Complete the following. Clearly mark your answers. YOU MUST SHOW YOUR WORK TO RECEIVE CREDIT.

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1 HE 3 ame Exam Spring 015 omplee he following. learly mar your answers. YOU UST SHO YOU O TO EEIE EDIT. arm-up (3 poins each). 1. The _hermal conduciviy deecor (TD) uilizes a series of heaed filamens in is deecion mechanism.. The _ovas reenion index scales he reenion characerisics of a compound o hose of aliphaic hydrocarbons and provides a means of comparing chromaographic resuls beween labs. 3. _Elecrophoresis describes he movemen of charged species in he elecric field during an E experimen. 4. The _pariion coefficien (disribuion coefficien)_ is an equilibrium consan ha describes he endency for a solue o exis in he saionary phase relaive o he mobile phase during a chromaographic separaion. You mus complee problem 5. (15 poins) 5. Perhaps he single mos imporan advance in separaions science in he pas few decades has been he maing of mass specromeric deecion wih HPL. hy has his been such a significan developmen? ha wo ey challenges mus have been overcome o mae L-S a realiy? The coupling of HPL wih S allows he user o ae advanage of he abiliy o HPL o separae mixures of a variey of classes of compounds (many more han can be separaed using G) wih he powerful quaniaive and qualiaive analysis capabiliies of he S. hile GS had been a worhorse echnique for small molecules, LS opens he door o separaions of large molecules, biomolecules, and ions. The wo ey challenges in coupling he L and S focus on he inerface beween he wo componens. One challenge is ha he maerial ha exis he L mus firs be convered o gas phase ions for he S o be useful and mos forms of L are buil around he separaion of neurals dissolved in soluion. second challenge is he very differen condiions a which each insrumen operaes. In radiional HPL, eluen exis he column a ml/min flow raes, resuling in a large amoun of maerial exiing he L in a shor ime. If all of his maerial were inroduced ino he S, i would be impossible o mainain he high vacuum condiions required o provide a large mean free pah for he ions produced in he S. In mos L-S inerfaces (such as elecrospray and PI), he ions are generaed a or around amospheric pressure and only a fracion of he ions produced are allowed o pass hrough he aperure of a sampler cone ino a porion of he vacuum chamber ha is held a an inermediae pressure. fracion of he ions ha remain are passed hrough a simmer cone ino he main S vacuum chamber.

2 omplee five of he following. e clear and concise. learly indicae which problem is no o be graded. (15 poins each) 6. riefly describe he mechanism of separaion of a mixure of caions, anions, and neurals in capillary zone elecrophoresis. ha parameers can be changed o opimize separaion condiions in ZE? Separaion in capillary zone elecrophoresis resuls from wo phenomena, elecrophoreic mobiliy and elecroosmoic mobiliy. Elecrophoreic flow occurs as a resul of movemen of charges species in he elecric field ha exiss when a high volage is applied across he lengh of he capillary. s a resul of his elecric field, caions are araced o he cahode a he negaive end of he capillary, and anions are araced o he anode a he posiive end. The rae a which ions move depends on heir mass o charge raio (small muliply charged ions move more quicly han large singly charged ions). eurals are unaffeced by he elecric field and do no move as a resul of elecrophoreic flow. Elecroosmoic flow resuls from he general flow of solven (waer) oward he cahode end of he capillary due o he presence of deproonaed silanol groups on he capillary walls and heir aracion for solvaed proons in soluion. Since caions are more highly solvaed han anions, hey end o "drag" solven along as hey move oward he cahode. Since solvaion is a dynamic process, bul solven flow resuls, carrying all species (anions, caions, and neurals) oward he cahode. Eluion order is: caions, neurals, anions. Separaion condiions can be modified by changing he separaion volage, capillary lengh, and o some degree, running buffer composiion. 7. Selecion of a deecor for separaions ofen involves a radeoff beween universaliy (or seleciviy) and sensiiviy. riefly describe why his is so, using examples of specific liquid chromaography deecors o illusrae your poin. Ofen, deecors ha are paricularly sensiive are uned o probe a paricular propery of he analye. For example, fluorescence deecors in L offer wonderful sensiiviy, bu are limied o analyes ha fluoresce. In order o expand he range of analyes ha a deecor sees, i is ofen necessary o probe a propery of he analye ha is less unique o a paricular compound. ypical oucome of his choice is poorer sensiiviy. onsider he refracive index deecion in L. In order o be more universal, i responds o a change in he refracive index of he eluing soluion, however, his response is much less sensiive han ha of a fluorescence deecor. Even new universal deecors, lie he ELSD canno rival he sensiiviy of fluorescence. eally he only deecion scheme which approaches boh he opimum in sensiiviy and seleciviy is mass specromeric deecion, which comes wih is own se of challenges (see problem 5!).

3 8. learly describe one of he wo injecion mehods for chromaography lised below. Include a diagram and a discussion of he ey characerisics of he mehod in your descripion. a. Spli/Spliless injecion for G b. n injecion loop and injecion valve for HPL. a. Spli/Spliless injecion allows he user o deermine wheher all or only a fracion of a sample ha is injeced ino he G maes i ono he capillary. Spli injecion is primarily used o reduce he effecive size of he sample inroduced ino a G capillary. Since he dimensions of he capillary are so small, direc injecion of nea liquids can overload he column and lead o poor separaions. Spli injecions help minimize his by carrying a porion of he sample away prior o i reaching he capillary. Spliless injecion allows mos of he sample o ener he capillary and is useful for more dilue soluions. b. You should indicae ha he purpose for an injecion loop is o improve reproducibiliy (precision) of he volume of sample injeced in o he HPL. Your descripion should include he operaion of load mode, where he injecion loop is filled by he user while effluen from he pump bypasses he loop and injec mode where he mobile phase is allowed o flow hrough he loop and carry he sample on o he column. 9. onsider he an Deemer equaion. hich erm is liely o be he primary conribuor o band broadening in capillary G? Jusify your answer. Does he same argumen hold rue for L and E? hy or why no? The an Deemer equaion describes hree classes of phenomena ha conribue o band broadening in chromaography. The mulipah erm () is independen of flow rae and corresponds o he large number of possible roues and analye may ae as i moves hrough a paced column. s paricle size increases, he difference in lengh of hese roues is magnified, leading o an increased conribuion of he -erm. The -erm is inversely proporional o flow rae and deals wih he conribuion of diffusion of analye in he mobile phase along he axis of he separaion column (longiudinal diffusion). The erm is direcly proporional o flow rae and corresponds o mass ranspor of maerial ino and ou of he saionary phase. Since diffusion coefficiens are very large in he gas-phase, he longiudinal diffusion erm dominaes band broadening in G, regardless of wheher i is in a paced column or capillary. For capillary G, he absence of pacing eliminaes he erm. Fas diffusion also helps o minimize he conribuion of he -erm as well. Since diffusion coefficiens are much smaller in he liquid phase, he relaive conribuion of all hree erms are much more comparable. In E, he absence of saionary phase pacing and pariioning causes he and erms o become negligible. Therefore only longiudinal diffusion plays a significan role.

4 For problems 11 and 1, consider he chromaogram below ha was obained for a reverse-phase HPL separaion on a 5 cm column, using U absorbance deecion. Unreained compounds elue in 0.85 minues omplee he following. a. alculae he number of heoreical plaes for componen. b. alculae he seleciviy facor of compound D over compound. c. alculae he resoluion of compounds and. d. hich compound is he mos polar? Jusify your choice. a: For compound : b: c: = (4 r ) = (4 x 5.63 min) = 10,500 plaes (0. min) = D = ( r ) c - m = ( )min = 1.08 ( r ) - m ( )min s = Z = ( )min = 1.56 = ( )min Or: s = () 1/ (-1) = (10500) 1/ (1.08-1)(5.6) = ) 4*1.08*(1+5.6) d: In reverse-phase separaions, he saionary phase is less polar han he mobile phase, meaning nonpolar compounds will be more srongly reained, while more polar compounds will elue firs. Soooooo, is he mos polar compound. 11. Your boss loos a he chromaogram and maes he following saemen: ell, i is clear o me ha compound is presen a abou 5 imes he concenraion of compound and ha compound is caffeine since i elues a 5.6 minues under hese condiions. Discuss he validiy of his saemen. hile i is rue ha he heigh (and maybe he area) of pea is abou 5 imes ha of pea, here is no guaranee ha he response of he deecor is he same for boh componens. Depending on he mode of deecion, he response may be quie differen. For example, if U absorbance deecion is used and compound has a much larger molar absorpiviy a he deecion wavelengh han compound, i could be he case ha he concenraion of is acually less han he concenraion of. The only way o be sure is o prepare calibraion curves for each componen. hile he reenion ime of a paricular compound is essenially consan under consan condiions, here is no guaranee ha anoher compound canno have he same reenion ime. If you have a good undersanding of he composiion of your sample, his asserion is beer, bu he only way o be sure is o have a deecion scheme ha can provide he ideniy of he compound (such as S). bsorbance Time (min) D

5 Possibly Useful Informaion = log(p 0 /P) = bc = S = L/H 4 L L H 1/.35 4 u u u u H m s s Z / / Z s v = ( e + eo )E = ( e + eo )/L D e eo