Anlytil Tehniques hromtogrphy hromtogrphy is n nlytil tehnique tht seprtes omponents in mixture etween moile phse nd sttionry phse. Types of hromtogrphy inlude: thin-lyer hromtogrphy (TL) plte is oted with solid nd solvent moves up the plte olumn hromtogrphy () olumn is pked with solid nd solvent moves down the olumn gs hromtogrphy (G) olumn is pked with solid or with solid oted y liquid, nd gs is pssed through the olumn under pressure t high temperture. The moile phse my e liquid or gs. The sttionry phse my e solid (s in thin-lyer hromtogrphy, TL) or either liquid or solid on solid support (s in gs hromtogrphy, G) In gs-liquid hromtogrphy G the moile phse is inert gs suh s nitrogen, helium, rgon. The Sttionry phse is liquid on n inert solid. Seprtion y olumn hromtogrphy depends on the lne etween soluility in the moving phse nd retention in the sttionry phse. A solid sttionry phse seprtes y dsorption, A liquid sttionry phse seprtes y reltive soluility If the sttionry phse ws polr nd the moving phse ws non- polr e.g. exne. Then non-polr ompounds would pss through the olumn more quikly thn polr ompounds s they would hve greter soluility in the non-polr moving phse. (Think out intermoleulr fores) Retention times nd Rf vlues re used to identify different sustnes. Method: Thin-lyer hromtogrphy ) Wering gloves, drw penil line m ove the ottom of TL plte nd mrk spots for eh smple, eqully sped long line. ) Use pillry tue to dd tiny drop of eh solution to different spot nd llow the plte to ir dry. ) Add solvent to hmer or lrge eker with lid so tht is no more thn m in depth d) Ple the TL plte into the hmer, mking sure tht the level of the solvent is elow the penil line. Reple the lid to get tight sel. e) When the level of the solvent rehes out m from the top of the plte, remove the plte nd mrk the solvent level with penil. Allow the plte to dry in the fume upord. f) Ple the plte under UV lmp or if using mino ids spry with ninhydrin in order to see the spots. Drw round them lightly in penil. g) lulte the Rf vlues of the oserved spots. Wer plsti gloves to prevent ontmintion from the hnds to the plte penil line will not dissolve in the solvent tiny drop too ig drop will use different spots to merge Depth of solvent if the solvent is too deep it will dissolve the smple spots from the plte lid to prevent evportion of toxi solvent Will get more urte results if the solvent is llowed to rise to ner the top of the plte ut the Rf vlue n e lulted if the solvent front does not reh the top of the plte dry in fume upord s the solvent is toxi UV lmp used if the spots re olourless nd not visile R f vlue = distne moved y mino id distne moved y the solvent N Goly hemrevise.org
olumn hromtogrphy () Simple olumn hromtogrphy A glss tue is filled with the sttionry phse usully sili or lumin in powder form to inrese the surfe re. A filter or plug is used to retin the solid in the tue. Solvent is dded to over ll the powder. The mixture to e nlysed is dissolved in minimum of solvent nd dded to the olumn. A solvent or mixture of solvents is then run through the olumn. The time for eh omponent in the mixture to reh the end of the olumn is reorded (retention time) solvent glss tue filled with sli omponents PL stnds for high performne liquid hromtogrphy nd it type of olumn hromtogrphy ommonly used in industry. PL: sttionry phse is solid sili PL: moile phse liquid filter Gs-Liquid hromtogrphy Gs-liquid hromtogrphy n e used to seprte mixtures of voltile liquids. The time tken for prtiulr ompound to trvel from the injetion of the smple to where it leves the olumn to the detetor is known s its retention time. This n e used to identify sustne. Flow ontrol In gs-liquid hromtogrphy, the moile phse is gs suh s helium nd the sttionry phse is high oiling point liquid sored onto solid. Smple in oven Some ompounds hve similr retention times so will not e distinguished. disply Bsi gs-liquid hromtogrphy will tell us how mny omponents there re in the mixture y the numer of peks. It will lso tell us the undne of eh sustne. The re under eh pek will e proportionl to the undne of tht omponent. rrier gs olumn detetor Wste outlet It is lso possile for gs-liquid hromtogrphy mhine to e onneted to mss spetrometer, IR or NMR mhine, enling ll the omponents in mixture to e identified. G-MS is used in nlysis, in forensis, environmentl nlysis, irport seurity nd spe proes. Most ommonly mss spetrometer is omined with G to generte mss spetr whih n e nlysed or ompred with spetrl dtse y omputer for positive identifition of eh omponent in the mixture. N Goly hemrevise.org
Infrred spetrosopy ertin onds in moleule sor infr-red rdition t hrteristi frequenies using the ovlent onds to virte ABVE 500 m - Funtionl group identifition omplited spetr n e otined thn provide informtion out the types of onds present in moleule BELW 500 m - Fingerprinting omplited nd ontins mny signls piking out funtionl group signls diffiult. This prt of the spetrum is unique for every ompound, nd so n e used s "fingerprint". e.g. = 640 750 m - - (id) 500-00 m - Use the IR sorption tle provided in the dt ook to dedue presene or sene of prtiulr onds or funtionl groups Bond Wvenumer A omputer will ompre the IR spetr ginst dtse of known pure ompounds to identify the ompound use spetr to identify prtiulr funtionl groups limited to dt presented in wvenumer form e.g. n lohol from n sorption pek of the ond, - 000-00 = 640-750 - 850-00 - roxyli ids 500-00 Very rod N- 00-500 - Aohols, phenols 00-550 rod 000 500 Spetr for utnl = Asorption or trough in etween 640-750 m - rnge indites presene of = ond Alwys quote the wve numer rnge from the dt sheet - sorptions tend to e rod Asorption or trough in etween 500-00 m - rnge indites presene of - ond in n id Spetr for ethnoi id = N Goly hemrevise.org
Mss spetrometry Mesuring the M r of n orgni moleule If moleule is put through mss spetrometer it will often rek up nd give series of peks used y the frgments. The pek with the lrgest m/z, however, will e due to the omplete moleule nd will e equl to the M r of the moleule. This M pek is lled the moleulr ion Spetr for 4 0 Mss spetrum for utne 4 9 Moleulr ion 4 0 + 58 M+ pek A smll pek will often our in mss spetrum next to the moleulr ion pek. This is due to the presene of the isotope in the moleule. The reltive height of this M+ pek ompred to the M pek n e used to lulte the numer of rons in moleule Use the following eqution to lulte the numer (n) of rons in moleule n = 00 x height of M+ pek. x height of M pek Lern this eqution Exmple A hydroron showed rtio of heights of the M : M+ peks of 9. : 0.5. lulte the numer of ron toms present in one moleule of this hydroron n = 00 x height of M+ pek. x height of M pek n = 00 x 0.5. x 9. =4.89 Round to 5 rons M+ pek If ompound ontins hlorine or romine tom then M+ pek will our due to the two nturlly ourring isotopes of hlorine or romine. hlorine exists s l 5 (75%) nd l 7 (5%) Bromine exists s Br 79 (50%) nd Br 8 (50%) l will hve m/z vlue of M of 50 l 5 nd M+ of 5 l 7 The rtio of heights M:M+ will e : Br will hve m/z vlue of M of 94 Br 79 nd M+ of 96 Br 8 The rtio of heights M:M+ will e : If ompound ontins two hlorine or romine toms then M+ nd M+4 pek will our 4 l will hve m/z vlue of M of 98 4 l 5 l 5, M+ of 00 4 l 5 l 7 nd M+4 of 0 4 l 5 l 7 The rtio of heights M:M+: M+4 will e 9:6: 4 Br will hve m/z vlue of M of 86 4 Br 79 Br 79, M+ of 88 4 Br 79 Br 8 nd M+4 of 90 4 Br 8 Br 8 The rtio of heights M:M+: M+4 will e :: Reltive undnes l 5 l 5 = 0.75 x0.75 = 0.565 =>9 l 5 l 7 = 0.75 x0.5x= 0.75 =>6 l 7 l 5 l 7 l 7 = 0.5 x0.5 =0.065 => smllest to get whole numer rtio l will hve m/z vlue of M of 4 l 5 l 5 l 5, M+ of 4 4 l 5 l 5 l 7, M+4 of 6 4 l 5 l 7 l 7 nd M+6 of 8 4 l 7 l 7 l 7 The rtio of heights M:M+:M+4:M+6 will e 7:7:9: Reltive undnes l 5 l 5 l 5 = 0.75 x0.75x0.75 = 0.49 =>7 l 5 l 5 l 7 = 0.75 x0.75x0.5x= 0.49 =>7 l 5 l 7 l 7 = 0.75 x0.5x0.5x= 0.406 =>9 l 7 l 7 l 7 = 0.5 x0.5x0.5 =0.056 => smllest to get whole numer rtio N Goly hemrevise.org 4
igh Resolution Mss spetrosopy igh resolution mss spetrometry n e used to determine the moleulr formul of ompound from the urte mss of the moleulr ion For exmple, the following moleulr formuls ll hve rough M r of 60, ut more preise M r n give the moleulr formul. e.g. M r = 60.0 moleulr formul = 4 M r M r = 60.0575 moleulr formul = 8 = 60.05 moleulr formul = 4 N igh resolution mss spetrosopy n mesure the mss to 5 d.p. This n help differentite etween ompounds tht pper to hve similr Mr (to the nerest whole numer) Aurte msses of toms: =.0078 =.0000 = 5.9949 N = 4.00 Exmple A ompound is found to hve n urte reltive formul mss of 46.047. It is thought to e either or N N. lulte the M r of eh ompound to 4 deiml ples to work out whih one it is. = (.0000 x ) + (5.9949 x ) + (.0078 x6) = 46.047 N N. = (.0000 x ) + (4.00 x ) + (.0078 x6) = 46.050 Frgmenttion When orgni moleules re pssed through mss spetrometer, it detets oth the whole moleule nd frgments of the moleule. Moleulr ion formed: M [M] +. + e The moleule loses n eletron nd eomes oth n ion nd free rdil Severl peks in the mss spetrum our due to frgmenttion. The Moleulr ion frgments due to ovlent onds reking: [M] +. X + + Y. Reltively stle ions suh s rotions R + suh s + nd ylium ions [R-=] + re ommon. The more stle the ion, the greter the pek intensity. This proess produes n ion nd free rdil. The ion is responsile for the pek The pek with the highest mss/hrge rtio will e normlly due to the originl moleule tht hsn t frgmented (lled the moleulr ion). As the hrge of the ion is + the mss/ hrge rtio is equl to Mr. Mss spetrum for utne 9 4 4 0 = 58 Eqution for formtion moleulr ion 4 0 [ 4 0 ] +. + e m/z 58 Equtions for formtion of frgment ions from moleulr ions [ 4 0 ] +. [ ] + +. m/z 4 [ 4 0 ] +. [ ] + +. m/z 9 Mss spetrum for utnone The high pek t 4 4 due to stility [ ] + of yl group Eqution for formtion moleulr ion [ ] +. + e m/z 7 Equtions for formtion of frgment ions from moleulr ions [ ] +. [ ] + +. m/z 57 9 [ ] + [ ] + 57 [ ] +. 7 [ ] +. [ ] + +. m/z 4 [ ] +. [ ] + +. m/z 9 N Goly hemrevise.org 5
NMR spetrosopy Different types of NMR NMR spetrosopy involves intertion of mterils with the low-energy rdiowve region of the eletromgneti spetrum NMR spetrosopy is the sme tehnology s tht used in mgneti resonne imging (MRI) to otin dignosti informtion out internl strutures in ody snners There re two min types of NMR. NMR. (proton) NMR There is only round % in orgni moleules ut modern NMR mhines re sensitive enough to give full spetr for The spetr is simpler spetrum thn the NMR The rdio wves used in proton nmr use the hydrogen nuleus to hnge its spin stte. Equivlent ron toms. In NMR spetrum, there is one signl (pek) for eh set of equivlent toms. d d 4 peks peks 4 peks, dinitroenzene, dinitroenzene,4 dinitroenzene N l peks peks N 4 peks peks d peks d e N 5 peks N Goly hemrevise.org 6
Equivlent ydrogen toms. In n NMR spetrum, there is one signl for eh set of equivlent toms. In ddition the intensity (integrtion vlue) of eh signl is proportionl to the numer of equivlent toms it represents. Ethnol hs groups of different hydrogen toms d d 4sets of equivlent s: rtio 6::: signl d sets of equivlent s: rtio ::9 sets of equivlent s: rtio :: sets of equivlent s: rtio :: Br d 4 sets of equivlent s: rtio ::: Solvents Smples re dissolved in solvents without ny toms, e.g. l 4, Dl. This mens tht in the NMR the solvent will not give ny peks The sme solvent is used in NMR nd in this se there will e one pek due to the solvent tht will pper on the spetrum. owever, it is known where this pek is so it n e ignored. In the exm it is likely this pek will not our on the spetr. lirtion nd shift A smll mount of TMS (tetrmethylsilne) is dded to the smple to lirte the spetrum TMS is used euse: its signl is wy from ll the others it only gives one signl it is non-toxi it is inert it hs low oiling point nd so n e removed from smple esily Si tetrmethylsilne The sme lirtion ompound is used for oth nd NMR The spetr re reorded on sle known s the hemil shift (δ), whih is how muh the field hs shifted wy from the field for TMS.. The δ is mesure in prts per million (ppm) is reltive sle of how fr the frequeny of the proton signl hs shifted wy from tht for TMS. 0 9 8 7 6 5 4 d hemil shift (ppm) 0 N Goly hemrevise.org 7
NMR shift The δ depends on wht other toms/groups re ner the more eletronegtive groups gives greter shift. δ ppm Proton exhnge using D δ ppm If D is dded to smple then proess of proton exhnge hppens with the in ny - nd N- onds. This hs the effet of removing the peks from the - NMR spetr. This n help with the identifition of nd N peks on the spetr. Addition of D to the smple of Propnoi id would mke the pek t δ =.7 (ppm) in the ove spetrum dispper N Goly hemrevise.org 8
NMR shift 60-0 0-45 50-90 0-45 It will not e possile to identify the ext ron orresponding to eh pek if severl rons re in the sme rnge ppm It is not possile to distinguish etween similr shifts for eh ron in enzene ring. In this exmple it should e possile to work out there re four different rons in the enzene ring nd these orrespond to the four peks etween 0-45 4 peks etween 0-60 N Goly hemrevise.org 9
Spin-Spin oupling in NMR In high resolution NMR eh signl in the spetrum n e split into further lines due to inequivlent s on neighouring toms. Splitting of pek = numer of inequivlent s on neighouring toms + signl singlet doulet triplet qurtet quintet pperne Split numer of peks numer of neighouring inequivlent toms 4 5 0 4 reltive size : :: ::: :4:6:4: Nulei in identil hemil environments do not show oupling mongst themselves! The pek due to group will e triplet s it is next to ( ron with s) The pek due to group will e qurtet s it is next to ( ron with s) The pek due to group will e singlet s it is next to ron with no s) For 6 split peks use the term hextet or multiplet The pek due to group will e triplet s it is next to ron with s Shift 0.7-. Integrtion tre ppm The pek due to group will e singlet s it is next to ron with 0 s Shift.-.6 Integrtion tre The pek due to group will e qurtet s it is next to ron with s Shift.7-4. Integrtion tre ydrogens onded to Nitrogen or xygen usully do not ouple with other protons nd pper s singlets on the NMR spetr The pek due to group will e triplet s it is next to ron with s Shift 0.7-. Integrtion tre The pek due to group will e qurtet s it is next to ron with s Shift.7-4. Integrtion tre The pek due to group will e singlet s the ydrogen is onded to n oxygen nd this does not split Shift 0.5-5.0 Integrtion tre Ethnol You will not e sked to interpret splitting ptterns for the protons tthed to enzene ring N Goly hemrevise.org 0
Bringing it ll together. Work out empiril formul Elementl nlysis 66.6%.8%.9%. Using moleulr ion pek m/z vlue from mss spetrum lulte Moleulr formul moleulr ion pek m/z vlue= 44 66.6/.8/.9/6 =5.555 =.8 =.86875 =4 =8 = Mr empiril formul 4 8 = 7 If Mr moleulr formul 44 then ompound is 8 6. Use IR spetr to identify min onds/funtionl group 8 6 ould e n ester, roxyli id or omintion of lohol nd ronyl. Look for IR spetr for = nd - onds There is = ut no - sorptions, so must e n ester. - = 4. Use NMR spetr to give detils of ron hin 4 peks only 4 different environments. singlet of re 9 At δ =0.9 Mens groups 9 Pek t δ 4 shows Are suggests Qurtet mens next to Pek t δ. shows = Are suggests Singlet mens djent to with no hydrogens Pek t δ. shows R- Are mens Triplet mens next to 5 4 δ ppm Put ll together to give finl struture N Goly hemrevise.org