If diluted 5/500 = 100 REfE If diluted 5/100 ml = 20 If diluted 5/1000 = RENCES 200 1. Somogyi, M., J. BIOL. CHEM., 160, 61 (1945). 2. Cundiff, R. H., RDR, 1954, No. 4(JULY 6). 3. Cundiff, R. H., RDM, 1958, No. 4 (JAN. 10). 4. Cundiff, R. H., RDM, 1962, No. 73 (AUG. 10). -0~-0 3 5 6 % 8 9 1_0' 20 30 4 0 50 60 70 ~80_ 90 1'00 200_300 23~567B _9rt~1020 30~JO50fi0_70_80_90 100 200 300 MH ~1 2 3~! 5 6 7^8 9 1~~ 30 40 0 «50 _60 70 f30 90.~100200 300 y
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R. J. REYNOLDS TOBACCO COMPANY ' RESEARCH DEPARTMENT Analytical Method No. 2008 DETERMINATION OF AMMONIA BY KJELDAHL DISTILI._ATION F}tINCI:PLE The sample is made basic by adding MgO, and the ammonia removed by distillation, trapped in 2% boric acid solution, and the amount present determined by titration with standard 0.1 N hydrochloric acid. ~ APPARATUS AND REAGENTS ' FLASKS, Kjeldahl, 800 nil capacity. FLASKS, Erlenmeyer, 500 ml capacity DISTILLATION ASSEMBLY, electrically heated. MAGNESIUM OXIDE, ACS grade. ~ HYDROCHLORIC ACID SOLUTION, 0.1 N. Prepared and standardized as described in Analytical Method No. ST-2. BORIC ACID SOLUTION, 2%. Dissolve 20 g of boric acid in a liter of water. INDICATOR, N-Point (Trade Name), Matheson, Coleman, and Bell. PROCE[)URE Accurately weigh 5-10 grams of sample into a 800 ml Kjeldahl flask, and add 400 nil of distilled water. Allow to stand for about one hour. Add 5 grams of MgO, mix, and place on distillation assembly. Pipet 50.0 ml of 2% boric acid solution into Erlenmeyer flask and place beneath the condenser so that the tip of the condenser extends below the surface of the solution.' Distill until 150 ml of distillate is collected. Wash down the condenser tubes and titrate the distillate with 0.1 N HC1, using 0.5 ml N-Point indicator, through gray warning color to red end point. CALCULATION °G N[{3 ~ (Factor) x (Titration) Sample Weight ' Factor =(0.017) x (Normality of FICI) x (100) :..,~__.__.._ S 2 3 4 5 6 7 8 9 U 20 30 40 50 60 70 8 0 90 100 200 3U0 '~R_fi 3 4 5 6 7 8 9 10112 0; 3 0 40 '~0 60 70 -R0 90 1'00 200_~300 ~ ~ 1411 '~1 2 3 4^ 5 6 78 9 10~2(~ 30 40 50 60 70 f3090100 200 300 ~ ti T
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R. J. REYNOLDS TOBACCO COIMPANY RESEARCH DEPARTMENT Analytical Method No. 2009 DETERMINATION OF NITROGEN IN WATER SOLUBLE AND 4JATER'INSOLUE~L[:_F~:ACTIONS TF TOE3ACC0~ PRINCIPLE Tobacco sample is extracted with water and the nitrogen content determined by Y.jeldahl digestion and distillation. APPARATUS AND REAGENTS FILTER PAPER, S&S 604 KJELDAHL APPARATUS, see method No. 2003 REAGENTS SAME AS METIIOD N0. 2003 - PROCEDURE ~ ~ Accurately weigh 1.0lg dried and ground tobacco and transfer to - 150-ml beaker. Add 100 ml distilled water. Stir and let stand for one hour with occasional stirring. Filter through S&S 604 filter paper. Pipet 50 ml filtrate into Kjeldahl flask and determine total nitrogen using Method No. 2003. CALCULATION % Soluble Nitrogen = Where: V x N x 0. 014 x 100 rWt. of Sample-Cgj Volume of 0.1 N hydrochloric acid required N = Normality of acid % Insoluble Nitrogen = (Total Nitrogen) - (Soluble Nitrogen) % Protein = (6.25) (% Insoluble Nitrogen) ~S 1?_ 3 4 20 5 G 7 f 9 f 0` _ _ - 30 40 50 GO 70 £30 90 100 ?00 300 ~ RT 1 _ , .. _ . ~__ __ .._ _ _ 45 6 7_£3_9_ 1-U ?.U .3G; 40 5U_GO __70_80 9U 1 U0 200_300_ + ~ M11 1 2 5 6 7 £3 9 3 4 10000D 40 50 60 70 80 90 100 200 300
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R. J. REYNOLDS TOBACCO COMPANY RESEARCH DEPARTMENT Analytical Method No. 2010 DETERMINATION OF pEl OF TOQACCO' PRINCIPLE Tobacco is placed into beaker with water and the resulting pH of the mixture determined by the use of a pH meter. APPARATUS AND REAGENTS pH METER, Corning, Model 12, or equivalent, equipped with glass reference electrode and calomel'electrode BEAKER, 250 nil capacity PROCEDURE Weigh 5.0 g of tobacco into 250-ml beaker. Add 100 ml distilled water, stir well, and allow to stand for one hour. Standardize the pH meter using pH 7.0 buffer. Immerse the electrodes into the water and record the pH displayed by the meter. , - S~~ 0 3 4 5~0_7 8 9 1'U 20 30 40 50 60 70 8U 90 100 2.OU 300 ^ RT-F2 3-4-~5:% 6 I T9 (1) '20 30 40 50 60 %0'80 90 100 200 300 ~I1 ~ 345~678910200040506070 80"90100200_30M" ~
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R. J. REYNOLDS TOBACCO COMPANY RESEARCH DEPARTMENT Analytical Method No. 2011_ DETERMINATION OF MOISTURE IN TOBACCO PRINCIPLE Weighed samples of ground tobacco are dried in a forced-draft oven, cooled and reweighed. APPARATUS AND REAGENTS OVEN, forced-draft type; temperature maintained at 99-100° C. WEIGHING BOTTLES, 30 ml capacity with ground glass outside caps, .60 mm high and 30 mm inside diameter. PROCEDURE Adjust the oven to maintain a temperature of 99 to 100° C. Place the weighing bottles in the oven for one hour, remove to a desiccator, allow to cool and weigh. Transfer approximately 5 grams of finely ground tobacco into a tared weighing bottle, cap the bottle.and weigh accurately. Remove cap and place weighing bottle and cap in the forced-draft oven for 3 hours, remove the dried samples to a desiccator, allow to cool, cap the weighing bottles and again accurately weigh. Calculate the loss in weight as percent moisture. Percent Moisture = Loss in weight x 100 Wt. of Sampl e C0 ~;~T~~ ~o~'IG 8~6 Do 1ou zoo 300 `1~}1 -T7.`-3- ~ 7~~ST-~0-30 ~6 5U-~U /0 f30 90 -10U 200300 - -_.~__
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R. J. REYNOLDS TOBACCO COMPANY - RESEARCH DEPARTMENT Analytical Method No. 2012 DETERMINATION OF NICOTINE, NORNICOTINE, ~ AND TOTAL ALKALOIDS IN TOBACCO 4 PRINCIPLE The alkaloids are extracted from an alkaline mixture of the test sample with a benzene-chloroform solution. One aliquot of the extract is titrated with 0.05 N perchloric acid in glacial acetic acid, a second aliquot is first treated with acetic anhydride, then titrated with the perchloric acid. The total alkaloids, nicotine, and nornicotine contents are calculated from the two titration values. APPARATUS AND REAGENTS ---- ----- . ~ BURET, Miachlett automatic, 10 ml capacity, equipped with•Teflon stopcock. PIPET, automatic measuring with overflow, 25 nll capacity, Teflon stopcock. PIPET, Machlett automatic, 100 ml capacity. WRIST-ACTION SHAKING MACEITNE, Model BB, Burrell • Corporation, Pittsburgh, Pennsylvania. SUGAR TUBES, Nash, graduated. Kimble No. 47116. VORTEX, JR. MIXER, with foot pedal control. CRYSTAL VIOLET INDICATOR 0.5%. Dissolve 0.5 g crystal violet in 100 nil glacial acetic acid. PERCHLORIC ACID SOLUTION, 0.01 N. Prepare as described in Analytical Method No. ST-8. Standardize by dissolving 15-20 mg potassium acid phthalate;in 15 nil glacial acetic acid, heating gently to dissolve. Cool, add 1 drop of indicator, and titrate to a blue-green end point. Titrate a blank solution and correct the volume accordingly. Calculate normality as described in Analytical Method No. ST-8. ACETIC ACID ACETIC ANHYDRIDE BARIUM HYDROXIDE, Ba(OH)?•8H20, ACS grade. BARIUM HYDROXIDE SOLUTION, saturated aqueous solution. BENZENE-CHLOROFORM SOLUTION. Mix 900 ml of benzene with 100 ml off chloroform. CELITE, analytical filter aid, technical grade, Johns-Manville Corp. POTASSIUM ACID PHTHALATE, primary standard grade. PROCEDURE Accurately weigh 0.5 g tobacco into a 125 nil Erlenmeyer flask. Add 0.2-0.4 g solid E3a(OH)2•.8H70 and 5 mi saturated barium hydroxide solution. Swirl the flask until the tobacco is wetted, then pipet in 25 nil benzene- chloroform solution. Stopper tightly and shake 5 minutes on the shaking machine. Add 0.5-1.0 g filter aid and filter approximately 6-7 ml of the hydrocarbon phase through Whatman No. 2 paper, or equivalent, into Nash graduated test tubes.
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2 Analytical Method No. 2012 ] A. Determination of Total_Alkaloids Only Adjust the volume in the tube to the 5.Q ml mark by siphoning off the excess hydrocarbon solution. Add 1 drop of indicator and titrate in the tube with 0.01 N HC10t, to a green end point using the Vortex mixer. Run a reagent blank and correct the volume of titrant accordingly. B. Dete rmination of Nicotine and Nornicotine Content Filter portions of hydrocarbon phase into each of two graduated test tubes, and adjust the volunne in each tube to 5.0 ml by siphoning off excess hydrocarbon solution. Pass a strearn of air into one tube for 2 minutes; add 0.5 nil acetic anhydride "to the second tube. Add one drop of indicator to each tube and titrate to a green end point with 0.01 N HC104. Run reagent blanks and correct the volumes of titrant accordingly. CALCULATIONS % Total alkaloids, as nicotine = V x N x 40.56 x moisture correction ~ '- . Sample wt. in grams (2V2-V1) x N x 40.56 x moisture correction ~' Sanipl e wt. i n grams ~ 2(.V1-V~ x N x 37.05 x moisture co,rrect.ion _ ~ Sample wt. in grams Vi V2 Milliliters of 0.01 N perchloric acid required for non-acetylated aliquot. Milliliters of-0.01 N perchloric acid required for acetylated aliquot. N - Normality of 0.01 N perchloric acid. NOTES 1. Reagent blanks need only be determined when standardizing the titrant, and when any of the other reagents are changed. 2. If the nornicotine content exceeds 25% of the nicotine content, reanalyze using Analytical Method No. 2085. REFERENCE 1. Cundiff, R. H., RDM, 1963, No. 27 (MARCN 18). _^ S ~I 2 3 4~~6j1 8 9 10 20 30 40 50 60_7 0 80 90 10 200 300 T 6(11 8 9 10-2U 30 QO 50 60 _7_0~80 90-fUO 200_300 _ ~ PiH ' 1 2 3 4 5-G~7~8 9 ~'10 20 30 ~l0 50 EO 70 £~0 90 1 OU 200 300~
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R.. J. REYNOLDS TOBACCO COMPANY RESEARCH DEPARTMENT Analytical Method No. 2013 DETERMINATION OF SAND IN TOBACCO PRINCIPI.E The organic matter in the sample is destroyed by dry ashing; the ash is treated with hydrochloric acid to render the Si02 insoluble and alkali soluble silica is removed by treatment with sodium carbonate. solution. APPARATUS AND REAGENTS DISH, platinum, flat-bottomed. FURNACE, electric muffle, thermostatically controlled. FILTER PAPER, S&S 604. HYDROCHLORIC ACID, concentrated, ACS.grade. PROCEDURE Accurately weigh and ignite 2.5 g of sample in a tared flat-bottomed platinum dish in a muffle furnace, at a temperature of 600° C., until the ash is white or nearly so. (Platinum dishes must be used with caution in ashin lant materials high~in iron; for such ma~:erials use well glazed procelain dishes and run a blank determination.) Add cautiously 10 nil of concentrated HC1 - the dish should be partially covered with a watch glass to prevent loss by spattering, and evaporate on a steam bath to dryness. After evaporation is completed,.heat on steam bath for 3 hours. Moisten residue with 10 ml of concentrated HCI, add approximately 10 nil of distilled water and heat on steam bath 5 minutes. Filter through S&S 604 filter paper and wash with three small portions of dilute HC1 (1 volume of HC1 i 4 volumes of water), and finally with hot water about three times until free of chloride. Fold filter paper with residue, put folded paper in platinum dish, and then heat in muffle furnace at 600° C for three hours. Cool to room temperature in desiccator, weigh, confirm residue as sand by microscopic examination and calculate as percentage of sand on a moisture-free basis, as follows: Percent Sand (M-F basi s) = 4!eit of Sand x 100 Wt. of Sample ~~-F) ~
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2 Analytical Method No. 2013 REFFRC:NCE Methods of Analysis, A.O.A:C., 8th Ed., p. 99 (1955). , S 1 2 3 4 5 6 7 8 9 1 U 20 30 40 50 60 70 80 90 100 200_ 300 _l.~ !_ W-77-T45?i 7 8 9 1 U 2U 3U 40 5U 60 70_80 ~~~100 200 30U ~ _ _.~.. TfH--1 lTAi ~ 6 7 8 9 U) ~U 3U ~0 50 GO 7U ~U 90 100 2UU ~300
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R. J. REYNOLDS TOBACCO COMPANY RESEARCH DEPARTMENT Analytical Method No. 2014 DETERMINATION OF CHLORIDES IN TOBACCO PRINCIPLE Chlorides are extracted from tobacco by suspending in water, and titrated potentiometrically with standard silver nitrate. APPARATUS AND REAGENTS i ...~~. -..~~. .~__. j . pH METER, Precision-Sheel, Leads & Northrup or equivalent. SILVER ELECTRODE, Beckman No. 1261 GLASS ELECTRODE, Beckman -No. 1190-42. BURETTE, Machlett type, 10 ml capacity, fabricated from amber glass. SILVER NITRATE SOLUTION, 0.1 N. Standardized as described in Analytical Method No. ST-7. NITRIC ACID, dilute. Add one volume of concentrated nitric acid to nine volume of water. DISTILLED WATER. PROCEDURE Accurately weigh 2 g. of tobacco dust into a 250-m1, electrolytic beaker. Add 100 ml. of water, a small amount at first so that the tobacco is thoroughly wetted, then the remainder. Allow to stand at least five , minutes at room temperature, stirring intermittently. Pipet 5 nil. of the dilute nitric acid into the mixture and place the beaker in position under the clean electrodes. Start the stirrer and continue the stirring throughout the titration at a rate sufficient to produce vigorous agitation without spattering. Titrate potentiometrically with the standard silver nitrate solution to the potential previously established as the equivalence point. Read the burette and record the volume of titrant. CALCULATIONS °~ Chloride = V x N x 3.5457 "Sample ~~i;. = Volume of 0.1 N silver nitrate solution. = Exact normality of silver nitrate solution.
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2 NOTES Analytical Method No. 2014 . The equivalence point potential may be established graphically after making several potentiometric titrations on one or more tobacco samples. This value should be rechecked occasionally, and must be redetermined when either of the electrodes is replaced. Harrell (1), using glass and silver electrodes, found the equivalence point potential to be -105 millivolts, but pointed out the potential coUld vary with different electrodes and with different pH meters. Nelson (2), using a Fisher Titrimeter equipped with silver and silver-silver chloride electrodes, recommends an equivalence point potential of +560 millivolts. REFERENCES 1. Harrell, T. G., RDR, 1954, No. 10 (OCT. 6). 2. Nelson, R. A., J. ASSOC. OFFIC. AGR. CHEMISTS, 43, 518 (1960). S 1 2 3 4 5 6 7 8 9 1Y20 30 ~~0 50 60 70 80 90_100 200 300 nT!'-f ~'O~ F3 '9 10~2 U 3 40 5 0 00 7 0 8 0«U 100 2 00 3 00 ~ ----- _ 0 9 10 20 30 Ii 0 50 60 70 8 0 90 100 200 300 m F~ w
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R. J. REYNOLDS TOBACCO COMPANY RESEARCH DEPARTMENT - Analytical Method No. 2015 DETERMINATION OF SULFATE CON T ENT OF TOBACCO PRINCIPLE The organic matter in the sample is destroyed by dry ashing; the ash is treated with hydrochloric acid to put the sulfate into solution so that it can be separated from the ash by filtration. The sulfate is then precipitated using QaCI solution, and the amount of sulfate present determined gravimetrically from the E3a2S04 precipitate. APPARATUS AND REAGENTS DISH, Platinum, flat-bottom FURNACE, electric muffle, thermostatically controlled PRE-ASH FURNACE, electric, 3000 watt .FILTER PAPER, S&S No. 604 and S&S No. 576 HYDROCtiLORIC ACID, concentrated, ACS grade' BARIUM CHLORIDE, 10% solution PROCEDURE Accurately weigh 2-3 g of dried and ground sample into a tared, plat-bottom platinum dish and place into pre-ash furnace until sample stops smoking, then ash the sample in muffle furnace at 6000 C for 2 hours. Remove and place dish on steam bath. 'Treat sample cautiously with 10 ml concentrated HC1 and evaporate to dryness. Again add 10 ml concentrated HC1 and heat to near boiling on steam bath. Remove dish from steam bath and filter througb S&S 604 filter paper. Wash dish and residue twice with 1:4 HC1 : H20 and'twice with distilled H20 to catch about 100-150 ml of filtrate. Bring filtrate to boil and add drop-by-drop 15-ml of 10% BaCI solution while stirring. Let filtrate stand until it cools to room temperature. Pour mixture into S&S 576 filter paper and then wash beaker and residue with distilled water. Place filter paper and residue into platinum dish and ash in muffle furnace for 2 hours. Remove dish and cool in desiccator before weighing. _ CALCULATION °6 SO,, = Wt. of PPT X 41.15 Sample Wt. S 1 2 34 5 lG. 7 8-9 _10 20 30 40 5060 70 &U 90 100 20 300 ~ RT 1 0 3_4 5 008 ~9 10 20 30 40 50 _60 !0 80 -_90 ;'I t)0~ 200 300 ~ 30U ~~ 9 1 O~t- UO 30 40 ~50 60 70 £~0 901 00 200
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R. J. REYNOLDS TOBACCO COMPANY RESEARCH DEPARTMENT Analytical Method No. 2016 DETERHI1NATI0N OF STARCH IN TOBACCO PRINCIPLE Starch is separated from the tobacco by the formation of the soluble perchlorate, then by centrifuging to remove the insoluble solids. The amount of starch is determined by colorimetric investigation of the starch- iodine complex. APPARATUS AND REAGENTS CENTRIFUGE, capable of 15,000 RPM. SPECTROPHOTOMETER, Beckman Model B or equivalent HOTE WATER BATH, Magni-Whirl 1120A-1 or equivalent. METHANOL-WATER SOLVENT SYSTEM, dissolve 80 g NaCI in 250 ml distilled water. Add 750 rnl'~methanol. Let stand until clear, then filter to remove excess NaCI. PERCHLORIC ACID, 47.7%. Add 300 ml 70-72% perchloric acid to 224 nil di.sti l 1 ed water. KI-I SOLUTION, Dissolve 2.0 g of iodine in 20 ml of a solution containing 20 g of potassium iodide, then dilute to one liter TOBACCO STARCH STANDARD, See RDM, 1970, No. 6. PREPARATION OF STANDARD CURVE Weigh 0.1000 g(corrected for moisture) of tobacco starch standard into 100-ml volumetric flask. Add 10 nil 47.7% perchloric acid. Mix and let stand ten:minutes. Dilute to volume and pipet 1.0, 2.0, 3.0, 4.0, and 5.0 nil, respectively, into each of five 100-ml volumetric flasks, already containing 5.0 ml 47.7% perchloric acid, and make to volume with distilled water. This will yield 100, 200, 300, 400,,and 500 pg starch per 10 ml of solution. To 10 ml of each solution, add 0.5 ml KI-I.solution. Mix and measure absorbance at 600 nm, using 1.0 cm cell and 10 ml distilled water with 0.5 ml KI-I solution as reference. Plot absorbance at 600 nm versus ug starch on linear graph paper to form concentration curve. Use this curve to determine ti,g starch in the tobacco sample. I PROCEDURE . I ~ ~ Accurately weigh a catchweight of about 0.4000 g iof tobacco ground to 60 mesh or lessI into a glass centrifuge tube with ~crew cap. Add 25 nil of inethanol-water solvent system. Miix and heat in 70° C water bath for ten minutes with cap lightly in place, then cool to room temperature in
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2 Analytical Method No. 2016 centrifuge at 15,000 rpm for 15 minutes. ice bath. Centrifuge at 2000 rpm for 10 minutes, then decant and discard liquid phase. Add 10.0 ml 47.7% perchloric acid to the residue, mix and let stand for ten minutes. Add 10.0 ml distilled water, mix, and in sample from concentration curve. Pi pet 1.0 Illl of supernatant i nto each of two tubes, A and B. Add 9.5 ml distilled water to tube A, mix and measure absorbance at 600 nm, using 1 cm cell, and distilled water as reference. To tube B, add 9.0 ml distilled water and 0.5 ml KI--I solution, mix, and measure absorbance as before, using a solution containing 10.0 ml distilled water and 0.5 ml KI-I solution as reference. Use the net absorbance to determine pg starch CALCULATION Net Absorbance =(Absorbance, Tube B) - (Absorbance, Tube A) °6 Starch = (ug Starch from Curve)(2) Tobacco I~e pht (mj~ NOTE: If absorbance of tube B is greater than 0.750, dilute 5.0 ml of supernatant to 25 ml with distilled water. Pipet 1.0 ml of diluted solution into tubas A and B and repeat the analysis. % Starch = (uq Starch from Curve)(10) -Tobacc'o Wei ght mg ) REFERENCE 1. Sensabaugh, A. J., and Rush, K. L., RDM, 1970, No. 6 (JAN. 1970) (. 5~~1^2 3 4 5 7 0 9 10 ' 0 30 40 50 60 70 80 90 100 2.00 300 Rl~ 1 3~4 5 6 7 8 9 10-VU>30'~+0 50 60"70 £~0 ~i0 100 20~~300~' 10~~' ~~ ~~ _~6~6~~b_f10 'zi(5'100~?UO 300 _T 1-111 ~ 1' 2' i~4 5 6 7£3 9.
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R. J. REYNOLDS TOBACCO COMPANY RESEARCH DEPARTMENT Analytical Method No. 2017 DETERMINING TOTAL ASH IN TOBACCO PRINCIPLE A weighed quantity of tobacco is ignited in an electric muffle furnace at 600° C until ashing is complete. APPARATUS AND REAGENTS PRE-ASHER, electrically heated, 3000 watt FURNACE, electric muffle, ther;-,~Dstatically controlled. DISH, glazed porcelain, 85- mrn diameter and 90 nil capacity. PROCEDURE i - ~ Place approximately12.5 grams of tobacco in a tared porcelain dish and determine its net weight. Heat in pre-asher until sample stops smoking, taking care that the tobacco does not flame. Ash in an electric. muffle furnace at 600° C for 2 hours, or until ashing is complete. If ashing is not complete, as indicated by the presence of black particles of unburned carbon, remove the sample from the furnace, allow to cool, disintegrate the ash with a glass stirring rod, and ignite for another hour. Cool in a desiccator to room temperature, determine weight of the ash and calculate as the percent of total ash on a moisture-free (M-F) basis as follows: Percent Total Ash (M-F basis) = Weicht_of Ash x 100 Wt. of Sample M-F REFERENCE 1. METHODS OF ANALYSIS, A.O.A.C.; 6th Ed., p. 405 (1945). "S 1'? 3~4-~DT6-7 8 9 1U 20 39 40 50 60 70 80 90 100 200 300 i4 5T6 7 0 9 10 0 20 :~q0 50 60 70 f30 90 100 l0U' 3U0 ~ t~1H 1'2 3 4 _5 6-_ 8 9 10 2.0 50 ~IO 50 60 70 80 ;0 100 200 300 7 ~
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c. R. J. REYNOLDS TOBACCO COMPANY RESEARCH DEPARTMENT Analytical Method No. 2020 DETERI•1INATION OF ALCOHOL SOLUBLE MATERIAL IN TOBACCO - PRINCIPLE A sample of tobacco is placed iri a extraction thimble and the alcohol soluble material removed by soxhlet extraction.. The solvent is evaporated and the residue determined gravimetrically. APPARATUS AND REAGENTS SOXHLET EXTRACTION, Assembly. THIMBLE, extraction, Alumdum, 80 x 25 mm. OVEN, electric, forced draft, thermostatically controlled. ETHANOL SOLUTION, 71 parts absolute.ethanol and 4 parts distilled water. PROCEDURE Accurately weigh 5.0 g of dry and ground tobacco into extraction thimble and insert cotton plug into mouth of thimble, covering tobacco. Obtain tare weight on 125-m1 extraction flask containing 3-4 boiling chips. Place thimble and flask into Soxhlet extraction apparatus, add 75 nil of ethanol solution, and adjust heater to a setting of 80. Allow extraction to proceed for 24 hours. Remove flask from Soxhlet assembly Evaporate solvent by using heater until large bubbles appear. Place flask into oven set at 103° C. for 2 hours. Remove flask and cool in desiccator. Weigh flask and residue. CALCULATION % Alcohol Soluble Material ~ (W2 4J)_) (100) 3 Where: W1. = Weight of flask and chips. ._- W? = Weight of flask, chips, and residue. W3 = Weight of sample. ~ 6 5~ 6 b-`I ~0 g 0~~0 ~ 300 S~-T 2 '3 0 2 0 3 ~`1 2 3 4 5 6 7 8 9 10 2.0 30-~10 ~~ j8~0 90 100(200)300 .......__..____--~.~-._...--_~._. ..._.~._.~...._.-.._..a-. _.._.-..~..__..c. ... __ __ _-__ _._.a~, .....~.-___" _ ~ I~ll I~ 2 3 4 5 6 7 8 9~l020 30 ~10 5U 60 I~ c30 9U' 100 1.U0 i00 ~ T
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R. J. REYNOLDS TOBACCO COMPANY RESEARCH DEPARTMENT Analytical Method No. 2021 DETERMINATION OF WATER SOLUBLE MATERIAL IN TOBACCO PRINCIPLE Extract the water soluble materials from tobacco by mixing with warm water, filtering out the insolubles, and gravimetrically determine the residue after evaporation of the solvent. APPARATUS AND REAGENTS OVEN, electric, forced draft, thermostatically controlled. STEAM BATH, low pressure steam. DISH, glazed porcelain, 100 mm, 125 nil capacity. FILTER PAPER, S&S 604 ~ FLASK, Erlenmeyer, 2000 ml. PROCEDURE Accurately weigh 10.0 grams of dried and ground sample. Place in 2000 ml Erlenmeyer flask and add 1000 ml luke-warm (120° F) water. Swirl until sample becomes completely wetted. Let solution stand for one hour, swirling occasionally. Filter mixture through S&S 604 filter paper. Pipet 100 ml of filtrate into tared porcelain dish, and place on steam bath until all visible solvent has been evaporated. Place dish and residue into oven set at 103° C for twelve hours, then cool in desiccator' and weigh. CALCULATION % Water Soluble Material - ({l~I-1)(10) W3 where: W1 = Tared weight of dish (g) W2 = Weight of dish and residue (g) W3 = Weight of sample (g) S ~ 34 5 8 9 1 ~0 30~0 50 f 0 70 9-0 90~. QU 2U0 300 ~ _ RT 1 1 3_4_ 5 67 8 9 1~20 30' 40 ~50~G(~` 70 80 90 (1 UU? 200 300 ~1FI-1~2 3 4 5 6 7£3 9~1(l 2~ 4U 5 70 80 90 100 2.00 300 ~ ~
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R. J. REYNOLDS TOBACCO COMPANY RESEARCH DEPARTMENT - Analytical Method No. 2021-E3 DETER_MINATION OF WATER SOLUBLE MATERIAL IN TOl3ACC0, AE3E;REVIATCfROCLDTRE ' r PRINCIPLE Extract the water soluble materials from tobacco by mixing with warm water, filtering out the insolubles, and gravimetrically determine the residue after evaporation of the solvent. APPARATUS AND REAGENTS OVEN, electric, forced draft, thermostatically controlled STEAM BATH, 1 ow pressure steam. SHAKER, E3urrel l Wri st-Act-ion DISH, glazed procelain, 100 mm, 125-=ml capacity. FILTER PAPER, S&S 604 FLASK, Erlenmeyer, 2000 ml. PROCEDURE Accurately weigh 1.0 grams of dried and ground sample. Place in 250 ml Erlenmeyer flask and add 100 ml warm (450 C) water. Swirl until sample becomes completely wetted. Shake on wrist-action shaker for 10 minutes with a shaker setting of 5. Filter mixture while hot through S&S 604 filter paper. Let cool to room temperature and pipet 20 ml of filtrate into tared procelain dish, and place on steam bath until all visible solvent has been evaporated. Place dish and residue into oven set at 103°;C for one hour, then cool in desiccator and weigh. CALCULATION °6 Water Soluble Material ~ 1W_z° W 30-(500) W3 where: W1 = Tared weight of dish (g) W1 = Weight of dish and residue (g) W3 = Weight of sample (g) y--_-_ __ -f-------- - RT ~~1 2 3 4 5 6 J 8 9 ~?0 (30; 40 50----60_T0- 80 90~ 1()0 L(10 300 _ M i TY3T-5 G774~( ~J2~ 0`10~ 0 a U G tl-~ 0~() 9 U TO ~~ GU ~~ 6U IU) 20 30 40 50 (00 7bb0 90 fbbZ00Ju0 S 4.~> b 8 9 ~
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R. J. REYNOLDS TOBACCO COMPANY RESEARCH DEPARTMENT Analytical Method No. 2022 UANTITATIVE DETERMINATION OF RE:SIDUAL *FREON FLUOROCARf30Nj 11 IN EXPANf)Ef) TO[3ACC0 PRINCIPLE Residual Fluorocarbon 11 in expanded tobacco is distilled from an aqueous suspension of the tobacco and trapped in hexane. The hexane- Fluorocarbon solution is analyzed by gas chromatography employing an electron capture detector. APPARATUS AND REAGENTS GAS CHROMATOGRAPH, Elarber-Colman, Model 5000, Selecta-System or equivalent with electron capture detector. CARRIER GAS - Nitrogen. FLASK, boiling, 500 nil, 1 24/40 joint. HEATING MANTLE, Glas-Col brand or equivalent, for 500-m1 flask. STOPPER, hollow, glass, 1 24/40. BOILING STONES TUBE, connecting, distilling, 1 24/40 joints, with arms at 75° and 105° (Sci.entific Glass Apparatus Co., JA 4830). CONDENSER, West 300-mm, ~f 24/40 joints. ADAPTER TUBE (Corning Glass 8945 Connecting Tube), modified by a glassblower to eliminate the 105° angle and give a vertical (180°) configuration. BOTTLE, Gas Washing, Tall Form, 500-ml, T 24/40 neck (Ace Glass Inc. 5516-34). ICE BATH, efther a pl asti c pail or 4-1 i ter beaker. HEXANE, free of interfering impurities. EMPTY GELATIN CAPSULES, size 11 (1/2-ounce), Michigan Capsule Co., Detroit, Michigan. EMPTY GELATIN CAPSULES, size 000. STANDARD SOLUTIO14S - Approximately 80 mg Fluorocarbon 11 per 25 ml of hexane. Example - 0.0714 g Fluorocarbon 11 per 25 ml hexane Step 1- 71.4 mg - 2.9 mg per nil 25 ml Step 2 - Dilute Stop 1 - 1:100 0.029 nig per ml or 29 v,g per nil
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2 Step 3- Dilute Step 2- 1:100 0.29 g/ml or 290 NG/ml or 290 PG/ul Step 4- Dilute Step 3 (a) 1:10 (b) 2:10 (c) 3:10 Standard (a ) 29 NIG/ml or 29 PG/I,1 .(b ) 58 NG/ml or 58 PG/ul (c) 87 NG/ml or-£37 PG/pl (_1 PROCE[)URE The gas chromatographic instrument conditions are as follows: Column - 5' X 1/B" SS, Porapak Q(50/t30) Injector Port Temperature - 200° C Detecto r Bath Temperature - 23J° C Column Bath Temperature - 150° C isothermal Carrier Gas - Nitrogen Flow Rate - 100 ml per minute Put boiling stones and 200 ml of water into the 500-ml glask. Add 10 ml of hexane. Aft~r a few seconds, close the flask with the 24/40 stopper. According1to the expected Fluorocarbon content, use a sample si.ze as follows: Under 100 ppm - 1 to 4 grams or l to 4 cigarettes 100 to 20,000 ppm - 0.2 to 1 gram Over 20,000 ppm - less than 0.2 gram Weigh the sample of tobacco or the cigarettes to the nearest milligram. Lift the stopper of the prepared flask, drop in the weighed sample, and replace the stopper. Some batches'of tobacco are losing Fluorocarbon at a rapid rate. This is true of tobacco that is warm, or moist, or has just been processed. These types of tobacco can be closed in a gelatin capsule to slow the escape of Fluorocarbon. For samples under 10,000 ppm, use a'size 11 capsule. Weigh the empty capsule and write an identifying number on it with a felt-tipped pen. Put the tobacco into the capsule, weigh the encapsulated sample and drop it into the prepared flask. The capsule dissolves. - ., For samples over 10,000 ppm, use both a small (size 000) and a large (size 11) capsule. Obtain a tare weight on the!two capsules together. Put the tobacco sample into the small capsule, and quickly close the small capsule into the large capsule. Weigh the sample and drop it into the prepared flask as soon as possible. ~
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3 4 Put the gas washing bottle in the plastic pail and surround with ice. Add measured quantity of hexane to the bottle. Use 100-ml or 250-ml depending on Fluorocarbon content of sample. Insert adapter tube into bottle. The end of the tube should dip into the hexane. Connect lower end of condenser to top of adapter tube. Connect 105° arm of connecting tube to top of condenser. Place flask with sample on heating mantle. Remove stopper from flask°and insert 75° arm of connecting tube. Caution: Make sure all joints of the distillation system are.tight:. Onneci; heating mantle (100 to 110 volts) to start di:stillat''ton. Distill for 45 minutes to 1 hour, or until about half of the water has distilled into the gas washing bottle. Decant hexane contai.ning the Fluorocarbon 11 from the water layer. Dilute with hexane, if necessary. Inject 2-ul into the gas chromatographic column. Construction of Standard Cu rye Shoot 2-ul shots of standards a, b and c and plot on a graph. Plot peak height vs. concentration of standards. 0 Calculations NOTE: Due to high concentration, the sample may he diluted. Example: Sample weight 0.752 grams. . Distillate diluted 1:100 Read the sample results from the standard curve and calculate. Sample reading i.s 80 NG/mI on the curve. 801 100 = 8000 NG/ml or 8.Oug/ml 260 ml'(vol. of hexane) X 8.0 = 2080 ug 2080 0.752 (sample weight) = 2766 ppm Fluorocarbon The fluorocarbon result represents the amount of Fluorocarbon 11 in the tobacco at the time it was dropped into the flask. If a capsule was used, the result represents the quantity of Fluorocarbon in the tobacco at the time it was put into the capsule. -'S ~'1-2 3 ~5'~ T-0 ~ 1Z) 20 30' 40 50 60 70 80 90 100?00 300 ___RT_~T23 4 5 ~ ff~Sl (~~6i~+0~0 60'J0 ~30 90 171TI-?00,1~0 ~"M}1i~2--S-4 `~5 ~~T F3 9 7~fT2030~U~U ~U 70f30 ~ OrO~ 20 a~0--_ ~
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R. J. REYNOLDS TOBACCO COMPANY RESEARCH DEPARTMENT Analytical Method No. 2025 DETER1,fINATION OF CITRIC, MALIC, AND OXALIC ACIDS IN_TOQACCO PRINCIPLE A dry sample is simultaneously extracted and esterified with 10% sulfuric acid in absolute methanol. The methyl esters formed from this reaction are extracted into chloroform and determined gas chromatographically. APPARATUS AND REAGENTS GAS CHROMATOGRAPfI, Hewlett-Packard, Model No. 7620, with FID. SHAKER, E3urrel 1 t-!ri st--Acti on FLASK, Erlernneyer, 125-ml capacity FUNNEL, separatory, 125-ml capaci ty ,, EXTRACTION SOLUTION,'10% H?SO4, 600 nil of absolute methanol is put into a dry one-•lit~r volumetric flask containing a magnetic stirring bar. The flask and contents are placed in an ice bath over a magnistir. One hundred ml of concentrated sulfuric acid are added slowly while mixing. 'The solution is cooled and diluted to volume with absolute methanol. CHLOROFORM, reagent grade. STANDARD ACID SOLUTION (STOCK), 5.000 g dl-malic and oxalic acid dihydrate, and 2.7346 g of citric acid monohydrate, all of reagent quality, are accurately weighed into a dry 100-ml volumetric flask, and made to volume with extraction solution. This solution is prepared at the same time the sample analyses are begun. SODIUM SULFATE, anhydrous. Gas Chromatograph Conditions Column •• 4' X 1/4" copper tubing packed with 2.5% Carbowax 20M-TPA on Chromosorb G-AW (70/80 mesh ) Injection Port temperature, 230° C Detector Temperature, 250° C Carrier Gas Flow Rate, 30 ml/min. Oven Temperature, Isothermally, Oxalic acid, 120° C, Citric and Malic acids, 200° C " i PROCEDURE I 1. Preparation of Standard Acid Curves i Aliquots of 1, 2, 3, and 4 ml of the stock standard acid solution were transferred to dry 50-ml volumetric flasks. The flasks were diluted to ~
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4 0 Analytical Method No. 2025 volume with the extraction and esterification solution, mixed, and allowed to stand overnight at room temperature. A 25-rnl aliquot of the standard solution was transferred to a 125-mi separatory funnel and 50 nil of distilled water was added. The separatory funnel's contents were mixed by swirling, and extraction of the methyl esters was effected by four succesive extractions with 10-m1 portions of chloroform. The chloroform extracts were (trained through a cotton plug into a50-ml volumetric flask and diluted to volume with chloroform. Residual water was removed by shaking with about 0.5 g of anhydrous sodium sulfate. These standard solutions were equivalent to 0.5, 1.0, 1.5 and 2.0 mg/ml malic and oxalic acids and 0.25, 0.50, 0.75 and 1.0 mg/ml citric acid. Triplicate 4 U1 injections were made into the gas chromatograph. The responses in peak heights (mm) plotted versus mg/mi concentrations for the individual acids. Linear plots are obtained. 2. Saq)le Preparation Solid samples were ground to pass a 20-mesh sieve and were dried three hours at 110° C in a forced draft oven. Semi-solid samples were diced, dried overnight at 110° C;, ground to pass, a 20 mesh sieve and then dried an additional three hours;at 1100 C. Liquid samples were evaporated to near dryness on a hot pla:te and then dried three hours at 1100 C. 3 ~ Analysis A sample containing from 25-200 mg of each acid (usually about two grams in the case of tobacco) was weighed into a 125 ml Erlenmeyer flask. Fifty millilite rs of the extraction and esterification solution were added by pipet, the flask stoppered and shaken for thirty minutes on a mechanical shaker and then allowed to stand overnight at room temperature. Solids were removed,by filtering through glass w-ool and a 25-m1 aliquot was transferred to a 12.5-m1 separatory funnel. At this stage the sample was treated as in the preparation of the standard curve from which the individual acids were determined in mg/ml. CALCULATION The percentage of each acid was calculated as follows: % acid = m9/ml acid'X 100 Final sample concentral,lon in 6g/m Citric acid was calculated as anhydrous citric acid and oxalic acid was calculated as the dihydrate. - '; REFERENCES 1. Harvey, W. R., Hale, R. W., and Ikeda, R. M., The Determination of Oranic Acids in Plants and Food Products,;TOBACC OCO SCI., l~ T19/0j. S ~1 -34-7i G / F3_970 ?0-30 4() 50 GC %0 ~ _~~ ~0~ 20 'TUO _ .~ -- ----- - R1 '10_60 60 70 U 90 l0 10-2 00431J0 ~ ~tFH ~~ ^l 3 4 5^ 6 7 8 9 10 20 30 0 5U (T/0 8b,)-6,0 100 lUU 300 _~
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R. J. REYNOLDS TOBACCO COMPANY RESEARCH DEPARTMENT Analytical Method No. 2026 DDETERMINATION OF GLYCERINE,. PROPYL_ENE GLYCOL, AND TRIETHYLENE GLYCOL^ON CASED TOBACCO BY GAS CHROMATOGRAPHY PRINCIPLE Sample is extracted with methanol to isolate the humectants on cased tobacco. The quantities of the individual hinnE!ctants present is determined by gas chromatography using anethole as an internal standard. APPARATUS AND REAGENTS GAS CHROMATOGRAPH, F;&M Model 720, equipped with programmed temperature oven and FID. I FLASK, Erlenmeyer, 250-m1 capacity.'--- SHAKER, Burrell Ulris:t-Action. METHANOL, anhydrous,! reagent grade. ANETHOLE STOCK STANDARD. Accurately weigh 20.00 g of U.S.P. anethole into a 100-ml volumetric flask and dilute to volume with anhydrous methanol. EXTRACTING SOLUTION. Dilute 10-ml of the anethole stock standard volumetrically to one liter with anhydrous methanol. PROPYLENE GLYCOL STOCK STANDARD. Accurately weigh 5.00 g of U.S.P. propylene glycol into a 100-ml volumetric flask and dilute to volume with extracting solution. GLYCERINE STOCK STANDARD. Accurately weigh 10.00 g of U.S.P. glycerine into a 100-ml volumetric flask and dilute to volume with extracting solution.` TRIETHYLENE GLYCOL STOCK STANDARD. Accurately weigh 5.00 g of triethylene glycol into a 100-mi volumetric flask and dilute to volume with extracting solution. HUMECTANT STANDARD SOLUTIONS. Into each of four 100-ml volumetric flasks, pipet 1.0, 2.0, 3.0 and 4.0 nil respectively of the propylene glycol, glycerine and triethylene glycol stock standard solutions. Dilute each solution to volume with extracting solution. Each solution.will contain the following: Soln. V ycol Propylene G1 Glycerine Trieth~Ilene Glycol 1 - 50 mg./100 ml. 100 mg./100 nil 50 mg./l00 ml. 2 100 200 100 3 150 300 150 4 200 400 200 L7
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Analytical Method No. 2026 Gas Chromatogra~ph Conditions CHROMATOGRAPHJC COLUMN, 42 in. x 3/16 in. copper tubing packed with 5% Carbowax 20M-terephthalic acid on 60-80 mesh 1:1 Diatoport S- (Hewlett-Packa.rd, Inc.) - Chromosorb 101 (Applied Science Lab., Inc.). To prepare the column packing, place a mixture of 15.0 g of Diatoport S and 15.0 g of Chromosorb 101 in a 500-ml round-bottom flask equipped with aI joint. Add a solution of 1.50 g of Carbowax 20M-TPA in 150-m1 of chloroform and slurry. Remove the chloroform under reduced pressure using a rotary evaporator and allow the column packing to air dry overnight at room temperature. Condition a new column for two hours at 240° C. before using (Note 3). Helium Flow: 60 ml/min. (Note 1) Attenuation: Cha rt Speed: 4 (Note 2) 6 in./hr. ~. Detector Bridge: 140 ma. Injection Temperature: 265° C. Detector Temperature: 280° C. Column Temperature: 90-240° C. @ 15°/min. PROCEDURE Accurately ~reigh 10.0 g of cigarette tobacco, or 5.0 g smoking or chewing tobacco, or 2.5 g G-10 or G-11, into a 250-m1 I Erlenmeyer flask. Pipet 100 ml of extracting solution into the flask and stopper. Shake on a shaking machine for one hour. Allow to settle for a few minutes until the supernatant is clear (Note 4). Prime the instrument before,starting the run (Note 3). . Inject a 30-u1 sample of the supernatant methanol extract into the chromatograph and obtain the chromatogram. Then inject 30 ul of a humectant standard solution into the chromatograph and obtain its chromatogram. Continue the analyses, alternately injecting extracts and standard solutions. Determine the peak heights of propylene glycol, anethole, glycerine and triethylene glycol from each chromatogram. Calculate the peak height ratios of propylene glycol:anethole, glycerine:anethole and triethylene_glycol: anethole for each standard and sample. Plot the peak height ratios vs. polyol concentrations for the standard solutions and construct a standard curve for each humectant. Determine the concentration in mg/lOO ml of propylene glycol, glycerine and triethylene glycol in the unknown solutions from the respective standard curves. CALCULATION % Propylene Glycol = mg/lOO ml (from std. curve) X 0.01 % Glycerine = mg/lOO ml (from std. curve) X 0.01 % Triethylene Glycol = mg/lOO ml (from std. curve) X 0.01 ~
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3 Analytical Method No. 2026 NOTES 1. The helium flow may be altered slightly if necessary to facilitate separation. 2. Attenuation may be varied according to the sensitivity of the individual chromatograph. Selection should be made so as to yield peak heights of sufficient size for accurate measurement. The same attenuation should be used for all standards and samples. 3. It is recommended that. a new column also be "primed" by injecting three successive 30--ul samples of a tobacco extract in the same manner used for sample analyses. This procedure ordinarily results in an improved baseline for the chromatograms. 4. Shaking for 30 min. is sufficient if the sample is to stand overnight before chromatography. S y1 _ 2 3 4,; 6 7£3~0 10 _0 30 40 50 60 70 80 9U 100 2 00^ 3U ~. Rl~~ 0 2 3 4~ 5 i G 1 7 8 ; 0 0 20-~0' ::7 60 10; ~0 9U 100 200 300 _____ . . . .--~ ..M _-_------- ---._------- ~~- IiI1 1 2 3 4 5 6 7£~ 9 10 20 30 40(bW60 70 80 90 100 200 300 ~
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R. J. REYNOLDS TOBACCO COMPANY - RESEARCH DEPARTMENT Analytical Method No. 2030 DETERMINATION OF STATIC BURNING RATE OF CIGARETTES - PRINCIPLE A fixed length of cigarette rod is allowed to burn without puffs being taken and the time required recorded. APPARATUS AND REAGENTS BURNING RATE APPARATUS, RJR 778121 PROCEDURE Weigh 4 cigarettes, filters and all,(see NOTE) and place them on the burning rate apparatus. String each cigarette to place 47 mm between the'two strings; the first string to turn the tinier on and the second to turn the timer off. Turn the apparatus on, light the cigarettes by use of the vacuum, then turn the vacuum off after the cigarettes are lit. When the cigarettes have finished burning, read timeys individually and record seconds of burning. Take the length (mm) of the cigarettes used and compute the factor for that length. Multiply the weight of the four cigarettes by this factor and divide by the total observed burning time for the four cigarettes. Report the values as milligrams of tobacco burned per minutes. Average the burning time of 4 cigarettes to obtain minutes per cigarette burning time. CALCULATION Factor - 1000 x 60 x 47 Lenyi:Fi of Ci gar ette mm Mg Tobacco Qurned/min ='Weight x Factor Burning ~firme Minutes/Cigarette - Sum of Times for 4 Cigarettes in Seconds NOTE If the filter is not cellulose acetate filter (i.e., charcoal, plastic, etc.), treat as follows: ~
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lip Remove the filter from the cigarette and measure the length of the tobacco rod. Obtain the weight of tobacco in 2 cigarettes, and multiply by 2 to arrive at the weight of tobacco in 4 cigarettes. Use this weight as the weight of the cigarettes and the length of the tobacco rod as the length to determine the factor. The remainder of the procedure is the same as for regular cigarettes. Analytical Method No. 2030 ~l 8 9 ~10 20 Tb -WTjU71071F 00"?~630o 00 300 u ~R'1'~~i 9 l~) ~b 4Q ~030~7b f36 y0~'I60 ~6U 30U ~~-' ~- ~' -- f~Cl~ 84 5 6 7£3 9 10 200 9 f 2 30 050 60 7 00)0 100 200 300
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R. J. REYNOLDS TOBACCO COMPANY RESEARCH DEPARTMENT Analytical Method No. 2033 DETERMINATION OF ACID-DETERGENT FIBER, ASH, CELLULOSE, AND LIGNIN PRINCIPLE The sample is extracted step-wise with various solutions to remove each desired product in turn. The amounts of each material removed is determined gravimetrically. APPARATUS AND REAGENTS FURNACE, muffle, electrically heated, thermostatically controlled. OVEN, force draft, electrically heated, thermostatically controlled. CRUCIf3LES, sintered glass, 50 nil, Kimax 40 M. CONDENSER, reflux, fitted with 250-rn1 flat bottom flask. ACID-DETERGENT SOLUT!ION. Add 20 g cetyl trimethylammonium bromide to l liter of 1 N sulfuric acid. Mix until dissolved. SATURATED POTASSIUM PERMANGANATE. Dissolve 50 g reagent grade KMnOL, i n.1 1 i ter di sti 1 l ed water. Keep out of di rect sunl i ght. BUFFER SOLUTION. Dissolve 6.0 g Fe(N03)3•9H2O reagent and 0.15 g AgNO3 i n 100 nil di sti 11 ed water. Combi ne wi th 500 ml gl aci al acetic acid and 5.0 g potassium acetate. Add 500 nil tertiary butyl alcohol and mix. COMBINED PERMANGANATE SOLUTION. Combine and mix 2 parts saturated potassium permanganate and 1 part buffer solution, v/v, before use. DEMINERALIZING SOLUTION. Dissolve 50 g oxalic.acid dihydrate in 700 nil 95% ethanol (3A). Add 50 ml 12 N HC1 and 250 ml distilled water. Mix. ETHANOL. About 80% - mix 200 nil distilled water and 800 m1 95% ethanol (3A).. PROCEDURE Weigh 1 g ground, dry tobacco into a 250 ml reflux flask. Add 100 nil cold (room temp.) acid-detergent solution and 2 ml decalin. Heat to boiling in 5-10 min; reduce heat to avoid foaming as boiling begins. Reflux 60 min. from start of boiling. I While still hot, filter through previously weighed and dried sintered gl ass cruci bl es . Wash wi th hot, water. Wash r•ti th acetone unti 1 no more color is removed. Dry at 1000 C eight hours or overnight. Cool in a dessicator over phosphorus pentoxide and weigh. i
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Analytical Method No. 2033 Take crucibles with samples from acid-detergent fiber step. Fill with combined permanganate solution and mix with a short glass stirring rod. Place crucibles in a shallow baking dish containing enough distilled water to be level with frit. Allow to stand for 60 minutes. A brown color indicates exhaused permanganate solution. Add more if necessary. Filter under vacuum and add more permanganate. Allow to stand i-o r 30 minutes. Filter, place in a clean baking dish and fill approximately one--half full with deminera.lizing solution. After 5 minutes, filter. Repeat addition of demineralizing solution. Let stand 20 to 30 minutes. reported as ash. 1-1 Fi l ter and wash cruci bl e and contents wi th 80% ethanol three ti mes . Suck dry and wash twice with acetone. Dry overnight @ 100° C and weigh. The loss in weight from acid- detergent fiber is reported as lignin. Ash @ 500° C for three hours, cool, and weigh.' Calculate cellulose as the loss in organic matter upon ashing. The amount of residue is CALCULATIONS % Acid-Detergent Fiber = w3 4J2 wl % Lignin = w4 - w3 wi % Cellulose, = w5 - w4 wl % Ash = w5 - w2 w 1 Where: wl = weight of sample w2 ~ weight of sintered glass crucible w3 = weight of dry crucible and residue after refluxing u ~
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3 w5 - Analytical Method No. 2033 weight of dry crucible and residue after permanganate treatment and acetone washing weight of dry crucible and residue after ashing at 500° C. S 1 2 3 4 5Q7 8 9 10 20 30 40 50 60 70 80 190 100 200 300 - ftT 1 2 34 5 6 7 10 20 30 1 o 750 ;60 70 30 90 1~0O: 2 0 0 (306 8 9 ~ l~ 2 3 4 5'yG ST IIN _ _ ~,.~.~ _ ,B_9 10 2030 _ iU 5() b0 70 8 0 90_ 1UO 200 300^ ~_ w
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R. J. REYNOLDS TOBACCO COMPANY RESEARCH DEPARTMENT Analytical Method No. 2036 DETERMINATION OF PECTIN IN TOBACCO r PRINCIPLE Pectin is isolated from tobacco using ammonium oxalate-oxalic acid solution with reflux followed by ethanol precipitation. The isolated pectin is converted to galacturonic acid, which is determined colorimetrically by carbozole complex. APPARATUS AND REAGENTS HEATING MANTLE, Labline, Inc. MAGNETIC STIRRER-HOT PLATE, Gyratherm IIa, Will Scientific. f3UCl-INER FUNNEL, 9 cm. diameter. FILTER PAPER, Carl Schleicher & Schuell Co., No. 589, 9 cm., red ribbon. SPECTROPHOTOMETER, Cary 14, Varian. TEST TUBES, 19 x 150 mm with stoppers. AMMONIIJM OXALATE-OXALIC ACID SOLUTION, Mallinckrodt, reagent grade. 0.25% each (w/v). Weigh 2.5 grams of each into a one-liter volumetric flask. Bring to volume with disti'lled water. ETHANOL, U.S.I., absolute. ETHANOL, 70% (v/v) SOLUTION. Add 700 ml of absolute eth.anol to 300 ml of distilled water. PECTIN (POLYGALACTURONIC ACID METHYL ESTER), Sigma Chemical Company, Grade I. SODIUM HYDROXIDE, Mallinckrodt, reagent grade, pellets. PECTIN (DE-ESTERIFIED). Weigh 2 grams of pectin. Place the compound in a 500 Ml beaker containing 300 ml of sodium hydroxide solution (pH 11.5). Stir vigorously for one hour. After mixing, add 50 ml of concentrated sulfuric acid and mix thoroughly. Filter the substance, and wash the precipitate with four 25-m1 portions each of water, methanol and ether. Allow the material to air dry. Then store the substance in a closed container. PECTINASE, Sigma Chemical Company, purified from Aspergillus Niger. This material should be purified by dissolving 0.15 gram in 80 ml of ethanol 3A. After stirring for 1/2 hour, add 70 ml of ethanol.. Filter the solution and store the filtrate in the dark. CARBAZOLE SOLUTION, 0.15% (w/v), Eastman Organic Chemicals, reagent grade. [)issolve 0.15 gram of carbazole in 100 ml of ethanol 3A. Solution is slow and stirring is required. The solution should be stored away from light. When the solution turns blue, it should be discarded. SULFURIC ACID, Fisher Scientific Company, reagent A.C.S. METHANOL, Mallinckrodt, reagent grade, re-distilled. ETHYL ETHER, Mallinckrodt, analytical reagent.
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2 'i Analytical Method No. 2036 PROCEDURE l. Preparation of Sample Weigh 0.5 g of dry and ground tobacco into a 250-mi flat bottom flask. Add 100 nil of ammonium oxalate-oxalic acid solution and refiux for one hour. Set reflux heater on 50 until boiling starts, then cut back to setting of 10. After refluxing, filter hot liquid through #i2 Whatman filter paper into 250-m1 beaker. Add 125 ml of ethanol to filtrate and cool in ice bath for 15 minutes. Set up assembly for vacuum filtration. Place S&S No. 589 filter paper in [3uchner funnel and wet with water. Turn on the vacuum and spray paper with ethanol. Vacuum.filter the.chilled filtrate and wash with a minimum of cold ethanol. Quantitatively transfer the gelatinous precipitate and paper to 250-m1 beaker. Wash funnel and paper with about 150 nil distilled water to remove and dissolve the gel. Stir until gel dissolves (about 5 minutes). Transfer the solution to'500 nil volumetric flask contai*ning 0.5 g (or 2 nil 25%) sodium hydroxide. Dilute to vQlume. The solution should contain 50 to 300 ug pectin per nil. 2._ Analysis of Samles Pipet 12 ml of concentrated sulfuric acid into•each test tube to be used. Place the tubes in an ice bath and allow to cool. Into these tubes, pipet 1 nil of sample and 1 ml of pectinase solution. Mix thoroughly. Heat the tubes on a boiling water bath for exactly 10 minutes. Cool the tubes and pipet 1 ml of carbazole solution into each. Mix thoroughly. Place the tubes in a boiling ti•;ater bath for 15 minutes. Then place .in an ice bath and allow to cool to room temperature. Determine the absorbance values for each solution at 530 nm. These samples are measured against a blank containing 12 ml of H?S04, 1 ml of pectinase solution, 1 ml of carbazole reagent and 1 nil of distilled water. Use a standard curve to obtain concentration of;pectin in the samples. The concentration range of standards should be from ~~0 to 300 ug/ml. ~
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3 CALCULATION % Pectin =(A x B)/C x 1Q^`' A B C Analytical fjethod No. 2036 ug/ml pectin from standard curve. dilution volume in mi1liliters. weight of sample. REFERENCE 1. Nege, Robert B., RDM, 1973, No. _ , (JAN. 18 S 1 2 3 4 5 6 7 8 9 10 20 _30 40 50 60 70 &0 90 100 200 300 R1, 1 23457f3 ~9102()07---- 09U 100200300 1411 M 1 ' 2~~ ~~~G 00 9~ 0 20 ~:~~~10~~0 60 IO.yf~O 90 7()0 . 2U0 300- N
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R. J. REYNOLDS TOBACCO COMPANY RESEARCH DEPARTMENT Analytical Method No. 2045 DETERMINATION OF METALS IN TOBACCO USING ATOMIC ABSORPTION PRINCIPLE This method provides for the determination of various metallic ions, such as aluminum, calcium, copper, magnesium, etc. (A complete list is placed at the end of this procedure.) The metal is put into solution by elution or wet ashing, then treated to reduce the effect of interferences. The end determination is accomplished by atomic absorption. APPARATUS AND REAGENTS ATOMIC ABSORPTION SPECTROPHOTOMETER, Perkin-Elmer Model 214 with accessories ,--~ CHROMATOGRAPHIC TUBES, 20 X 250 mm FLASK, Volumetric, 2000-m1 capacity FLASK, Volumetric, 1000-m1 capacity FLASK, Volumetric, 100-m1 capacity PIPET, 10-m1 capacity, to deliver BEADS, solid glass, 3 mm (Fisher Scientific #11-312A) TUBES, digestion, 200 min X 25 mm, 24/40 1 joint. CELITE, 545, acid washed NITR-IC ACID, concentrated, reagent PERCHLORIC ACID, 70%, reagent HYDROCHLORIC ACID SOLUTION, 3 N, add 250 nil concentrated hydrochloric acid to distilled water, and dilute to one liter. LANTHANUM SOLUTION, 20,000 PPM. Dissolve 46.9120 g La203 and 71.5 ml concentrated hydrochloric acid in distilled water and dilute to 2000 nil. PROCEDURE A. Sample Preparation by Acid Elution This method can be applied to the leaching of only calcium, potassium, sodium, magnesium and manganese from both plant material and paper. In order to analyze for additional metals, it is necessary to use the Wet Ashing Procedure to prepare the sample`. In every series of sample to be run, weigh every tenth sample in duplicate. Also prepare one blank by placing about 2.4 grains (both dippers full) of the acid-washed Celite 545 in a chromatographic tube for treatment throughout the entire procedure just as if it were a sample. . m ~ ~
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Analytical Method No. 2045 Accurately weigh 0.5 gram samples of dried tobacco dust, weighed to the nearest 0.1 mg, into a tared weighing pan. Record the sample weight and remove the sam 1e pan from the balance. Add approximately 0.7 grams (small dipper full~ of acid-washed Celite 545 directly into the weighing pan contai ni ng the sampl e. Careful 1y mi x the Cel i te and sampl e together by stirring with a small stainless steel spatuli. Introduce the mixed sample and Celite through a glass funnel into a 20 X 250 mm chromatographic tube. Use adequate brushing to insure a complete transfer of the sample. Tap the tube1gently to settle and level off the column of sample, and then introduce through the same glass funnel another 1.7 grams (large dipper full ) of Celite in a manner to aid in washing down the sides of the funnel. This 1.7 grams of Celite should be sufficient to form a layer about one-half inch high on top of the sample - Celite mix. Tap the tube brishly to compact the sample - Celite mix in the column. Place the chromatographic column in the neck of a 1000 ml volumetric flask already containing some 400-600 ml of distilled water. Introduce by means of a"Cal ab" di spenser 40 ml of 3(d ..HC1 , i n such a manner as to wash down the sides of the tube as completely as possible, yet without causing a turbulent mixing of the Celite up into the eluent. As the last drops of eluent are eluting through the sample, before the top of the columrn runs dry, add 50 ml of distilled water by means of another Calab dispenser. When most of this water h?s eluted through, a second portion of 50 ml of distilled water is added and allowed to clute completely through. The last portion of liquid is blown through the sample - Celite column by means of a compressed air tube being connected to the top of the column. Caution: Wear safety glasses and leather gloves to protect eyes and hands in case a column is weak and should break. Remove the chromatographic tube from the neck of the volumetric flask, rinse the liquid`from-the tip of the tube into the flask with distilled water, and fill the flask almost to the mark. Allow the flask to stand until' the eluate is in equilibrium with room temperature and make the final adjust- ment to volume and shake. The sample solutions should not be allowed to stand in the liter flask more than three days before proceeding to the final step. Withdraw by pipette a 10-ml aliquot of the well-mixed eluate obtained from the liter flask, and introduce into a 100 nil volumetric flask. Add to the same 100 ml volumetric flask a 5 ml portion of 20,000 ppm Lanthanum solution by Calab dispenser, and finish filling the flask to volume with distilled water at room temperature. Stopper and shake well before the samples are analyzed for the suggested metals by atomic atisorption spectro- photometry. ~
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Analytical Method No. 2045 All standards should be prepared fresh each day from a 100 ppm stock standard solution. The standards to be used in the analysis must contain the same amount of acid and Lanthanum that the dilute samples contain. A volume of 100 mis of standard is sufficient to set the instrument up for analysis. All glassware must be cleaned with 1% Micro-soap. It will be necessary to remove the soap from the glassware with hot water and rinse several times with distilled water. Most of the material in the chromatographic tubes can be removed by back flushing with water before washing with 1% Micro-soap. Q. Sample Praration~t-let Ashi.nq Procedure c. This method involves the use of concentrated nitric acid (5 ml) and 70% perchloric acid (2 nil) for the digestion of plant material, cigarette paper and liquid samples. Caution: It will be necessary to decide before the acid is added t.o the sample, if the perchloric acid will react in an explosive manner with material in the sample. Al~,s wear a•face shield and apron when handling the perchloric acid to prevent any possible injury due to an explosion. , Plant samoles should be oven-dr'ied for one hour-at 100° C, cooled, andgrourid into a ftne homogeneous powder. Cigarette_pa~er samples should be cut into small squares approximately 4 mm x 4 mm. The person that prepares the paper should protect the sample from contamination of inorganics, such as sodium, which might be present on the hands. Contamination of sample can be prevented by wearing disposable vinyl medical gloves that have been rinsed with distilled water. l_iq_uid samples should not exceed a 5-ml aliquot to be placed in the digestion tubes. If the volume needed will exceed 5 ml, then it would be necessary to evaporate the volume chosen down to approximately 5 nil or less before placing the.liquid in the digestion tube and completing the ashing procedure. When the volume exceeds 5 ml in the digestion tubes, the sample has a tendency to bump out of the digestion tube. Weigh one-gram samples, weighed to the nearest 0.1 mg, and transfer to a digestion tube. A blank should be prepared and carried through the procedure in the same manner as the samples. Pipet concentrated nitric acid (5 ml) into the tubes, rinsing the tube sides clean of any sample. Add two glass. boiling beads (size 3 mm) to each tube to prevent bumping during the heated digestion. The digestion should begin at a low heat setting for the first .30 minutes. The tubes can then be heated for one hour at a temperature to provide gentle boiling of the digestion solution. The heat can then be increased in order to further decrease the volume of nitric acid down to approximately 2 nil. Add 70%
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4 Analytical Method No. 2045 perchloric acid (2 ml') to each digestion tube and boil until dense white fumes of perchioric acid exist. Fifteen minutes or less of additional fuming is necessary to assure complete absence of nitric acid. The solutions are allowed to cool to room temperature and made to the desired volume. This volume is usually 50 nil since the digestion tubes are calibrated to a 50-m1 volume. Make the desired dilutions when needed. The time required to digest twelve samples including blanks is approximately 4 hours. ; When a metal is to be determined'for the first time in a wet ashed sample, it is necessary to run the ashed sample against stock standards and by doing a standard addition to the sample. The two methods will allow one to decide if the metal to be analyzed is being interfered with by acids or other materials in the wet ashed solution. , If both answers are the same', it should be safe to assume that one may compare samples against the stock standards without the.danger of interference. All glassware must be cleaned with fiot 1% Micro-soap. It will be necessary to remove the soap from the glassware with hot water and rinse several times with disti1le d water. ' C. End_ Determination ~y Aiomic Absorption Set the appropriate parameters, such as wavelength, light source, oxidant, fuel, etc., for the element of interest according to the Perkin- Elmer operations handbook. Have samples, standards, and blank solutions that can be easily aspirated through the nebulizer. At least 2 ml of solution should be available for each measurement. Prepare standard solutions having known concentrations of the metal to be determined in:the same solvent as that of the sample. The standard concentrations should bracket the expected concentration in the sample. If very dilute standards are needed for a period longer than one day, stock solutions in concentrations greater than 500 iig/mi should be made up. The stock solutions can then be stored and diluted as required when standards are needed. This will avoid changes in concentration (as much as 2 to 3% in 3 days) that occur when very dilute solutions are left standing. When all sample and standard solutions have been prepared, determine the absorbance values for the sample and standard solutions. Normally, standards are analyzed at the beginning and end of a run, and periodically during longer runs. Blank or solvent is normally run between each sample or standard to verify baseline stability.
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5 Analytical Method No. 2045 The method of determining concentration depends upon the instrument used. With instrument such as Model 214, reading directly in absorbance or concentration, the readout may be calibrated to read directly in concentration if the concentrations of the samples and standards are within the linear working range. Calibration can then be made with only a reagent blank and a standard at the upper end of the linear concentration range. This can be done simply by first.aspirating the blank into the nebulizer and setting the read out to 0. Next, a standard solution of'known concentration is run, and the read-out set to proper ppm reading. Standards of other concentrations can now be run to check for linearity. Then, the samples can be run and the concentration of the element of interest read directly. A calculation can then be applied to determine the amount of the element present in the original sample. For some samples, it is difficult to prepare standards which are sufficiently similar. This may occur when the samples contain high and variable concentration of matrix materials, or when the samples contain very high solids whose effect on absorption is hard to duplicate. In such instances, the method of additions should be used. , Take three aliquots of the sample. Dilute the first to a known volume with solvent. Make up the second and third aliquots to the same volume with suitable quantities of known standards added so that•the final solutions contain different additions of the metal to be determined. Determine the absorbance for each solution, expanding the scale if necessary to improve readibility. Plot the absorbance readings obtained against the added concentration. Extrapolate the resulting straight line through zero absorbance. The intercept on the concentration axis gives the concentration of the metal in the diluted sample solution. For an accurate determination by the method of additions, the working curve must be linear over the concentration range covered by the sample plus additions. Additions should be of approximately the same concentration as that anticipated for the diluted sample solution. CALCULATION % o Metal (Concentration, i~/L)(Uolume of Solution, L) = ~Weighi:+o~ Sample, g)(l0~ ~~ CID f12/ 3 4 5 6_7 _8 9~ 10'20 30 40 50 60 70 80 90 100 200 300 .~. ~_.._....._..~-.,._....__ -_._ .-._., {Z'f 1 7~ 3 A, aC6 >£3 9~I0_20 30 40 50 60 :70;&0 90 100 200 300 ~---------_ _----.-~-0--_ - ~Irl 1 _ 2:3 4_~G /~3 9~ 10 20 30 410 50 6~,~f30 90 100 20300 T
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' METALLIC IONS DETECTABLE W ITfI-70T11TC -FAMF r'TTUid-SPE"CT RO S C OP Y• • ELEMIENT Actinium ~ Aluminum - Amerl C1 Ufn * Antimony Argon ** Arsenic Astatine *E3arium Berkelium * Beryl 1 i um * Bismuth *Boroii Bromine *Cadmium *Calcium Californium Carbon Cerium *Cesium Chlorine *Chromium *Cobalt *Copper Curium *Dysprosi um E.insteiniurn Erbi um Europium Fermium Fluorine Francium Gadolinium Gallium Germanium *Gol d Hafmium Helium Holmium *Hydrogen dium I n .t ~ ; Iodine j~ Iridiurn _ *Iron uq/ml FOR 1 ~ ABSOftPI 10 il DETECTION SYM,QOL LIi•iiTS rg%ml ) INTERFERENCES Ac Al 6.0 0.90 0.03 Yes Am' Sb 0.60 . 0.10 Yes Ar As 2.0 0.01 Yes At ~ Ba 5.0 0.30 0.02 Yes Bk Be 0.20 0.03 0.001 Yes Bi 0.80• 0.04 No B 250.0 40.0 ~ 2.5 No Br ~ Cd 0.25 0.001 No Ca 0.07 0.05 0.001 Yes Cf - C• Ce Cs 0.50 0.05 Yes C1 Cr 0.10 0.003 Yes Co' . 0.15 • 0.01 No Cu 0.10 0.002 No Cm Dy 0.80 0.20 Yes Es Er 0.90 0.10 Yes Eu ' 0.60 0.04 Yes Fm F Fr Gd 6.0 .0 es Ga 2.5 1.2 0.10 No Ge 2.5 _--1. 0 No Au 0.20 ~ 0.02 Yes }if 0.15 8.0 Yes He Ho 1.5 0.10 Yes H In 0.90 0.05 . Yes I Ln Ir 12.0 2.0 Yes m Fe 0.10 0.01 Yes ~ ~ ~~ m
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J,c ml FOR 1 ~w AE;SORI0'N DETECTION ELEMENT SYMBOL ACE T-7I R /tMU-NzO LI ~i1ITKT11g/m1) INTERFERENCES.. Krypton Lanthanum Kr La 30.0 2.0 Yes *Leud Pb 0.50 0.02 No *Lithium Li 0.03 0.0006 No Lutetium - Lu 6.0 3.0 Yes 4-hfagnesium Mg` 0.007 0.0001 Yes *Manaanese Mn 0.05 0.002 No Mendelevium **h',ercury Md Hg 10.0 0.50 Yes *Molybdenum Mo 0.90 0.60 0.03 Yes Neodymium Nd 1*3.0 2.0: Yes Neon Neptunium *Nickel Ne Np Ni 0.10 0.01 No Niobium Nb 20.0 1.0 Yes Nitrogen N ~ Nobelium Osmium No Os .5.0 -1.0 0.5 No Oxygen 0 *Palladium Pd 0.20 0.02 Yes *Phosphorus P 250.0 100.0 Yes *Platinum Pt 2.5 10.0 0.10 Yes Plutonium Polonium *Pot.assi urn Pu Po K 0.02 0.005 Y,es Praseodsrniium Pr 22.0 ' 10.0 Yes Promethium Protactinium Radium Radon Rhenium Pm Pa Ra Rn Re 2.0 .0 o Rhodium Rh 0.50 0.03 Yes *Rubidium Rb 0.05 0.005 Yes Ruthenium Ru 0.50 0.30 Yes Samarium Sm ' 7.0 2.0 Yes . Scandium Sc 0.65 0.10 Yes **Selenium Se 2.0 0.10 Yes *Si l i con Si 2.0 0.08 No *Silver Ag 0.06 0.002 Yes *Sodium Na 0.015 0.002 Yes *Strontium Sr 0.15 0.09 0.01 Yes Sulfur Tantalum S Ta ' 40.0 2.0 Yes Ln Technetium Tc 3.0 0.9 Yes i- m . m w
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i,q ml FOR 1~ A6SOr'`l~ 10 N DETECTION ELEMENT SYMBOL ACET-1CI_R 1`,~E'I`-W20 1.It1I`fS ug/ml ) INTERFERENCES *Tellurium Te 0.50 Terbium Tb Thallium T1 0.50 Thorium Th Thulium Tm *Ti n Sn 3.0 *Titanium Ti *Tungsten W . Uranium U *Vanadium V 1.5 Xenon Xe *Ytterbium Yb Yttrium Y +k Zinc Zn 0.015 Zirconium Zr • , c 0.09 No 8.0 3.0 Yes 0.03 No 0.70 0.20 Yes 2. . 0 0.02 Yes 2.0 0.09 Yes 20.0 3.0. No 50.0 30.0 Yes 0.06 Yes 0.20 0.04 Yes 2.0 0.30 -Yes 0.002 No 6.0 5.0 Yes *Sources on hand in our laboratory. **Capable of determining Mercury at levels of less than 0.001 ug/ml in solutions by means of Flameless Mercury Accessory. **The. detection limits for Arsenic and Selenium using the Perkin-Elmer High Sensitivity As/Se Sampling System are 0.001 ug/ml for Arsenic, 0.0015 pg/ml for Selenium.
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R. J. REYNOLDS TOBACCO COMPANY RESEARCH DEPARTMENT Analytical Method No. 2051 DETERMINATION OF MOISTURE BY KARL FISCHER PRINCIPLE Cigarette cut tobacco is extracted with d ry methanol and the moisture content of the methanol is determined by titration with Karl Fischer reagent. APPARATUS ANf) REAGENTS MAGNISTIR, Cat. No. 1250, Labline Instrun-ients STIRRING BARS, magnetic, Teflon coated, one-inch long AQUATRATOR, Precision Scientific Company, or an equivalent titrometer for the determination of moisture by the Karl Fischer method. KARL FISCHER REAGENT, stabilized single solution. METHANOL, A.R., A.C.S., or Karl Fischer grade. SODIUM TARTRATE, reagent, (15.66 percent ti?0) suitable for standardi- zation of Karl Fischer reagent. PROCEDURE Place Karl Fischer jars for aquatrator and stirring bars in oven at 103° C and dry at least overnight. Remove jars, seal with screw-type lid and cool to room temperature. Place stirring bars in a dry, closed container and allow to cool. Turn on aquatrator assembly with Karl Fischer (K-F) jar in place. Add stirring bar,and pipet 50 ml methanol (A.R. grade) into the jar. Start magnistir unit and immediately titrate with Karl Fischer reagent to a point slightly over the K-F end point. Leave the apparatus in this manner for a 30-minute warm-up period. Occasionally check the meter and add Karl Fischer reagent as needed to maintain the blank at the end point setting. At the end of the warm-up period, remove the jar and contents, and place another 50 nil methanol blank into position. Titrate as before to the K-F end point. Stop the titration and measure the time required for. the needle to drift below the end point. If this time is less than 30 seconds, add titrant to end point and time again as before. If needle does not drift below end point, in 30 seconds, discontinue titration and record volume of titrant required. This value will be used as blank value. ~
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2 Analytical Method No. 2051 Next, for solid samples, accurately weigh 0.1 g into dry K-F jar. Add stirring bar and place jar into aquatrator assembly. Pipet 50 nil of methanol (A..R. grade) and immediately proceed with titration in the same manner as was done for the blank determination, except omit the.warm-up procedure. Record the volume of titrant required as titration value. For liquid samples, proceed through the blank titration step, zero the buret, and then pipet an amount of the sample into the jar containing the blank. Adjust the amount of sample based on-expected water content. Titrate as before to a 30-second stable end point. Record the volume of titrant added as titration value. To standardize the titrant, %%,eigh 0.1000 g sodium tartarate standard into K-F jar. Add 50 ml methanol and titrate as for solid samples.' CALCULATION K-F Factor = (Wt. Sodium Tartarate ( )) (156.6) MO-ume of Ti trati on) ~ Vol uiiie of Bl ank °6 H20 (Solid Samples) = [(Titration Val_ue) - (Blank Value)] [K-F Factor] ! ~~ 1leight of Sample (g)_ ( mg H20/ml (Liquid Samples) _(Titration Value) (K-F Factor) Volume of Sample REFERENCES 1. Mitchell, J., and Smith, D. M., Aquametry, Interscience Publishers, Inc., New York. 2. Laurene, A. H., and Sullivan, B. N., Jr., RDR, 1956, No. 5 (JUNE 29). I S h_ 3 5_ 6_7 8 9 10 20 30 40 50 60 70 HU 90 100 200 300 t~~~1 L'3 ~ 6 7 8 9 10 ~6' 33 4'0 50 60 _70 830 90 100 200 300 ~ _ThII -1 -'234 1567^8910~0;3040 5060708 09010U20 3U0^
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R. J. REYNOLDS TOBACCO COf1PANY RESEARCH DEPARTMENT Analytical Method No. 2051-1 DETERMINATION_ OF MOISTURE IN PUFFED_ TOBACCO BY ~~ GAS CIiROMATOGRA('fiY _ PRINCIPLE Tobacco samples are extracted with methanol containg ethanol as an internal standard. The extracts are analyzed directly by injection into a gas chromatograph. ; APPARATUS AND REAGENTS ~ GAS CHRO!•1ATOGRAPH, Victoreen 4000 or equivalent equipped with a thermal conductivity detector. SHAKER, f3urrel 1 bJri s t-Acti on . , FALSKS, Erlenmeyer, 50 ml, with rubber septum stoppers CARRIER GAS HUPr1IDIFIER, as described by Sloan and Sublett (1) is critical for reproducible analyses. SYRINGE, Hamilton, 10 microliters METHANOI., reagent grade, anhydrous. ETHANOL, absolute. ' EXTRACTION SOLUTION,.prepared by dissolving 20 ml absolute ethanol in 1000 ml reagent grade methanol. DISTILLED 1JATER Chromatoqraphic Conditions Column - 6 ft. x 3/16 in. copper containing Porapak Q (50/80 mesh) Column Oven;temperature - Isothermal at 145° C Injector temperature - 260° C Detector temperature - 260° C Bridge Current - 130 ma Carrier gas - Helium at 60 ml/min. Attenuation - 8 Calibration Three standards containing 30, 60 and 90 mg water per 40 ml extracting solution are prepared and injected into the gas chromatograph (5 lil). A solvent blank correction may be necessary if the methanol contains traces of water. In practice, calibration and analysis is accomplished by on-line GC-computer operation. However, a calibration curve may be constructed manually by plotting the ratio of the peak areas of water to ethanol versus mg of water added. N ~ ~
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2 Analytical Method No. 2051-1 PROCEDURE Weigh accurately about 0.8 g tobacco into a throughly dried and tared 50 nil Erlenmeyer flask. Add 40 nil of extracting solution and stopper with a rubber serum cap. Extract the tobacco by shaking for one hour on a Burrell Wrist-Action Shaker. Analyze the sample by injecting 5 ul of the solution into the gas chromatograph. The amount of water may be determined by on-line computer or from a previously constructed calibration curve. CALCULATION _. Measure accurately the peak areas obtained for water and ethanol. Determine the peak area ratio of water to ethanol. Read the mg water present in the sample from the calibration curve. , % F2~ _ ~6 N20 = mg Sampl e X 100 REFERENCES 1. Sloan, C. H., and Sublett, B. J., TOB. SCI. 9, 70 (1965). ~ _____ ~i~_ i ~ I E3 9_1 Q/ 20 30 40 50 60 JO ~80 90 100 200 300 ~-'-- ~ Rl~~~ 2 3~I 5 6 7 8 9T0067_,~U ii[~; 50 60 702Q 90 100-200- 300 ~...~..~.~------------.._._ -___.__._.--------- ~I1li 1 2 3~i 5 6 J8 3 9-1 0?6 ~~50 60 %0 &0 90 -1 UO 200 300 __ ~
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R. J. REYNOLDS TOBACCO COMPANY - RESEARCH DEPARTMENT Analytical Method No. 2061 DETERMINATION OF ALKALINITY, WATER-SOLUBLE ASH ~ ~ PRINCIPLE ANO WA7EEZ~If~SOLU[3LE ASH ~ The alkalinity of an ignited sample probably represents its content of carbonates, bicarbonates, and hydroxides of alkali elements. It is determined by back titration with standard sodium hydroxide to a phenol- phthalein end point after a measured amount of standard hydrochloric acid is added to the sample. APPARATUS AND REAGENTS PORCELAIN CRUCIBLE; flask, volumetric, 100 ml capacity; flasks, Erlenmeyer, 250 ml capacity; beaker, 500 ml capacity, filter paper S&S No. 604. SODIUM HYDROXIDE SOLUTION, 0.1 N; prepare and standardize as described in Analytical Method No. ST-1. HYDROCHLORIC ACID SOLUTION, 0.05 N; prepare and,standardize as described in Analytical Method No. ST-2. PHENOLPHTI-IALEIN INDICATOR SOLUTION. PROCEDURE 1.~ Alkalinity Wash ash from the ash determination (1) into a 250 ml Erlenmeyer flask with distilled water (carbon dioxide-free). Add 15 ml of 0.05 N hydrochloric acid solution and 2 drops of phenolphthalein indicator. Allow to stand for 20 minutes. If there is no pink colo r in the flask, place on a steam bath for 10-20 minutes. Titrate the solution with 0.1 N NaON to a permanent pink coloration. CALCULATION (V1N 1) - (V2N 2) We ght of ash Volume of 0.1 N HCl/g. ash
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2 Where: V1 = Volume of 0.05 HC1 required N1 = Normality of standard HCl V2 = Volume of standard NaOH V2 = Normality of NaOH 2. Soluble and Insoluble Ashes Analytical Method No. 2061 Place the residue from the ash determination (1) in a 500-ml beaker. Add approximately 40 ml water. Allow to stand for 30 minutes during which time break the ash apart with a rubber policeman. Filter through filter paper S&S No. 604 and continue washing until a volume of 300 nil water has passed over the residue. Take the residue and ash. Dry in oven at 100°-105° C., cool in desiccator and weigh. Report increase in crucible weight as insoluble matter. , CALCULATION REFERENCE Weicht of 2nd ashinq x 100 - % Ash (1) - % Insoluble ash = % Soluble ash Weight of tobacco sample = °6 Insoluble ash 1. Analytical Method No. 2017. S 1 2 3 4(5)6_7 8 9 10 20 30 40 50 60 70 80 90 100 200 300 Ln RT 1'-2 3~4 (~ (~ 1 f? 9'(R?)10 30 40 ~0 60 70 80 ~90 100 200 300 ~ -~ -~ --- '~1i1~`~~'2 3 4~5 G 7&~9 1~U 2(~3(1-~40~ 50 60 70 ~0 9U 1 OC) 2U0 300 ~ G CO m
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R. J. REYNOLDS TOBACCO COMPANY RESEARCH DEPARTMENT Analytical Method No. 2064 DETE:RMINATION OF WATER SOLUBLE FREE ACIDITY OF TOBACCO PRINCIPLE The water soluble free acidity of tobacco indicates the amount of excess acidic materials that can be extracted from tobacco with water. It is determined by extraction with water and then titration using 0.1 N sodium hydroxide solution. APPARATUS AND REAGENTS pH METER, Corning, Model 12, or equivalent, equipped with glass reference and calomel electrodes. FILTER PAPER, S&S No'. 604 ~ E3URET, 10 nil capaci ty. SODIUM HYDROXIDE SOLUTION, 0.1N. Prepare and standardize as described in Analytical Method No. ST-1. PROCEDURE . Accurately weigh a catchweight of 2.5 grains of ground tobacco into a 250-ml beaker. Add 200 nil distilled water, mix, and allow to stand for 2 hours with occasional stirring. Filter to remove tobacco. Pipet 100 nil of fi 1 trate i nto 250 nil beaker. Standardize pll meter with pH 7.0 buffer. Immerse electrodes into filtrate and titrate with 0.1 N NaOH solution to pH 8.10. Record titration value.' Report values as ml 0.1 N NaOH per g tobacco. CALCULATION ml 0.1 N NaOH per g Tobacco _ Titration Valuex Normalit x 10 4,ei ght of Tobacco'~g j~ ~ _ _ ~ S_~ T? 3 q fri 6 7 8~ 10 20 30 40 50 6 O 70 F30 ~0 100 200 _ 3U0 72 0~~1~~~~~60 _70 &f) 9~~1 UO 2U0 300~ ,~___ _ 111 -~ 1' 2 3~ ~ 6 7 8 9 tl 0.% 20 3U 40 50 6(1 70~&090 `1 ~0~2OU 300~
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R. J.• REYNOLDS TOBACCO COMPANY . RESEARCH DEPARTMENT Analytical Method No. 2064-1 UETE_R1,1INATION OF. ACIDS WITH TETRABUTYAMMONIUM HYDROXIDE PRINCIPLE Acids are dissolved in neutral or basic anhydrous solvents and titrated with tetrabutylannnonium hydroxide. APPARATUS AND REAGENTS See Analytical Method No. ST-9. PROCEDURE 1. Potentiom^tric Titration Usinq 0.1 N Tetrabutlammonium Hydroxide • Accurately weigh 0.8-1.0 milliequivalent of the unknown acid into a 250 ml electrolytic beaker. Add 40 ml of the desired solvent and place beneath the electrodes. Titrate potentiometrically, under nitrogen, using either the automatic or a manual titrator, recording volume of titrant and millivoltage. Plot the volume of titrant vs. millivoltage and determine the end point from this curve. Perform a blank titration and correct the volume of titrant accordingly. 2, Potentiometric Titration UsinQ 0.02 N Tetrabut lammonium Hydroxide Accurately weigh 0.15-0.20 milliequivalent of the unknown acid and proceed as described above when using the 0.1 N titrant. CALCULATION ~ `~ = ~~illigrams of Sample Neutral izatio n Equivalent VXN• Where V= N = Corrected Volume of Titrant Normality of Titrant % Purity = V x N x Millieduivalent 1_!eight x 100 ~ ~ Milligrams of Sample or - ~ Neutralization E.quivalent (Themo) Ne-ZFdlization Equivalent CFound) NOTES Indicators may be used for visual determinations provided a particular acid is being assayed on a routine basis. Weigh the amount of sample listed above and fo11 ow the procedures outl i ned i n Analyti cal Method No. ST-9.
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2 Analytica) Method No. 2064-1 CO Several specific procedures for acidic materials are described in this manual. Their use is recommended wherever available. Some acidic materials are difficulty soluble in the nonaqueous solvents commonly employed. In these cases weigh approximately 20 times the recommended sample weight into a 100--ml volumetric flask. Dissolve in 5-10 ml water, then dilute to volume with pyridine or one of the other solvents. Transfer a 5-m1 aliquot-of the dilution to a 250-m1 electrolytic beaker, acld 40 ml of solvent.and titrate by the usual procedure. Perform a b1ank'titration using the same volume of solvent and change the calculation to account for the dilution. In determination of strong acids or strong acid mixtures, always use pyridine as solvent. REFERENCES 1. Cundiff, R. H., RD(:, 1955, No. 6 (March 29). 2. Cundiff, R. H., RDR, 1958, No. 9-(June 13). 3. Cundiff, R. H. , RDR, 1958, No.,11 (June 13). 4. Cundiff, R. H., RDR, 1961, No. 46 (Nov. 7). 5. Sensabaugh, A. J., and Cundiff, R. H., RDR, 1957, No. 21 (Dec. 17 S TT_3 _4_) Ci I i3 9~~ 20-3U ~I U 50 GO 70 f;0 90 100 200 30~ ~ 4 , ~5)_.6 7 .__ 0 ~.____.._-----____.__..__.__.___.~.-..--..__..__..._..______._--- -- ~RT 1 2 3 H 9 1 20 3U 4U 50 60 70 ~30 y0 l 0U 'l00 300 MH 7 2 3~I- 5 6 7 f3 9 10 2U 30 40 50 60 7U £iU 9U 100 ?.UO 300 ~
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R. J. REYNOLDS TOBACCO COIMPANY RESEARCH DEPARTMENT Analytical Method No. 2072 DETERMINATION OF PHOSPHORUS IN TOBACCO AND WASTE WATER PRINCIPLE This method_may be used to determine phosphorus present as ortho- phosphate I(P01,) j,] only. Ammonium molybdate and potassium antimonyl tartrate react in an acid medium with dilute solutions of phosphorus to form an antimony--phosphate-molybdate complex. This complex is reduced to an -i ntensely bl ue-col ored complex by ascorbi c acid. The color i s proportional to the phosphorus concentration. APPARATUS AND REAGENTS PHOTOMETER. A spect'rophotometer or filter photometer suitable for measurements at 880 mu, and providing a light path of 1 inch (2.54 cm) or longer, should be used. ACID-WASHED GLASSWARE. All glassware used in the determination should be washed with hot 1:1 HCl and rinsed with distilled water. The acid-washed glassware should be filled with distilled tirater and treated with all the reagents to remove the last traces of phosphorus that might be adsorbed on the glassware. Preferably, this glassware should be used only.for the determination of phosphorus and after use it should be rinsed with distilled water and kept covered until needed again. If this is done, the treatment with 1:1 HC1 and reagents is only required occasionally. Commercial detergents should never be used. • - - SULFURIC ACID SOLUTION, 5N. Dilute 70 ml of conc. H2SOa with distilled'wai.er to 500 ml. POTASSIUM ANTIMONYL TARTRATE SOLUTION. Weigh 1.3715 g K(Sb0)C4H,,Oc•1/2 H20, dissolve in 400 ml distilled water in 500 ml volumetric flask, dilute to volume. Store in glass-stoppered bottle. A(1i40iflUM MOLYt3DATE SOLUTION. Dissolve 20 g(0)s(-1o7024•`'H20 in 500 ml distilled water. Store in a plastic bottle at 4° C. ~ ASCORBIC ACID, 0.1M. Dissolve 1.76 of ascorbic acid in 100 ml m of distilled water. The solution is stable for about a week ~ if stored at 4° C. m CONit3INED REAGENT. Mi x the above reagents i n thq fol l owi ng proporti ons for I co 100 ml of the mixed reagent: 50 ml of 5N H?5W, 5 ml of potassium ~ antimonyl tartrate solution, 16 ml of ar,:moniud molybdate solution, and 30 ml of ascorbic acid solution. Mix after addition of each rea~rent. All reagents must reach room temperature before they yy are rnix~~d and must be mixed in the order given. If turbidity formslin the combined reagent, shake and let it stand for a few minutes until the turbidity disappears before proceding. The reagent is stable for one week if stored at 4° C. STRONG•-ACID SOLUTION. Slowly add 310 ml conc. H2SO4 to 600 rnl distilled water. When cool, dilute to 1 liter. AMMONIUM PERSULFATE. STOCK SOLUTION. Dissolve in distilled water 0.2197 g of potassium dihydrogen phosphate, HK 1P0 Y, whi ch has been dri ed i n an oven at 105° C. Di l ute the sol uti on to 1,000 ml ; 1.00 ml equal s 0.05 rng P.
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STANDARD SOLUTION. Dilute 10.0 nil of stock phosphorus solution to 1,000 nil wi th di sti l l ed water; 1.00 nil equal s 0.5 ug P. Using standard solution, prepare the following standards in 50.0-m1 volumctric flasks: ml of Standard Solution Conc., mq 1_ ~ p 0.00 1.0 0.01 3.0 0.03 5.0 0.05 10.0 0.10 20.0 0.20 30.0 ~ 0.30 40.0 0.40 50.0 : 0.50 PROCEDURE Add 1 ml of strong-acid solution to a 50 ml sample of waste water, or 0.5'g dried and ground tobacco in 50 ml distilled water, in a 125--m1 Erlenmeyer flask. Add 0.4 g of ammonium persulfate. Boil gently on a pre-heated hot plate for 30-40 minutes, or until aa final volume of about 10 ml is reached. Do not allow sample to go to dryness. Cool to room temperature and add 4.0 nil 3 N sodium hydroxide. Add 1 drop phenolphthalein and adjust sample to pink color with l N NaOH. Bring back to colorless with one drop of strong acid solution. Cool and dilute the sample to 50 ml. Pipet an aliquot of the prepared sample into a 50-ml volumetric flask. The size of the aliquot may be varied depending upon the expected range that the phosphorus content may attain. Add 8.0 nil of combined reagent to sample and mix thoroughly. Dilute sample to 50 ml volume. After a minimum of ten minutes, but no longer than thirty minutes, measure the color absorbance of each sample at 880 mu'with a spectrophoto- meter, using the reagent blank as the reference solution. CALCULATION Prepare standard curve by plotting absorbance values of standards as ordinates and the corresponding phosphorus concentrations as abscissas. Process standards and blank exactly as the samples. Run at least a blank and two standards with each series of samples. If the standards do not agree within a_ 2% of the true value, prepare a new calibration curve. Obtain concentration value of sample directly from prepared standard curve. Report results as P, niq/1.
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REFERENCES 1. Murphy, J. and Riley, J., "A Modified Single Solution Method for the Determination of Phosphate in Natural Waters." ANAL. CHEM. ACTA. , 27, 31 (1962). 2. Gales, M., Jr., Julian, E., and Kroner, R:, "Method for Quantitative Determination of Total Phosphorus in 4later." JOUR. AWWA, 58, No. 10, 1363 (1966). _ S 1_ 2 3 4(5)6 7 8 9 0 20 30 40 50 60 70 80 90 1_00 200 300 _..-- -~1~~'T ~ 3 44 .i 6 78 9 10 20 30 40 50 60 70 '8 90~100~-?_UO 300 - J760 --- ---__• .--300- Ii!{ 1 2 3 4 5 6 7 ~3 9 10 20 30 40 50 60 pU"8U 90 200-
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R. J. REYNOLDS TOBACCO COMPANY RESEARCH DEPARTMENT Analytical Method No. 2085 DETERMINATION OF TERTIARY AMINE, SECONDARY AMINE, AND ~TOTIIL 'AL1QKi_O1DS IN MIXED ALfAL()ID-TYPE~ 10E3ACCOS_ PRINCIPLE Tobacco is acidified with dilute' acetic acid, benz_ene-chloroform solution added, then concentrated sodium hydroxide. After extraction two aliquots of the benzene-chloroform solution are titrated, with and without the addition of acetic anhydride, with perchioric acid. The total, secondary amine and tertiary amine alkaloid values are calculated frorn the two titration values. REAGENT AND APPARATUS , See Analytical Method No. 2012 BENZENE-CHLOROFORM SOLUTION. Mix equal parts by volume of benzene and chloroform and saturate with distilled water. SODIUM HYDROXIDE SOLUTION, 36%. Dissolve 500 g sodium hydroxide in water and dilute to 1 liter. ACETIC ACID, 5%. Dilute 50 ml acetic acid to one liter with water. CRYSTAL VIOLET INDICATOR. Dissolve 0.5 g crystal violet in 100 nil acetic acid. PERCHLORIC ACID, 0.025 N. Prepared and standardized as described in Analytical Method No. ST-8. FILTER-CEL, Johns-Manville Products, Inc. PROCEDURE Accurately weigh 2.5 g finely ground tobacco into a 250 nil Erlenmeyer flask. Add 15 ml 5% acetic acid and swirl until the tobacco is thoroughly wetted. Pipet 100 ml benzene-chloroform solution into the flask, then add 10 ml 36% sodium hydroxide solution. Stopper flask tightly and shake 20 minutes, using wrist-action shakers. Add 4.5-5 g Filter-Cel, mix, and filter most of the hydrocarbon layer through Whatman No. 2 paper into a second flask. If the filtrate has any turbidity, add 2-2.5 g addit.ional Filter-Cel, and refilter through~' Whatman No. 2 paper. Filtrate must he clear. Pipet 25 nil aliquots of the filtrate into each of two 125 ml Erlenmeyer flasks. Pass a stream of air ove r the surface of the solution in the first flask for 5 minutes, add 2 drops of crystal violet indicator and titrate to
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Analytical Method No. 2085 a green end point with 0.025 N perchloric acid. Add 1.0 nil acetic anhydride to second flask and let stand at least 15 minutes. Add 25 nil acetic acid and 2 drops of crystal violet indicator, and titrate to a blue--green end point with 0.025 N perchloric acid. Take the first appearance of the blue- green color throughout the solution as the end point. For each series of .analyses, perform blank titrations and correct the respective volumes of titrant. CALCULATIONS % Total Alkaloids (as Nicotine V x N x 32.45 Sampi 1 e ~Wt. % Tertiary Amine Alkaloids (as Nicotine) _(?ls=~-'.1~ x f~ x_3?_.45 Sample Wt. % Secondary Amine Alkaloids (as Nornicotine) =?(VI-Va x N x 29.64 Sample L-Jt. V1 = Volume of titrant for nonacetylated aliquot. V2 = Volume of titrant for acetylated aliquot. N = Normality of perchloric acid. NOTES 1. The reagent blank values, both in standardization of the titrant and in analysis of tobaccc, are quite small; however, they should be applied for the most accurate results. If not applied in standardization of the titrant, they should not be applied in analysis of tobacco samples. 2. It is'most critical that a clear filtrate be obtained from filtration of the hydrocarbon.extract, otherwise results will be high. The hydrocarbon layer is easily separated from the tobacco-alkali residue after addition of Filter-Cel. 3.. The visual end point in titration of the acetylated solutions is transitory. Once the blue-green end point is reached, the color changes back to blue on very short standing. The first blue-green color discerned throughout the s'olution is the true end point. ~
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3 Analytical Method No. 2085 REFERENCES 1. Cundiff, R. H., and Markunas, P. C., J. ASSOC. OFFICIAL AGR. CI{EMISTS., 43, 519 (1960). 2. Cundiff, R. H., RDR, 1959, No. 14 (JUNE 22). ~~S ^l 2 3 4 5~,~8 0 1O 20 30 q0 50 60 70 8 0i 90 100 2030' _20 0 50 60 70 20 . 9U~00 200 300' P11~1 ~1 03~ y62_8910~C)1304U502070809010020U300
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R. J. REYNOLDS TOBACCO COMPANY RESEARCH DEPARTMENT Analytical Method No. 3001 DETERMINATION OF TRIACETIN AND TRIETHYLENE GLYCOL_ DIACYTATE IN FILTER RODS BY GAS CHYO(JAIOGRA~PHY PRINCIPLE Triacetin and/or T.E.G. diacetate is extracted with methanol from cigarette filters. An aliquot of the extraction is injected into a gas chromatograph for analysis. ; APPARATUS AND REAGENTS GAS CHROMATOGRAPH, Hewlett-Packard, Model 720, or equivalent having a thermal conductivity detector and a recorder. MICROLIIER SYRINGE, 50 pl ~ FLASK, Erlenmeyer, 250 ml METf-IANOL , anhydrous. ~ SHAKER, Burrell 6~Jris -Action STANDARD SOLUTION. Accurate weigh 250-650 mg of triacetin or triethylene glycol diacetate into 100-m1 volumetric flask. Dilute to volume with anhydrous methanol. ChromatocLraph Conditions Column - 42" X 1/4" copper, 5% Carbowax T.P.A. on 1:1 Diatoport S 60/80 mesh-Chromosorb 101 Injection Port Temperature - 260° C Detector Temperature - 290° C Milliampere'- 110 . ' Temperature Program - 150° C - 240° C @ 15° C/min. Attenuator - 4 Shot Size - 10 microliter PROCEDURE Accurately weigh 20 (or less) cigarette filters afte r the filter paper has been removed and record the total weight on sample flask. Place the filter material into a 250 ml Erlenmeyer flask then add 100 ml of anhyd rous methanol. Stopper the flask and shake for one hour on the Burrell shaker. Then take a 10 microliter shot from the sample solution for injection into the gas chromatograph instrument. Also analyze 10 microliter of standard solution in the same manner. Record peak height of sample and standard solutions. I
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2 Analytical Method No. 3001 CALCULATTON a Triacetin - :_/ Peak Heicht of Sam le_(mm) X tJei~h tht of Std: (mc) ~~ - /ol. of Std. So I iati on ml ) (or T. C. G. D. ) ( PeaO I-lei qht of Std. (mm _- T Weight of Sample (g) (10 S -'0?3~4 a0 J_8-970 0304.050GO70£30 90 100 200 300 i R I-_ -`I ?_ 3 4 5 6/£3 9 10 20 30 ~1 ~' 50 60 70 &0 90 100 200 300 '~ 14i '^ 1 2 3 4 5 6 7_, 8 _9~ 10 20 L30j40 50 60^ 70 8;0 90 100 200 300
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R. J. REYNOLDS TOBACCO COMPANY RESEARCH DEPARTMENT Analytical Method No. 3002 DETERMINATION OF TOTAL ADDITIVES ON CIGARETTE FILTER PLUGS PRINCIPLE The additives are removed from cellulose acetate filte r rods by Soxhlet extraction using ethanol. The ethanol is then evaporated and the residue determined gravimetrically. , APPARATUS AND REAGENTS SOXHLET EXTRACTOR, 24/50 K joint to flask; condenser, 24/45 T joint; extraction thimble, 80 x 25 mm; flask, extraction, 125 ml capacity, 24/40 S joint. OVEN, VACUUM, electric, thermostatically controlled. ETHANOL, 26. ! ~ PROCEDURE Accurately-weigh two long or six short filter rods and place them in extraction thimble. Add 2-3 boiling chips to extraction flask and obtain a tare weight on flask and chips. Assemble Soxhlet apparatus with flask and thimble in place. Add 75 ml of 2Q alcohol. Adjust to heater setting of 80 and heat for two hours, with reflux and extraction. Remove thimble and boil off alcohol until 10-15 ml remains in extraction flask. Place flask in vacuum oven at 25 in. Hg and 55° C for two hours. Remove and cool in desiccator, then weigh flask, chips, and residue. CALCULATION 1 °6 Total Additives where: _ I ) (100) 143 ld, = Weight of extraction flak and boiling chips 412 = Weight of extraction flask, chips, and residue W3' = Weight of sample S~I 2 3 4 5G£3 9 10' 20 30 ~!U 50 60 70 £30~ 90 100 200 300 6~7 B 9 1 G 20 3U ~1-0!.~6i60 70 ~30: 90 1 GO 20U 300 ---L-- (4-0-'T "?'3456709i10;20304U506070830.90100?0U.300~
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R. J. REYNOLDS TOBACCO COMPANY RESEARCH DEPARTMENT Analytical Method No. 3003 DETERMINATION OF TRIACETIN IN FILTER PLUGS PRINCIPLE Triacet-in is extracted from filter plugs with ethanol, then determined colorimetrically by the formation of the ferric hydroxamate complex. APPARATUS AND REAGENTS SPECTROPHOTOMETER, Beckman B or equivalent. HYDROXYLAMINE HYDROCHLORIDE SOLUTION. Dissolve 10 g of hydroxylamine tydrochloride in 3A ethanol and dilute to one liter. F'ERRIC CHLORIDE SOLUTION. Dissolve 10 g anhydrous.ferric chloride or 16.69 g of FeCl;3•6H2O in 210 ml of concentrated HCl and dilute to two liters with'3A ethanol. ~ POTASSIUM HYDROXIDE ~OLUTION. Dissolve 15.5 g of 85% KOH in distilled water and dilute tb one liter. TRIACETIN, STOCK STADARD SOLUTION. Accurately weigh 1.000 g of triacetin into a 100-ml volumetric flask and dilute to volume with 3A ethanol. PROCE:DURE A. Prqparation of Standard Prepare a series of standards by pipetting 1, 2, 4, 6, 8, 10, and 12 of triacetin stock standard solution into separate 100-ml volumetric flasks and diluting to volume with ethanol. This will provide standards con taining 100, 200, 400, 600, 800, 1000, and 1200 ug/ml, respectively. E3. Determi nati on of Sampl es Accurately weigh a catch-weight of-about 1.5 g of cellulose acetate filter material into a 125-ml Erlenmeyer flask and add exactly 100 nil of 3A ethanol. Shake by hand until complete wetting is assured. Allow sample to stand for 10 minutes or longer and shake briefly just before sample. Analyze sample using Robot Chemist. IThe Robot Chemist program for triacetin is as follows: ml ~
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2 Analytical Method No. 3003 Robot Turntable Station Number, Operati on 0 Pipet 0.9 nil of sample or standard. 1 Pipet 0.9 nil of hydroxyl amine hydrochloride solution. 2 Pipet 0.9 ml of potassium hydroxide solution. 3 Stir. 41 fipe.t 3.6 ml HC1-FeC13 solution. 46 Withdraw sample and read at 500 nm The Robot Chemist is usually run on-1ine with computer. Hot-;,ever, calculations can be done manually, if,necessary, by using the absorbance values obtained by digital print-out. CALCULATIONS Concentration Factor % Triacetin - NOTE _ E (Concentrations)2 ~~ ~ Absorbances ~ (Absorbanc:e)(Concentration Factor) TVdeight of Samp1e, g~j10OT-i-- It is also possible to report results as mg triacetin per rod. To do do, place two rods into 125-m1 Erlenmeyer flask and proceed by same procedure for weighed samples. mg Triacetin/Rod REFERENCE (Absorbance)(Concentration Factor)(105) Number of Rods 1. Rush, Kenneth L. and Sensabaugh, A. J., Jr., RDM, 1969, No. 16. ~ S 1 7, 3 4 5 6 7 II 9 1 U) 20 30 40 50 60 /0 80 90 100 200 300 RT '~ _ ~23456_789 10('10,:30 40 50 40 70 80 9'0 100 200 300 ~~f~ ~ 2 0 4 5 6%~ J r 1~~- 20 30 u-0 50 60 70 BO 90 100 200 3U0~
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R. J. REYNOLDS TOBACCO COMPANY ' RESEARCH DEPARTMENT Analytical Method No. 3003-1 TRIACETIN CONTENT OF CELLULOSE ACETATE FILTERS AS MEASURED E3Y GAS CHROMOOGMPHY PRINCIPLE The triacetin content of cellulose acetate filter rods is determined by dissolving the rods in acetone and analyzing the solution by gas chromatography for triacetin content. APPARATUS AND REAGENTS COLUMN, 3/16" copper tubing 9 ft. in length maintained at 195° C. PACKING, 60-80 mesh Chromosorb W containing 10% Castorwax (hydrogenated castor oil). ~ CARRIER, Helium at 105 cc./minute. DETECTOR, Thermal conductivity cell (Gow Max W-2 filaments) operating with 190 ma on the bridge at 260° C. RECORDFR, Speedomax Model G(l_eeds and Northrup) at 2 mv. sensitivity. ACETONE, technical grade. INTERNAL STANDARD, dimethyl phthalate (DMP) in ethanol, 180 rng/rnl. KNOWN SOLUTION for gas chromatography analysis, 0.600 g triacetin 1.80 g dimethyl;: pht.halate diluted to 100 nil with ethanol. PROCEDURE , Remove the paper from the filter rod to be tested and weigh to nearest 1.0 mg. Place the filter rod in a test tube, add 1 ml of internal standard (180 rng dimethyl phthalate) and 25 ml of acetone. Seal tube with a rubber serum cap and shake (mechanical) for 20 minutes. An 8},l aliquot of the acetone solution is then analyzed for triacetin content by gas chromatography. CALCULATION Mg Triacetin/Filter Rod = Peak Heiqht Known D1iP Conc Unknown DMP Peak Height Unknown Ur~1P X Coric.~ l:nor~n Dff P~ X Peak Height Unknown Triacetin X Conc. Known Triacetin Peak' lici ght Known l ri aceti n Cle ~ Triacetin on Filter Rod = P-tg Triacetin X 100 ~Tlg~ii~of"~-i'f-ter~Ro~1,1g. ) -5-6* ~2*0-71;6~~0 ~~G E~-7b ~so ~0 ~i bb ~yFf{T" ~1 --Z- ~U 4C) !Y0 60 70-£~0~90 1 OU 607800 "~
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/ R. J. REYNOLDS TOBACCO COMPANY RESEARCH DEPARTMENT - Analytical Method No. 3007 DETERMINATION OF CaC03 IN CIGARETTE PAPER PRINCIPLE Standardized 0.2 N hydrochloric acid is added to the paper to extract and react with the CaC03. The excess HC1 is titrated with 0.1 N NaOfl to determine the amount of CaCO3 present. APPARATUS AND REAGENTS pH METER, Corning, Model 12, or equivalent, equipped with glass reference and calomel electrodes. WATER BATH, electric heated, thermostatically controlled BURET, 50 ml capacity. HYDROCHLORIC ACID, 0.2 N. Prepared-and standardized as described in Analytical Method No. ST-2. SODIUM HYDROXIDE SOLUTION, 0.1 N. Prepared and standardized as described in Analytical Method No. ST-1. PROCEDURE Accurately weigh 1.0 g of sample, tear into small pieces and place in 250-ml beaker. Add 75 ml oi' distilled water and 25 ml standardized 0.2 N hydrochloric acid solution. Stir well and place beaker and contents into 60° C water bath for one hour. Remove beaker and allow to cool. Standardize pH meter using pH 4.0 buffer. Immerse electrodes into beaker containing sample and titrate with mixing with standardized 0.1 N sodium hydroxide solution to a pH of 4.0 end point. If less than 10 ml of 0.1 N NaOH is required, add 10 Ml of 0.2 N HC1, stir well and continue titration to end point at a pH of 4.0. Record titration value. CALCULATION % CaCO where: [(VixNi) - (V2xNI)1x (5.004) Vi = Volume of 0.2 N fiCl added. V2 = Volume of 0.1 N NaOH required. Ni = Normality of HC1. N2 = Normality of NaOH. W = Weight of sample. S 1 234 567891)?2_0 30 40 50 60 7080_901_0O20OA~QQ___ 23 4^ 5 6 7 8 9 10_; 2Cl _30 4 i0 50 60_70 BO _9Q__UO ?00~00 µT{1^_ 1 2 3 4 5..6 7 8 9 I'(1 20 30 ~I0 50 6 0 _jQ~(~Q._LQQ~OQ__~•3 ,/QQ
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R. J. REYNOLDS TOBACCO COMPANY RESEARCH DEPARTMENT Analytical Method No. 4001 DETERMINATION OF MOISTURE IN LICORICE PRINCIPLE Moisture is determined .gravimetrically with oven drying. APPARATUS AND REAGENTS OVEN, forced draft, thermostatically controlled CRUCIBLE, Platinum PROCEDURE Weigh one gram of li6orice sample into a tared platinum crucible. Place in an oven overnight at 105° C. Remove from oven and when cool reweigh. Calculate percent moisture as follows: A ^ B A X 100 where M = percent moisture A = original sample weight B == weight of residue 20_30 0 50_60 7_0 ~30 90 10 200 300 ~ 1t U -~~fiiM 1- 1;3 4~7~~6 7~3 9~0 20 30 ~i 50 E~0 (70;}80 90 ti100)lb0 300
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R. J. REYNOLDS TOBACCO COMPANY RESEARCH DEPARTMENT Analytical Method No. 4002 DETERMINATION OF BUTTER FAT IN COCOA POWDER PRINCIPLE The butter fat is extracted from'cocoa with petroleum ether, dried, and weighed. APPARATUS AND REAGENTS PETROLEUM ETHER (Ligroine) AR grade, boiling point range 30-60° C. CHROMATOGRAPHIC TUBES, 20-x 400-or 20-x 200-mm. Coarse frit. FLASKS, 250 ml flat-bottomed with T neck. ASBESTOS, shredded, Gooch grade, acid-washed. PROCEDURE Prepare a 6-8 mn mat of asbestos over the frit of the chromatographic tube. Rinse with distilled water, acetone and petroleum ether. Accurately weigh receiving flask and place beneath the exit tube of the filter. Accurately weigh 2-3 g of the cocoa and add to the tube, washing the,-Areighing pan, the funnel, and sides of the tube with petroleum ether from a wash bottle. Break up any lumps of cocoa with a stirring rod, washing rod with petroleum ether when removed. Add sufficient petroleum ether so about 25 nil volume is above frit. Apply gentle air pressure to top of the tube forcing the ether into the receiver. Repeat the extraction in a similar manner three additional times. Evaporate the petroleum ether either on a steam bath or under reduced pressure on a manifold evaporator. Dry flask for 120 minutes at 100-105° C, allow to cool and reweigh. % Fat = Wt. fat x 100 .gramssample REFERENCE 1. Official Methods of Analysis, Ninth Edition, 1960, p. 152-'53, Method 12.022. Association of Official Agricultural Chemists, Washington, D. C. 2. Cundiff, Robert H., RDM, 1965, No. 34 (APRIL 30) 16 7 8 9 10i20 3D 40 50 60 70 80 90 100 200 300 S 1 2 3 4C R1 1 2 3~~ ~~ G/ 8 l_~ ?0 (3U% 4U 50 60 '/0 ~<~0 90 100 20~ :700 9 M 45 G 789 tl~,(k20 3040 50 60 70 8090 100 200 300
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R. J. REYNOLDS TOBACCO COMPANY RESEARCH DEPARTMENT Analytical Method No. 4003 DETERMINATION OF SPECIFIC ROTATION OF MENTHOL PRINCIPLE The apparent purity of.a menthol sample may be indicated by its specific rotation. This is determined by analyzing the sample in ethanol solution using a polarimeter made by O.C. Rudolph and Sons. APPARATUS AND REAGENTS POLARIMETER, Miodel No. 80 with Spectroscope-Monochromator Model No. 95, O.C. Rudolph and Sons.. POLARIMETER tubes, glass with metal end caps, 10 ml capacity, 8.5 rnm bore, 100 nim length, 7.0 mm end cap aperature. VOLUMETRIC FLASKS, 25-m1 capacity. At3SOLUTE ETHANOL, UiS.P. reagent quality, 200 proof. PROCEDURE Weigh a catchweight of 2.5 grams, accurate to 0.1 mg, and transfer quantitatively to 25 ml volumetric flask. Dissolve metnhol and make to volume with absolute ethanol. Stopper the flask, mix well and let flask stand overnight. Set the spectroscope-monchromator ori the sodium D-line (5893A) by adjusting the wavelength screw setting to 7.63 with the yellow filter in place. Turn the polarimeter on and set the zero point as described in the instrument manual. Set the spectroscope-monochromator with 1.0 mm slit width and green filter in place. Pour sample solution into polar- imeter tube, using normal care not to have a bubble.in the light path. Place cel'l into the polarimeter and determine the observed rotation. CALCULATION (a)t - Vw Where: (a)t = Specific Rotation V w Vol ume of sampl e i n)»l Observed rotation I ; Weight of menthol in grams ~~Y'_l 23~ ~ G7 8 9 10 20 30 40 50GQ70_£'_0 901 f)2Q030_0 ~ ._ Q.,^ RT ~ 1 2 3 4 5(i 7 F~ ~) 1il 20 70 ~l0 50 E_0 7U_!~,t;i)(~ ~~Of;~? ~ Q~)._3_90 J Q f l~; 20 30 40 5U 60 70 90 1(T0 2UQ 300 ~
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R. J. REYNOLDS TOBACCO COMPANY RESEARCH DEPARTMENT Analytical Method No. 4003-1 DETERMINATION OF SPECIFIC GRAVITY BY WESTPHAL BALANCE PRINCIPLE Specific gravity is determined directly by immersing the plummet of the Westphal balance i.nto the liquid and reading the scale on the counter-balancing weight device. APPARATUS AND REAGENTS BALANCE, Specific Gravity, Chain-Gravitometer, Westphal THERMOMETER, Plummet, Reimann CYLINDERS, Glass, 30 x 150 mm PROCEDURE ° First check the level bubble to assure that the balance is level. If adjustment is required, turn the screw-legs until bubble centers. Next check zero point on balance; pointer should remain at center point with weight and chain at zero, and plummet in place. If udjustment is necessary turn set-screw at right end of the beam until balance attains zero conditions. Carefully pour about 60 ml of liquid sample into the glass cylinder. Remove any air bubbles by heating in hot water bath, if necessary. Adjust the temperature of alcohol samples to 20° C. Immerse the plummet into the' liquid. Put the cylinder in place and hang the plummet on the beam, taking care that the plummet does not touch the wall of the cylinder. Adjust the weight and chain~until the beam balances with the pointer at the center point. Read the balance directly with the weight giving unit and first place and vernier giving second, third, and estimated fourth place. Record the temperature indicated by thermometer-Plummet. CALCULATION Weight/Gallon = (Specific Gravity) X (8.34) Ethanol Proo-f, See Table 1, % Isopropyl Alcohol, See Table 2 S_V1 j 2~~4 5 6 78 9 10 20_3U40 50 60 70 80 90 100 200 300 6 7 8 9 l U 20 30 40 50 60~70 80 fi0 100 200300 _ MI~ ~'~i 3~ 9'~B~ 9 l-0 20 30 ~0 50 60. 70 f,0 90 100 200' 300
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TABLE I 'SPECIFIC GRAVITY OF ETHANOL-WATER MIXTURES SPECIFIC GRAVITY @ 20° C VOL. % SPECIFIC PROOF GRAVITY @ 200 C. VOL. % PROOF .84581 85.0 170 .81984 93.0 186 .84428 85.5 171' .81796 93.5 187 .84274 86.'0 172 .81603 94.0 188 .84119 86.5 173 .81407 94.5 189 :83962 87.0 174 .81206 95.0 190 .83803 87.5 175 .81446 .95.5 191 .83643 88.0 176 .80834 96.0 192 .83480 88.5 177 .80620 96.5 193 .83349 89.0 178 .80445 97.0 194 .83183 89.5 179 .80265 97.5 195 .83015 90.0 180 .79985 98.0 196 82845 90.5 181 .79743 98.5 197 . .82670 91.0 182 ~ .79423 99.0 198 .82494 91.5 183 .79249 99.5 199 .82315 92.0 184 .79074 100.0 200 .82170 92.5 185
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TABLE 2 SPECIFIC GRAVITY OF ISOPROPYL AI.COHOL-WATER MIXTURES .SPECIFIC 20/20° C SPECIFIC 20/20° C SPECIFIC 20/20° C GRAVITY VOL. % WT. % GRAVITY VOL % WT. % GRAVITY VOL. % WT. % 1.0000 - 0.0 0.0 .9990 0.8 0.6 .9640 29.4 23.8 .9290 48.5 41.1 .9980 1.6 1.3 .9630 30.1 24.4 .9280 48.9 41.5 .9970 2.4 1.9 .9620 30.8 25.0 .9270 49.4 42.0 .9960 3.2 2.6 .9610 31.4 25.6 .9260 49.8 42.4 .9950 4.0 3.3 .9600 32.1 26.2 .9250 50.3 42.9 .9940 4.8 3.9 .9590 32.7 26.7 .9240 50.7 43.3 .9930 5.6 4.5 .9580 33.3 27.2 .9230 51.2 43.7 .9920 6.5 5.2 .9570 33.9 27.7 .9220 51.6 44.2 .9910 7.3 5.8 .9560 34.5 28.2 .9210 52.0 44.6 .9900 8.1 6.5 .9550 35.1 28.7 .9200 52.5 45.0 1 .9890 8.9 7.1 .9540 35.7~ 29.2 .9190 52.9 45.5 .9880 9.8 7.8 .§530 36.3 29.7 .9180 53.4 45.9 -.9870 10.6 8.4 . 520 36.8 30.3 .9170 53.8 46.3 .9860 11.5 9.1 ..510 37.4 30.8 .9160 54.2 46.7 r1l"l", ~9850 ` 12.3 9.8 .9500 38.0 31.3 .9150 54.7 47.2 _9840 . . 132 105 . 9490 . 38 5 . 31 8 91 40 55 1 47 6 .9830 14.0 11.2 .9480 39.0 . ... 32.3 .9130 55.5 48.0 .9820 14.9 11.9 .9470 39.6 32.8 .9120 56.0 48.5 .9810 15.7 12.6 .9460 40.1 33.3 .9110 56.4 48.9 .9800 16.6 13.3 .9450 40.6 33.8 .9100 56.8 "49.3 .9790 17.4 14.1 .9440 41.1 34.3 .9090 57.3 49.7 .9780 18.3 14.8 .9430 41.6 . 34.8 .9080 57.7 50.2 .9770 19.1 15.5 .9420 42.1 35.2 .9070 58.1 50.6 .9760 19.9 16.2 .9410 42.7 35.7 .9060 58.6 51.0 .9750 20.8 16.9 .9400 43.2 36.1 .9050 59.0 51.4 .9740 21.7 17.5 .9390 43.7 36.6 .9040 59.4 51.8 .9730 22.5 18.2 . .9380 44.2 37.0 .9030 59.8 52.3 .9720 23.4 18.8 .9370 44.7 37.5 .9020 60.3 52.7 .9710 24.2 .19.4 .9360 45.2 38.0 .9010 60.7 53.1 .9700 25.1 20.1 .9350 45.6 38.4 .9000 61.1 53.5 .9690 25.8 20.7 .9340 46.1 38.8 .8990 61.5 53.9 .9680 26.6 21.3 .9330 46.6 39.3 .8980 62.0 54.4 .9670 27.3 22.0 .9320 47.1 39.7 .8970 62.4 54.8 .9660 28.0 22.6 .9310 47.5 40.2 .8960 62.8 55.2 .9650 28.7 23.2. .9300 48.0 40.6 .8950 63.2 55.6 N
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TABLE 2 (CONTIN UE.D ) C, ECIFIC 20/20° C SPECT .FIC 20/20° C tAVITY VOL. % WT. % GRAVITY VOL. % WT. % .8940 63.6 56.0 .8590 77.00 70.75 .8930 64.1 56.5 .8580 77.36 71.17 .8920 64.5 56.9 .8570 . 77.72 71.59 .8910 64.9 57.3 .8560 78.08 72.01 .8900 65.3 57.7 .8550 78.44 72.43 .8890 65.7 58.1 .8540. 78.80 72.85 .8880 66.1 58.6 .8530 79.16 73.27 .8870 66.5 59.0 .8520 79.52 73.69 .8860 66.9 59.4 .8510 79.88 74.11 .8850 67.3 59.8 .8500 80.24 74.54 .8840 67.7 60.2 .8490 80.60 74.95 8830 68.0 60.7 .8480 80.96 75.37 . .8820 68.4 61.1 .8470 81.32 75.79 .8810 68.8 61.5 .8460 81.68 .~ 76.21 .8800 69.2 61.9 .84 50 82.04 76.63 °.8790 69.6 62.3 A 40 82.40 77.04 .8780 69.9 62.8 .84 30 82.76 77.45 (~770 70.3 63.2 .8420 83.12 77.86 ip)760 .8750 70.7 71.1 63.6 .8410 64.0 .8400 83.48 83.84 78.27 78.68 .8740 71.4 64.4 .8390 84.20 79.09 .8730 71.8 64.9 .8380 84.55 79.50 .8720 72.2 65.3 .8370 84.90 79.91 .8710 72.6 65.7 .8360 85.25 80.32 .8700 72.9 66.1 .8350 85.60 80.73 .8690 73.3 66.5 .8340 85.95 81.14 .8680 73.7 67.0 .8330 86.30 81.55 .8670 74.0 67.4 .8320 86.65 81.96 .8660 74.4 67.8 .8310 87.00 82.37 .8650 74.8 68.2 .8300 87.33 82.78 .8640 75.2 68.6 .8290 87.69 83.19 .8630 75.5 •69.1 .8280 88.03 83.60 .8620 75.9' 69.5 .8270 88.36 84.01 .8610 76.3 69.9 .8260 88.69 84.42 .8600 76.6 70.3 .8250 89.02 84.83 SPECIFIC 20/20° C GRAVITY VOL. % IJT. ~ .8240 89.35 85.24 .8230 89.68 85.65 .8220 90.01 86.06 .8210 90.34 86.47 .8200 90.67 86.88 .8190 91.00 87.29 .8180 91.32 87.70 .8170 91.63 88.10 .8160 91.93 88.50 .8150 92.23 88.90 .8140 92.53 89.30 .8130 92.83 89.70 .8120 93.13 90.10 .8110 93.43 90.50 .8100 93.72 90.90 .8040 95.42 93.27 .8030 95.69 93.66 .8020 95.96 94.04 .8010 96.23 94.42 .8000 96.50 94.80 .7990 96.77 95.18 .7980 97.04 95.56 .7970 97.31 95.94 .7960 97.57 96.32 .7950 97.83 96.70 .7940 98.08 97.08 .7930 98.33 97.46 .7920 98.58 97.84 .7910 98.83 98.22 .7900 99.08 98.60 .7890 99.33 98.98 .7880 99.58 99.36 .7870 99.83 99.74 .7863 100.00 100.00 ~ Gravity-temperature coefficient iof 100% alcohol, 20-22° C.: 1-.00086/0 C. Ln t- (s) N m (A)
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R. J. REYNOLDS TOBACCO COMPANY RESEARCH DEPARTMENT Analytical Method No. 4005 DETERMINATION OF REDUCING, NON-REDUCING AND TOTAL SUGARS I(J LICORICFy i PRINCIPLE Sugars are extracted from licorice and determined befo re and after inversion by the Somogyi procedure. APPARATUS AND REAGENTS ! See Analytical Method No. 2005 LEAD ACETATE SOLUTION, saturated; dissolve 65 g of neutral lead acetate in 100 ml of distilled water. SODIUM OXALATE, ACS grade. ~ PROCEDURE Accurately weigh 0.15-0.18 g licorice into a 100 ml volumetric flask. Add 75-80 nil of water and heat until licorice is dissolved. Cool, add 1.0 nil of neutral lead acetate solution and dilute to volume with water. Stoppe r, shake, allow to stand 3-4 minutes, then filter through S&S No. 588 paper. Add approximately 50-75 mg of sodium oxalate to the filtrate, mix, then refilter through Wahtman No. 2 paper. Pipet 5 nil aliquots into 25 x 200 mm test tubes and proceed exactly as described in Analytical Method No. 2005, determining the sugar content both before and after inversion. CALCULATION (a) % Total sugar as glucose (b) % Reducing sugar as glucose Vb-Vs)(G)(2) (Inverted Sample) Sample Wt. _ Vb-Vs)(G)(2) (Non-inverted Sample) Samp~ e 6Ji:. - % Non-reducing sugar as glucose = (a) - (B). Vb = Volume of thiosulfate for blank determination. Vs = Volume of thiosulfate for sample determination. G = Glucose factor. REFERENCES l. Cundiff, R. FI., RDR, 1963, No. 14 (Feb. 15). 7 8 9 10 20 30 40 50 60 70 80 90 100 200 300 _RT 1 23 4 5_G 7_t£,~ 9(10; 20_. 30_ _~I0__50 _6_0_ 70_80 90 _100._200 ,300 t~111 ~~ 1 ? 3~4~5 6 7~3~9 l.U rC1M30 40 50 f0 70 100 ?00 00 ~ ~ __~ ~__._0 9U ~.____._ _ ~ _
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R. J. REYNOLDS TOBACCO COMPANY RESEARCH DEPARTMENT Analytical Method No. 4007 DETERMINATION OF VISCOSITY BY aR00KFIELD VISCOSIMETER G (9 PRINCIPLE Viscosity is determined by measuring the d rag of the liquid on a rotating disk using a brookfield viscosimeter. APPARATUS AND REAGENTS VISCOSIMETER, Brookfield, Model RVF, equipped with RV 1-7 circular spindles. BEAKER, 600-m1 capacity. PROCEDURE shaft to protect its alignment. Attach spindle to loFVer shaft. It is best to lift the shaft slightly while it is held firmly with one hand while screwing the spindle on with the other. Care should be taken to avoid putting side thrust on the Insert spindle in the test material until the fluid's level is at the immersion groove cut in the spindle's shaft. With a disc type spindle it is sometimes necessary to tilt the instrument slightly while immersing to avoid trapping air bubbles on its surface. (You may find i_t more con- venient to immerse the spindle in this fashion before attaching it to the Viscometer). Care should be taken not to'hit the spindle against the sides of the fluid container while it is attached to the Viscometer, since this too can damage the shaft alignment. Level the Viscometer. The bubble level on all models will be of help in this respect. Depress the clutch and turn on the-Viscometer's motor: following the procedure of having the clutch depressed at this point will prevent unnecessary wear. Release the clutch and allo,,,j the dial to rotate until the pointer stabilizes at a fixed position on the dial. The time required for stabilization will depend on the speed at which the spindle rotates: at speeds above 4 RPM thi s wi l l general ly be about 20-30 seconds, while at lower speeds it may take the time required for one' revolution of the dial. It is possible to observe the pointer's position and stability at low speeds while the dial rotates but at higher speeds it will be necessary to depress the clutch and snap the motor switch to stop the instrument with the pointer in view. Very little practice is needed!to stop the dial at the right point.
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Analtyical Method No. 4007 If check readings are required, start the Viscometer with the clutch - still depressed, holding the ori.ginal reading, and then release. This will speed up readings by reducing oscillation of the pointer. If pointer does not stabilize, the material may be either thixotropic or its temperature may not be constant. Having the spindle at the temperature of the test material will eliminate the latter possibility. The viscosity of the test mate wial can easily be obtained by consulting the Factor Finder supplied with the Viscometer for use with all models. ~ rJ%~'3'~5 -i _~ 9 10 20 30 40 50 60 "/0 80 90 100 ?_00 300 _~i 5 6 7~ 9 10 ?.0 30 40~: 60 70_80 ~0~ 1U0-200 300 P4H _T ~2)3~k~>67£0910203U~}050fi0 70 8090~10U200300 ~..._.~_.___
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R. J. REYNOLDS TOBACCO COMPANY RESEARCH DEPARTMENT Analytical Method No. 4009 DETERMINATION OF REFRACTIVE INDEX PRINCIPLE Refractive index may be determined directly using refractometer. APPARATUS AND RFl1GENTS REFRACTOMETER, Bausch & Lomb, Model AE3QE-3L PROCEDURE Adjust temperature of unit to 20° C, if necessary.. Turn on lamp and swing away top prism. Place one drop of liquid sample on bottom prism face, being careful not to touch prism face with glass rod. Reposition top prism, turn coarse adjustment knob until light-dark interface appears. Adjust occluder to remove apparent color. Turn fine adjustment knob until interface intersects cross-hair junction. Press button and read refractive index directly from top scale. ~ ~ ' Y 3 A 5 G 7 8 9 10 2U 3Q ~0 50 GO 70 80 90 100 200 I S ( 1) ~ 300 _40 _50 _60 70 80 90_ 100 200 300 6~`/ 09_ 10 20 30 40 50 60 70 £;0 _90 100 200. 300 ~ ~
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R. J. REYNOLDS TOBACCO COMPANY RESEARCH DEPARTMENT Anal,yti cal Method No. 4009-1 DETERMINATION OF SUGAR SOLIDS BY REFRACTOMETER PRINCIPLE Percent sugar solids is deternJned directly by relating refractive index of aqueous solution, which is measured using refractometer. APPARATUS AND REAGENTS REFRACTOMETER, Bausch & Lomb, Model ABBE--3L PRINCIPLE Prepare sample to be read as in AM 4009. Read bottom scale to obtain sucrose sugar solids directly. To obtain true sugar solids, multiply % reducing sugars (AM 4005) by 0.022 and add to % sucrose sugar solids. ~-W- 6"7 £~-'f U 20 30-40 50 60 70 00; 90 100 200 300 ~ _ _ t2) 3 4 5 6/8 I 9 10 20 30 40 50 60 70 80' 90 100 200 300 ~ _ _ _ _ ~ MN~~`I 34 5 6 7 8 9 10 20 30 40 50 60 70 80 90 100 200 300 +
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R. J. REYNOLDS TOBACCO COMPANY RESEARCH DEPARTMENT Analytical Method No. 4014 DETERMINATION OF HOT AN[) COLD WATER SOLUBLES ~~ AND pH ON LICORICE 0 PRINCIPLE Licorice is extracted with hot or cold water, and the extracted residue determined gravimetrically after evaporation of the solvent. pH is determine directly using a pH meter. APPARATUS AND REAGENTS pH.METER, Corning, Model 12, equipped with calomel and glass reference electrodes. DISH, procelain .OVEN, forced draft, thermostaticalljr controlled FILTER PAPER, S&S No. 589. ° FUNNEL, Buchner. FLASK, Vacuum Filter, 250-•ml capacity.. PROCEDURE Accurately weigh 1.0 g licorice sample into 150-m1 beaker and add 50 ml distilled water (Ambient Temp.). Mix and let stand for about 4-5 hours. Standardize pH meter at pH 4.0 with buffer solution. Immerse electrodes into sample solution and measure pH. Rinse electrodes and dilute sample 'to 100 ml with disti.lled water. Mix and let sample stand overnight. ' Assemble Buchner Funnel and filter flask into vacuum filtration assembly. Moisten filter paper and apply vacuum, then pour sample into funnel to remove solids. Transfer filtrate into tared procelain dish and evaporate solvent on steam bath, then dry in oven set at 105° C for 2 hours. Remove dish from oven, cool, and weigh. This weight is used to determine cold water solubles. Accurately weigh 1.0 g licorice into 150-m1 beaker, and add 100 ml of boiling distilled water, mix and let stand overnight. Proceed to filter and weigh dried residue from filtratc-as for cold water solubles. . '~ CALCULATION : % Water Solubles (Hot or Cold) Wei_qht Resi due Y, 100 Weight Sample _~-~L ~34J(~ 6 7 8 9 10 2030 !10 50_60_70 80 90 10200 300 'ft1y_~1 2_3 4 5 6_7 8 ~9 10 7~ 30r40)'b 0 60 70 8 0 01_00 _ hiFf ~"~ 2 3 4 5.G 7 0 9 10~O!3Q ~ ~U 6U 10_80 90 l0U 2'b0_300 F-- .
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R. J. REYNOLDS TOBACCO COMPANY ' RESEARCH DEPARTMENT Analytical Method No. 4015 DETERMINATION OF ASH IN LICORICE. PRINCIPLE The residue is determined gravimetrically after the sample has been ashed at 600° C. APPARATUS AND REAGENTS MUFFEL FURNACE, electric, thermostatically heated. CRUCIBLE, Platinum. PREIGNITION APPARATUS, constructed from Nichrome wire.heating (electric oven) coil attached to an asbestos board and connected to a 220 volt power supply. The crucible containing the sample is placed under the coils so as to have approximately'one inch clearance and preignition is carried out so as to prevent splattering. PROCEDURE I Place the dry residue in the crucible from the moisture determination above under a preignition apparatus. Allow sample to remain until completely charred (approximately 45 minutes). Place crucible in a muffle furnace at 600° C for 4 hours. Remove from furnace and when cool reweigh. Calculate percent ash as follows: D = A X 100 where D = percent. ash A= original sample weight as above C = weight of residue from muffle furnace NOTE: Ash is reported on sample "as is" and not on a dry weight basis. S'-1 2 _3 4 i~6 7_8 9 10030 40 50 6-0 70 20 90 100 ?00 300 R'f~~~I~2 3~I 5 6 7 0~30 20 30 ~0 5076(~10 00 90 100 200 300 ~' r M0~? 3 4 5 6 7 8~9 j~' 2(I_ 30 ~LO 50 6U %0 &U 90 100 200300~
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R. J. REYNOLDS TOBACCO COMPANY RESEARCH DEPARTMENT Analytical Method No. 4019 COLORINiETRIC PROCEDURE FOR DETERMINATION OF GLYCYRRFIIZIC ACID IN LICORICf'~~ ~ PRINCIPLE Glycyrrhizic acid is hydrolyzed with acid to its aglycon, glycyrrhetinic acid. Glycyrrhetinic acid is extracted with chloroform, and after evapora- tion of the chloroform, a red color is developed by reaction with 80 percent sulfuric acid, ethanol, and vanillin. APPARATUS AND REAGENTS SPECTROPHOTOMETER, Beckman Model B, or equivalent. COREX CELLS, 1 cm., or equivalent.~. TEST TUBES, Lewis-Benedict, 20 x 200 mm. o CHRO1NITOGRAPHIC TUBES, 20 x 200, equipped with coarse frit. ICE BATH, 1-3° C. CONSTANT TEMPERATURE WATER BATH, 65 + 1° C. WATER BATH, 100° C. ~ DIOXANE-WATER SOLUTION, 50% (v/v). SULFURIC ACID, 12N. Add 336 ml of concentrated sulfuric acid to water, and dilute to 1 liter with water. CHLOROFORM, U.S.P., or equivalent. SODIUM BICARONATE SOLUTION, 2%. Dissolve 20 g sodium bica rbonate in 1 liter of water. SODIUM SULFATE, anhydrous, analytical reagent grade. GLYCYRRHET;INIC ACID, standard. ETHANOL, absolute, U.S.P.-N.F. ETHANOL, 200 proof, S.D.A., 3A. VANILLIN, 1%. Dissolve 1 g vanillin, refined, U.S.P., in 100 ml of absolute ethanol. Store in an amber bottle. SULFURIC ACID, 80%. Dilute 200 ml of water with 800 ml of, concentrated sulfuric acid. PROCEDURE si s l .~1 droly Accurately weigh 0.1 g'licorice into a 250-ml Erlenmeyer flask. Add 20 ml of 50 percent dioxane and connect to a reflux condenser. Apply heat and when the licorice is dissolved, add 20 ml 12 N sulfuric acid through the top of the condenser. Reflux vigorously for 1 hour, add 70 ml
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Analytical Method No. 4019 of water, then 100 nil of chloroform through the top of the condenser. Relfux an additional 15 minutes, cool, then transfer the contents of the flask to a 250-mi separatory funnel. Shake vigorously, allow the phases to separte, and draw the chloroform layer into a second funnel . containing 100 ml of 2 percent sodium bicarbonate.. Shake vigorously, and after phase separation, drain the chloroform layer into a chrornato- graphic tube containing 25 g of sodium sulfate, collecting the eluate in a 200 nil volumetric flask. Repeat the extractions with 75, then 25 ml of chloroform, finally adjusting the volume of eluate to 200 ml. 2. Color Development a._ Analvsis for Glvcyrrhetinic Acid_ Pipet 4.0 nil of the chloroform dilution into a 20 x 200 mm test tube, and 4.0 ml of chloroform into a second tube to serve as a blank. Add a boiling stone and place the tubes in the boiling water bath. Evaporate to dryness, removing the last traces of chloroform with a nitrogen stream. Add 5.0 ml of 80 percent sulfuric acid and replace the tubes'in the boiling water bath for 10 minutes. Cool in an ice bath and add 5.0 nil of 3A ethanol by allowing it to run down the side of the tube. Similarly, add 0.5 ml of 1 percent vanillin solution. Replace in the ice bath without mixing. When the ethanol and vanillin have been added to all tubes, mix the first tube and immediately place in a 65° C water bath. Repeat the mixing-of each successive tube and place in the 65° C bath. After the last tube is in the bath, allow all to stand 5 minutes, then remove all tubes and cool to 20-25° C. Determine the percent transmittance at 545 mu for each solution, setting the spectrophotometer at 100 percent transmittance with the blank solution. Determine the micrograms of glycyrrhetinic acid from the standard curve as prepared below. b._ Preparajion of Standard Curve Accurately weigh 100 mg of glycyrrhetinic acid into a 100 nil volumetric flask and dilute to volume with chloroform. Dilute 5.0 ml of this solution to 100 ml with chloroform. Pipet 0.0, 1.0, 2.0, 3.0,. 4.0 and 5.0 nil, respectively, of the dilute solution into 20 x 200 mm test tubes. This corresponds to 0, 50, 100, 150, 200, and 250 micrograms of glycyrrhetinic acid, respectively. Adjust the volume in each tube to 5.0 ml with chloroform, add a boiling stone, and evaporate the chloroform in a boiling water bath. Proceed as described above in analysis for glycyrrhetinic acid. Plot the percent transmittance against the concentration of glycyrrhetinic acid on semi-logarithmic graph paper to obtain the standard curve:
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3 Analytical Method No. 4019 CALCULATION Percent Glycyrrhizic Acid = u9 Glycyrrhetinic Acid from Curve x 5 x 1.75 ~ mg Licorice ~ NOTES It is essential that the blank remain yellow in color if accurate results are to be obtained. If a reddish tint is noted in the blank solution, prepare a fresh vanillin solution. The color developed is stable for at least 1 hour. It is preferable to prepare a new standard curve for each series of analyses in order to maintain close accuracy and precision. It is mandatory to prepare a new standard curve when any of the reagents are changed. REFERENCE 1. Cundiff, R. H., iRDR, 1963, No. 14 (Feb. 15 ~ I ~~`^1~~~t~ jf~}1 0 91'0 20 30 4U 50 6U_70 HU790 100 200 300 Rr~2 3 4 ~/ 0 9 l0 20 30 Ql)50 60 %0_80 90 100 ~200 300 8 9 _TI 0(2U~ 30 40 50 60 70 80~910 . 100 200 300
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R. J. REYNOLDS TOBACCO COMPANY . RESEARCH DEPARTfriENT Analytical Method No. 5002 DETERMINATION OF AMMONIA USING AN AMMONIA ELECTRODE PRINCIPLE: This method offers a direct determination of the ammonia concentration of a liquid by using an ammon-ia-sensitive electrode. It requires only that the electrode be immersed into the liquid and the resulting meter reading be recorded. This meter reading can then be converted to ammonia content. APPARATUS AND REAGENTS ! SPECIFIC ION METER,.Orion Model 407. AMMONIA ELECTRODE, Orion Model 95-10. MAGNETIC STIRRER AND STIRRING BARS. SODIUM HYDROXIDE SOLUTION, 10M. Dissolve 100 g NaOH in 175 ml water. Cool and di l ute to 250 nil. ~ AMMONIA STANDARDS. Standard A - Accurately weigh 2.5132 g NH1,Cl. 'Dissolve in water and dilute to 100 nil in a volumetric flask (solution contains 8.0 mg NH3 per nil). Standard B - Pipet 10 ml of standard A into 100 nil volumetric flask and dilute'to volume (solution contains 0.8 mg NH3 per ml). PROCEDURE FOR AMMONIA IN TOBACCO .Grind the tobacco sample to a fine particle size and accurately weigh approximately one gram. Transfer sample to a 150-ml beaker and accurately add 100 nil of water, initially introducing only a small quantity to wet the tobacco before adding the remainder of the water. Do not use alcohol or.other organic solvents to wet the tobacco. Allow the mixture to stand at least ten minutes with occasional stirring. Add 1 ml of 1011 sodium hydroxide solution; place beaker on stirrer and stir at a fixed rate (moderate, not fast). Immerse electrode to a depth of approximately one inch and move it rapidly from one side of the mixture to the other side once or twice. This is to free any air bubbles which may become entrapped under the electrode when it is immersed. With the calibration knob, adjust meter to center scale reading (-) on the green scale. Pipet 1 nil of the appropriate standard solution into mixture and read green scale of meter. Use standard A for burley tobacco samples and standard B for other tobacco types and for blends.
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Analytical Method No. 5002 CALCUI.ATION Meter Reading X Factor*_ % Ammonia = - Saimpfe Weight *Factor = 0.8 if standard•A is used, 0.08 if standard B is used. PROCEDURE FOR AMP-90NIA IN WATER Pour 100 ml of the sample into a 150-m1 beaker, then analyze in the same manner as described above for tobacco samples. Select a standard that has a known ammonia concentration near the expected ammonia concen- tration of the sample. CALCULATION % Ammonia = Meter Reading X Factor .NOTES 1. As an alternati4, rather than the Standard Addition procedure described above, sample measurements may be made and the results calculai:ed from a calibration curve prepared•from values on a series of standards. However, this requires frequent preparation of a calibration curve and the conditions in the following note (No. 2) are of greater importance. 2.. For best results, always make measurements at nearly the same temperature; use the same size beaker; use the same moderate sitrring rate, and immerse the electrode to approximately the same depth. 3. The pH of the sample-water mixture should be >11.0. . 4. For best results in the Standard Addition procedure, the aliquot of standard solution added should contain one to four times the quantity of ammonia in the 100-mi sample. 5. When not i n use, store the el ectrode i mmiersed i n 0.114 ammon i um chloride solution. Replace the electrode membrane and filling solution every month, or sooner if leakage is observed (detected by color in the electrode storage solution),! After replacing membrane and filling solution, soak the electrode in 0.11.1 ammonium chloride solution for at least one-half hour before use. ~ . I
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3 Analytical Method No. 5002 6. If the electrode is affected by movement of the operator (static electricity), turn the electrode body so that the refer- ence element faces away from the operator. If this does not aleviate the condition, wrap the electrode with aluminum foil• from approximately 4 cm above the bottom to the cable at top of electrode. REFERENCES 1. Harrell, T. Gibson, RDM, 1973, No. 18 (JUNE 1973). 3 4-~~ 0/ 8 9 ll 0; 20 Rfi - 1 2 30 4'0 50 60 70 80 00 100 .200 300 _ ~MI1' 1 2 3 4 5 6 1~ 9~1,O; 20 30 40 E~0 GO 70 BO 90 100 200 300 ~
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R. J. REYNOLDS TOBACCO COMPANY RESEARCH DEPARTMENT . Analytical Method No. 5003 DETERMINATION OF TOTAL AND VOLATILE SOLIDS PRINCIPLE A sample is analyzed gravimetrically by first evaporating the solvent to determine the total solids, then ashing in electric: muffle furnace to determine organic, or volatile, solids. APPARATUS AND REAGENTS OVEN, electric, forced draft, thermostatically controlled. FURNACE, electric muffle, thermostatically controlled. DISH, glazed porcelain, 100 mm diameter, 125 ml capacity. PROCEDURE , Place 100 nil of sample in an ignited and tared dish, and remove the visible solvent by heating on a steam bath. Dry to constant weight at 103° C, cool in a desiccator, and weigh. Drying for one hour at 103° C is usually sufficient. Place the dish and residue into a muffle furnace set at G00° C and ignite to constant weight, usually requiring about 15 to 30 minutes. Remove dish, cool in a desiccator, and weigh. Solids are reported as mg/L. CALCULATION Total Sol ids (mg/L) _ (-Wq=W. ) (.~O) Volatile Solids (mg/L) = (4'~~"W~~(1U00) v where; W, = Tare weight of dish. W? = Weight of dish and residue after 103° C heating. W3 = Weight of dish and residue after 600° C ashing. V = Volume of sample. ~S ~"~1 ~O~i~3o -~6-~ ~(r7o-T0~"'£0(G TZ-1"~"I~ 2~ 3 4 5 6 7 £3 9 0~ 0 30 ~4 O 5 0 6 0 J 0 f>' 0_9 U 1 U 0 2 U:) 3 U 0~' ~U 10 50 GO 70 870 ~ 90 "100 2UU T00
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R. J. REYNOLDS TOBACCO COMPANY RESEARCH DEPARTMENT Analytical Method No. 5006 DETERMINATION OF TOTAL NITROGEN IN WASTE WATER PRINCIPLE The organi c ni trogeneous materi a.1 i nwaste water i s changed to ammonia by Kjeldahl digestion and distillation, and the quantity of ammonia determined titrametrically using 0.1 N HCI. APPARATUS AND REAGENTS SAME AS IN METHOD 140. 2003 HYDROCHLORIC ACID SOLUTION, 0.02 N standardized PROCEDURE Shake waste water sample well to disperse insoluble material. Place 50b ml of sample into 800 ml Kjeldahl flask, add 40 ml sulfuric-salicylic acid mixture, and place on digestion rack at low heat. Concentrate sample by evaporation until about 40 ml of sample remains. .Turn up the heat and proceed using method No. 2003 except titrate with 0.02 N HC1. CALCULATION TKN (mg/1) _(Volume of Titrant) (Normali of Titr_an_t 14000) Voume of Sample -f;-~ _ _ r~~__ ~' ~S ~1i 3, 6 7 8 9 10) 20 3U U 5U GO IO £3U 9U 100 200 300 RT 1 2345G /B9 10120 30 40 50 60 7080 90 100 200 300~ __- .. _00__._ t1H^~ 1 2-3~4 567 0 9 10 40 ~~0 60 10 80 90 'i UU 2UU-3
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R. J. REYNOLDS TOBACCO COMPANY RESEARCH DEPARTMENT Analytical Method No. 5045 DETERMINATION OF METALS IN WASTE WATER PRINCIPLE This method provides for the direct determination of metals in waste water by atomic absorption. APPARATUS AP4D REAGENTS of the concentration ATOMIC ABSORPTION SPECTROPHOTOMETER, Perkin-Elmer Model 214. FLASK, Volumetric, 2000-m1 capacity. FLASK, Erlenmeyer, 125-ml capacity. FILTE:R PAPER, S&S 576, 12.5 cm. NITRIC ACID, concentrated, reagent. LANTHANUM SOLUTION, 20,000 ppm. Dissolve 46.9120 g La203 and 71.5 ml concentrated hydroChloric acid in water and dilute to 2000 ml. PROCEDURE This method can be applied to the analysis of metals in both t,,aste and natural waters by direct aspiration of the water sample into the flame. When the element to be determined exists in very low concentration, it may be desirable to concentrate approxiniately 100 ml of the sample down to a 10-mi volume to allow greater sensitivity. When the element to be determined exists in very high concentration, it will be necessary to make a proper dilution of the water sample in order to reduce sensitivity for the desired metal. Add to each'water sample 0.5 ml concentrated nitric acid, HN03, to prevent the growth of bacteria while the samples are waiting to be analyzed. If the sample appears to have foreign matter suspended in the water, it will be necessary to filter the water through S&S filter paper #576. Caution: Clean disposable vinyl medical gloves must be worn while the fi l ter___pa~~eri bei nU~reLard i n order to avoi d contami nati on from~the ~ an ds . ___ In a normal sample analysis, it is usually necessary to make a dilution of the water in order to determine calcium and potassium. Five nil of 20,000 PPM lanthanum solution which must be added to each 100 ml of volume when the calcium and potassium analysis is desired, may be added when the sample is diluted and before the sample is made to volume. ; Analyze the sample by atomic absorption by procldure described for end determination in Analytical Method No. 2045.
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2 Analytical Method No. 5045 CALCULATION Read the concentration of the element to be determined directly against the appropriate standards. The element that is determined is reported as ug/ml. Where a dilution is required, the concentration of the element is calculated as follows: (i, /ml n diluted solution)(Voiume of diluted solution in ml) ug/ml TVol ume of al i qu'ot taken for di 1 uti on i n rnl~ r _.,.,.y._ .~._.-.__~. _. .._. ~..~_-._ _..-.._ S~1 ?. 3 4`.5 6 7 8 9 1020 30 40 50 60 70 80 90 1,Q02Qp..30_Q_.__._~. T I"' "T~~~t 3~~~ £i 9 l U 2 C7 30 4 0 50 60 7 8090 .1 °`1~}i T'1.'3 _4-~6 T'~ 9~'I0'1Ui30 4U 50 GO 70 80
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