Caen ee OAC OFFICIAL METHODS OF ANALYSIS (2010) 4 psi pressure uni surtice stabilizes andthen 69a (10s. Clean cess konbent fn snfaces by vacuuen. bd sry of fier aid iit for 26m dep ad wash cole wth 500 mf 10 ad (O04 Sool ner which itmay be stored, BefOrEUse, sash ‘lum with 250 mn. 1.0, ¢Vaewaa operation may be wed, but ea (10 psi as esse is pele Flow rat of 8 mLxin at Commonly achieved (by Plates Coit with 280 0 thickness of siiea gol G Store le) Sent -erButanol CHCOOH HQ? +14 0. rat Dsalve Fn, riled anitine an 1 PO, As Itemative fo rediing anitne. roe as follows: Dissolve 1 hilin n $0 on acstne unl decotoize wth deeoloizing carbon rt Dans 6, Files dite w $0 mt. ad al diphenylamine Ind HPO Make fies diy or stare a °C inenylanine ICH in $0 ni, aetone aud add St (2) Pesaran of te solaon vortionin 10on S. beaker. Add HDnvL 11.0 0 dssolveand ple ‘tap ofcolumn Fase ina cul eth suction bu do nt Let Aya ly Rinse beaker wth two $ mL potions UO and Free nt ‘lan, Wish wit 300. 7 ‘oth Hm 0% aleool. Evajporte eluate in 50 a. beaker on tea bath in cute of ar oF eiragen, oF by oer apprapriae Trans esidue o 13 100 mes wit ota of fm H.O. porate 0 dryness in bh ca 60°C in current tr orig. Disave reside .1-02 LO (b) Cloymaiograply “Place solvent i) tank 15 min before seting ple. Apply 2 and 6 (2 3 oF 32) nfo test solution to Hate Appy contol pots oF pac and alters hone. prepares those Conta! solutions nay be peeserved by feezng ar dying lace pote plat ieee tank wi solvent feat approaches ‘op of plate Remove plate. dy, and spray thocoughly with col gent. (Cunrion Avoid contact with spray) Let aestone waporae, ad plage i oven at 90°-98°C anil spots ane we leveloped (ca 10 inh (e) Inerpreaton ~ Page honey ill show one oF 2 lage ne: ptay orb brownspotst R038. Any ae steaksorseres 1 spots extending fom onigin provide presunpsve evidence of sresenc of coun sr high cose cotn syrup UIECS,incling igh rutose ytupeciv fom plantscurce mer thaneoe, Its wc, aitional fest mast be cust to confi allortion Xepeat charcoal colunns pretcatnvent as follows: Prepare te excep ash col with 100 mL 25% alco teohol, Any blue Fiscal thi eto, and cash i the 5 ineaks or series of spots extending frow origin on TLC plat ‘llowing this treatment provide conclusive evidence of References: J104C 62, 919, 921(1979% 71, 4811988) Weigh 1 nearest ang Hg te“ ‘Sucans ano Scar PRODUCTS Chapter 4.932 44447 AOAC Official Method 978.17 Com and Cane Sugar Products in Honoy carbon tsotope Ratio Mass Spectrometric Method First Action 1978, Final Action 1979, Alternative |-Batchwise Method Meth is epced by 998.12 {sce 444. 1RAY 444.18 AOAC Official Method 991.41 (C-4 Plant Sugars in Honey Intornal Standara Stable Carbon Iotope Ratio Method ; First Acton 1991 C Final Action 1996 G Method is replaced by 998.12 (see 44.418, 44.4108 - , AOAC Official Method 998.12 G-4 Plant Sugars In Honey Internal Standard 5 Stable Carbon Isotope Ratio Method First Aetion 1998 Method suse dinate ear ane) sugasinboney Results othe ierlaatony sy Supporting aceepuance of the cto ange ~ 0.20 (2.1%) 0-1 9% (13.6 11.25 2.69. RSD, 9.22.90 0% RSD 5122075 Ms b20%6 A. Principle Stable carbon ot ovdes estan hich sable cara isotope ratio vale of the trhole honey iseompared.Thedllrence between these ves he ISCURA index) sa case he Ct sugar content of honey otis ney and protein st be analyzelon te sme instrument io vale Hips slated from honey Honey ‘ternative |-Batehwise Method Final Action 1979, 8. Apparatus 0) Comustion stem, Use oe oF te fallow () Crarg procedave- Vaught hiss manifold including ‘qari const taba ie with CuO in wr fue, bad nigogen tap. automatic Foepler pur, a bigl-vaeuam sure. To prepare HO Tr Crapo, pri Co {sie fm) by rig in lect funneces | hat 00°C. Storein closed bother cooking 12) Soferprocedore=—-Cobstion we: stun wal horse assem" 9m seaedat ne eal Bee we, pores hewing 1 at 380°C. To prepare CO far Sor procedure, ems CuO (ie Fe ¥ pass toh 15 ese ad et 2h at 750°C before we {b) Purificarion system (Craigh.--Glass manifold interconnected with combustion systom inelaing tap, colletion tube, and manoncter [see Figue 998-124 and Geachinicn et Casmochimicn Ate 3, 54-55.1953 (©2010 AOAG INTERNATIONALSUGARS AN SuGAR PRODUCTS Chapter 4,9. 38 [AOAC OFFICIA METHODS OF ANALYSIS (2010) ezeny ‘aa runmace | ease Tara ‘ie Figure 898.124. Carbon combustion and purification system for Craig procedure, (©) Mass spectrometer—Instrument expecially designed or ‘modified for isotope ratio measurement at natral abundance and capable of accuraey of 0.01% of abundance at mas 4, (2) Stondards.—For calibration purposes (available every 3 yeas in amount of 400 mg, except ol, | mL; graphite, 08 g sucrose Ig fom Of. of Standard Reference Materials, National Institute of Stndands and Technology, Gaithersburg, MD 20899, USAy: (I) NIST 19 Limestone BC = =1.95%e aginst Pee Dee Belemnite. (2) NIST 22 Crude Oil-~B°C = -29.73 + 0.0% (3) ANU sucrase—8"C = -10.87 + 0.13§Me. (4) USGS 24 Graphite °C = 15.9 + 0.13§Me. (5) PEFI polyethylene ©. Proparation of Honey (8) Craig procedure. Place 20-50 mg test portion, weighed to nearest 1 mig in erami bot, position Boa in tube, and evacuate system. Admit to 600 mm Hg, tank O putifed over CuO at 700°C fotlowed by liquid mitogen tap. Heat test portion to 2850°C in ‘manifold in tubular furace, condensing CO, inliguidnitrogen tap Recirculat gases over CuO 10-30 min at 850°C. Isolate collection trap and purification system from combustion system and Toepler np by valves, and punp off ©. Cool parfeation trap with solid {©0,- acetone; cool analyte tube with liquid nitogen, Lat coletion texp warn, condensing impurities in solid CO, tap and CO, in analyte tube By Few vere atmosphere Figure 998.128, Integrated system for continuous-flow method, (®OIDAORC TERNATIONAL‘AOAC OFFICIAL METHOOS OF ANALYSIS (2010) (0) Sefer procedure Use 9, (2.9 es) ste ie! Pe eg te pov on ade wal of prepared combustion Ns, sad thn in sip long axis of tbe, Avo pene are tin 3-4 em of open end. Cover tet portion wih 3S & ‘rat and et abe ean horizontal 25min. Place tube dingoven siege 65°C fr 2B. Remove tbes from oven, bad etl. od ‘pty tlodge CuO pice Som wall aca where seal wil= sade. “ite wbes are ail warm, place 1-6 analyte rubes on vac manitldandevscunte by mechanical pumnp3-~4mi,henseal nibs aaaiurch, Place tubes horizontally io oven with Tonse CVG wan test potion and btm ofeach ibe end-to-end Combs’ ‘crpoton ot 585"-590°C er. Lettbes cool inowen thn 18 wrefoo"C. tac ube to vacum purification ine, ands a open witha abe cracker (ee Anal Chem. 48, 1652(1975) ‘asi and analyae CO, as described in C(0) . Determinstion Operate mass spectrometer according 1° inmvetions. Calibrate with 22 standards, BOG), Comet vals ine for zero enrichment i inet syste mining Bete aaiag and sendard valves, ting of major ont mor Penk Signal and combustion of "0 to mass 45 signa Cale manufacturer's Berets standard -t\x 1000 convert aboratory analyse, relative to whatever stand was ‘usa to PDB base with the following eationsiP Boxe rowy” Been) Barut 10" Doxa ron vier By. Bo, ra ero aalyes of test OX) we Peed raativeto PDB, and e-om sanayssof standard {By elave to PDB, andl bx we in pari pe thowsan itrnative I--Continuous-Fiow tethod Final Aetion 1996 ‘A Principle “tent portion i ure by iin automated Damas conbostn, wee eatlyt and sofware alectd pole of Op parton ma chop eto fon source of ras epectrometr Consruous Noy aera dyn and purfcation process wis on-line measurement by “Gaerne op ass spectrometer is completely automate sa Ntware cone, with anayte BC value obtained ieeslY fom computer printout 8. Apparatus Integrated system —Equipped, with automated Dunes oer ren oyster with GC es puricaton, mass spctrometey Gexiped ce modified for isotope ratio measurement tt Tae! cranes, and 1BM-compatile computer software control for wemnion parameters and collation of results (PDZ, Curope Sautes Gene, Europa House, Blecra Way, Crowe, Cheshire CW! (62A, UK; se Figure 998.128). sycans AND SUGRK UU ‘Chapter 44. 9.38 6, Proparation of Test Potion seeps, scarey weigh 3g united honey 10 mast carga in cps, sel nd place on aoe of oie fron unit. piace working standard referenes s8n Ei {Cat aan 2 refrenc stands arin Alternative BO) following every # or fewer et portions. . Determination prt pte ascosting to manatee. ot Fee eo Linn, with compute-cotroed 15 mL pulse of apy (09 999% ied onan beat 1O0C SA varveton stage at 600°C and GC cosa 190°C Ton mens A se gona? are smutaneousy integrated corected or Trekground, 70 contbuion at ass 45, ao ey Git Powers aacnres Because oly one measrement canbe made fo eel ren pions deerined by measuring tt partons of wert Cue Oil apne itself a eferene, Compute printout may bein8°C units Protein ‘A. Apparatus Connfage With orzotl ead oar fr 50 mL ube 0 provide 1500 * & B, Reagents (a) Tungstic acid, sodium salt-—10% Na,WO, 2840. “ay Sarl ac ~0.33M, Die 881i 80440 100 aqueous solution of ©, Determination aprecabe amount of std ater ao presen sain hooey trot 100-150 mes yom stoking ras exe sts materiel heavier tan water wil conariate poten precipitate. Use one of the following oF purification: “Uy Repetitive washing procedare—Aad 4-140 0 TE yarn 0 ml canta tbe ix well, A208 Aer clon end 2.0 mL-0.335M H,SO, 0 sal es i aaereeitely add to honey solution; mix well Swit be in 2 Soec waterbath unl visible le forms with clear supemae, 4 ace roma, oF if superate remains cloudy, ad 0.235M JO in? mL inerernens,repeting esting between adios eth wae in, eae Sin. 1500 nd ent spat Repent was, isn, 2d cei ses ae ra potons of watt, thoroughly Sspersing pels ach time. Me) Dab procedre—Use cellulase dai ingen ane ol wt >12 00, 25mm (fia) * 30 em (Sigs 250-90 re) Hydrate tubing, closely We 2 Knots at one end. Het ee onsy to incipient bol (mzowave oven wsefl), 9048 Sear yo smi, place isc, ie2knotsatends anda gant rn tap water fr 216 hy Transfer contents of sb 10 $0 ge we and centtuge 5 min at 1500 *g, ent Pee se AL bake, end isa elle. Mix 0 mL 10% NeW/e se gral 0.33844 HO, and ato syste, Heat on ot ie a antl abe foe fons with leat supe, Ads sting of azid maybe needed. Transfert SO ra. cenbige incre ene 3 ain at 1500 = g- Discord soperate, SDSSe ‘be throuhy, il abe with wae wel an eas prions for protein isolation and {5 2570 NORE INTERNATIONAL eR‘SucaRs ano SuGaR PRODUCTS Chapter 4, p96. rr ‘AOAC OFFICIAL METHODS OF ANALYSIS 2010) Pace appropriate amount of ron in ceri combustion boat simi that used for honey est portions Combustprotiny sane soto wae for hony. If necessary tol for lr eto at analysis ther waster (Paste pi) washed pellet vith mi ‘evount of wate o sal vie, and pac in olin water? min, x ry protein atleast 3h inca 75°C oven, Clelate apparent C4 ugar content as follows =, BC, = 97) 7 Cl sugars, Y= where "Cp and 8°Cy ate 8C values, Yo, for protein and honey, respectively, and -9.7 is the average 8° value fr corm syrup, Hr. Report negative values from this calewation as OY Product is considered to conan significant C4 sugars (primarily com orcane) nly at or above a value of 7% References: AOAC 61, 46(1978); TL 81988); 72, 907(1989); 14, 6270991). AOAC Int. 78, $43(1992}; 76, 140199). Geochim et Cosmochim Acta 12, 133(1957). Spectroscopy 4, 42(1989), Anal. Chem. 48, 1651(1976); 82, 1389(1980), 444.19 AOAC Official Mothod 958.09 Diastatic Activity of Honey First Action 1956 Final Action 1977 (ote: Do not beat honey foe this determination.) A. Principle Buffered soluble starch-honey solution is inated! and time ‘equred to reach specified ead points determined photometia Results areexpressedasmL. 1% starch hydrolyzed by enzyme in |g honey in 1 1. Apparatus (2) Reaction vessel—~Atach seated side am, 18 x 60 mm, 0 18 + 175 mm test ube. Lower side of sidearm is atached 100 ram om botom ofrbe, making 45° angle wit lower portion of be. () Photoelectric photomerer—With 60 ued fer or 600 am interference filter nd | er ells, ©. Reagents (2) ladine stock soluion Dissolve 8.80 g resublimed ly in 30-40 ml. H,0 containing 22.0 g KI, and dilute to tL with HO. (©) fodine solution ~0.0007M, Dissolve 20 g KI and 5.00 fy Solution, (a), in HO and dilute to $00 mt. Prepare fresh every secon ay, (©) Acetate buffer soltion—pHt $3 (159M), Dissolve 87 g 'NaCH,COO:3H,0 in 400 mL H,0, add ea 105 «ik CH,COOH in ‘0, nd dilute to 500 mL. Adjust pH o 5.30 with NaCH,COO ot CH,COOH, if necessary (4) Sodium chloride solution. Dissolve 14.5 g NaCl in 10 and diate to 500 mt, (€) Storch solution —VWeigh 2.000 g soluble starch (special for Aiastaticpower determination, availabe Got several supplies, nd ‘mix with 90 ml. 1,0 in 250 aL Exlenmeyer, Rapa bring to bp, ‘S201 RONG TERNATIONAL ‘able 958.08. Absocbance values with corresponding olnt i ‘witing solution as muchas possible, Reduce ea! and boi gently 3 min, cover, and let cool to zoom temperate Transfer to 100 mt volumetric flsk and dilate co volume. Observe dtl elosly to limit variation in absorbance (4) values of starch blank, D. Standaraization Piet $ mstarh solution into 10 mil HjO and mix well. Ppet | nil ofthis solution int several 50 mL graduates containing 10m. dilue solution, Mix well and determine water dilaton necessary to produce d value of 0.760 + 0.02, This is standard dilation for ‘arch preparation used. Repeat when changing starch source . Determination Weigh Sg test portion into 20 mi. beaker, dissolve in 10-15 mL 10 and 2.5 ra buffer solution, and transfer to 25 mL. volumetic Mask containing 1.5 ml NaC solution. Dit o volume. (Solution rnust be buffered before addition to NaCl solution) Piet $ mi. starch solution into side atm of reaction tobe and 1 mL test solution into botom of tube, with ere not o mix. Place ‘ube in waterbath 1 minat 40" 02°C; thea mix contents by iting ‘ube back and forth several times. Stat stopwatch. AtS in remove 1 mL aliquot with 1 mL. serological pipet and add reply to 10.00 mt. cite fy solution in $0 mL graduate, Mix, dilute previously determined volume, and determine 4 in photometer. ‘Nottie from mixing of starch and honey to addition of aliquot to {yas reaction time, (Place | mi ppetin reaction tbe for reuse wien later aliquots are taken) Continue taking {ml liqots at intervals nll value of <0.235 is obtained. Table 988.09 shows absorbance values with coresponding end point times, F. Calculations Plot A against ime (in) on rectilinear pape; dea straight line thyough surting 4 and as many poinls sx posible. From graph, etermine time diutedreaction-I mixture reaches 4 of 0.235, Divide 300 by this time to obtain diastase number (ON). [Wotes: AS min reading is suficien! for approximating end point Df test solutions of high DN’ (38) if anlber value i taken soon ‘enough to obtain A of ca 0.20, For accurate results, repeat seterminaion, taking test solutions each min from start. With test solutions of low DN, another reading at 10 min will permit rection of end point by plotting the dita. No additonal readings need be taken until within few mines of end point. Only 2 such readings are needed, The Sin value wil not accurately predit low DNa References: Rood Res, 23, 446(1958), “AOAC 42, 344(1959); 47, 486(1968) Revised: February 2010