Separation. Ch. 23 Fundamentals of Analytical Separations. Extraction Efficiency

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Sepaaion Ch. 3 Fundaenals of Analyical Sepaaions Saples ae usually coplex ixues. In ode o idenify and quanify he coponens of a ixue, we have o sepaae he coponens in he ixue.

1 Sepaaion Ch. 3 Fundaenals of Analyical Sepaaions Saples ae usually coplex ixues. In ode o idenify and quanify he coponens of a ixue, we have o sepaae he coponens in he ixue. Sepaaion ehods Exacion Choaogaphy Elecophoesis Solven Exacion The ansfe of an analye fo one phase o a second based on he elaive solubiliy of he analye in wo iiscible liquids. [ S] ( q) / V K S q / V [ ] n V q V KV A equilibiu: K: he paiion coefficien fo disibuion of S beween he wo phases; q: he facion of S eaining in phase ; n: he # of exacions. If q /4, hen /4 eains in phase afe one exacion Exacion Efficiency A solue S has a paiion coefficien of 3 beween oluene and wae. If you have 00 L of a 0.00 M soluion of S in wae.()wha facion of he solue eains in H O afe a 500 L exacion wih oluene? () Wha facion of he solue eains in H O afe a 5-00 L exacions wih oluene? 00 q % 00 (3)(500) q (3)(00) % I is oe efficien o do seveal sall exacions han one big exacion. ph Effecs The chage changes of an acid o base is dependen on ph. Disibue coefficien (D): an alenae fo of he paiion coefficien. Toal conc.in phase C D Toal conc.in phase C [ B] KKa D K [ B] [ BH ] Ka [ H ] [ B] [ H ] [ B] ( Ka ; K ) [ BH ] [ B] α B [ HA] K[ H ] D Kα [ HA] [ A ] Ka [ H ] [ A ] [ H ] [ HA] ( Ka ; K ) [ HA] [ HA] HA α: facion of he species (P.9) ph Effecs K fo an aine B is 3.0 and he Ka fo BH is If L of 0.00 M aqueous aine is exaced wih 00 L of solven, calculae he % eaining he in aqueous phase in M a () ph 0.00; () ph KKa ph 0.00 : D Ka [ H ] V 50 q 0.5 5% V KV KKa ph 8.00 : D Ka [ H ] V 50 q % V KV

Exacion wih a Meal Chelao Usually neual coplexes can be exaced ino oganic solvens. Chaged coplexes (e.g. MEDTA - ) ae no vey soluble in oganic solvens.

Cown ehes can exac alkali eal ions and can bing he ino non-pola solvens.

M n is in he aqueous phase and ML n is in he oganic phase The disibuion coefficien (D) fo eal ion exacion depends on ph and

Choaogaphy A sepaaion pocess based on he vaious paiioning coefficiens of diffeen solues beween he wo phases.

Saionay phase: A solid o liquid ha eains in place.

2 Exacion wih a Meal Chelao Usually neual coplexes can be exaced ino oganic solvens. Chaged coplexes (e.g. MEDTA - ) ae no vey soluble in oganic solvens. Coonly used: dihizone, 8-hydoquinoline, and cupfeon. Exacion wih a Meal Chelao Coonly used: dihizone, 8-hydoquinoline, and cupfeon. Cown ehes can exac alkali eal ions and can bing he ino non-pola solvens. Exacion wih a Meal Chelao Each ligand can be pesened as a weak acid, HL. M n is in he aqueous phase and ML n is in he oganic phase The disibuion coefficien (D) fo eal ion exacion depends on ph and [ligand]. By selec a ph, you can bing he eal ino eihe phase. Choaogaphy A sepaaion pocess based on he vaious paiioning coefficiens of diffeen solues beween he wo phases. Involving he ineacion of solue(s) and wo phases Mobile phase: A gas o liquid ha oves hough he colun. Saionay phase: A solid o liquid ha eains in place. Type of Choaogaphy Based on he echanis of ineacion of he solue wih he saionay phase Type of Choaogaphy Based on he echanis of ineacion of he solue wih he saionay phase () Adsopion choaogaphy - Solue is adsobed on he suface of he saionay phase (solid). - The songe a solue adsobs, he longe i akes o avel hough he choaogaphy colun () Paiion choaogaphy -GC - he paiioning of solues beween a obile phase (gas) and bonded liquid saionay phase

Type of Choaogaphy Based on he echanis of ineacion of he solue wih he saionay phase Type of Choaogaphy Based on he echanis of ineacion of he solue wih he saionay phase (3) Ion-exchange choaogaphy -

(4) Molecula Size exclusion choaogaphy -size exclusion, gel filaion, o gel peeaion choaogaphy -sepaae olecules by size -lage olecules pass hough fase (hey do no ge caugh up in poes) Type of

saionay phase -Mos selecive (e.g. use anibodies o selec ou one poein fo a ixue of hundeds) The Choaoga A plo of deeco esponse wih ie. Volue flow ae (flow ae): vol.

3 Type of Choaogaphy Based on he echanis of ineacion of he solue wih he saionay phase Type of Choaogaphy Based on he echanis of ineacion of he solue wih he saionay phase (3) Ion-exchange choaogaphy - ionic ineacions o sepaae ions. - a saionay phase of anions will sepaae caions and vice vesa. (4) Molecula Size exclusion choaogaphy -size exclusion, gel filaion, o gel peeaion choaogaphy -sepaae olecules by size -lage olecules pass hough fase (hey do no ge caugh up in poes) Type of Choaogaphy Based on he echanis of ineacion of he solue wih he saionay phase (5) Affiniy choaogaphy -Specific ineacions of one kind of solue olecule o a second olecula ha is covalenly aached o he saionay phase -Mos selecive (e.g. use anibodies o selec ou one poein fo a ixue of hundeds) The Choaoga A plo of deeco esponse wih ie. Volue flow ae (flow ae): vol. of solven pass hough he colun Linea flow ae: he lengh of he colun passed hough by he solven : uneained obile phase avels hough he colun in he iniu possible ie : eenion ie, he ie fo each coponen needed afe injecion of he ixue ono he colun unil ha coponen eaches he deeco : adjused eenion ie, - V: eenion volue, volue of obile phase equied o elue a solue o a axiu fo a colun. V *flow ae The Choaoga Reenion Paaees Adjused eenion ie - Tie spen in he saionay phase (o s ) Relaive eenion - aio of adjused eenion ies fo any wo coponens - he geae he elaive eenion, he geae he sepaaion α Capaciy faco (o eenion faco): - he longe a coponen is eained by he colun, he geae is he capaciy faco. k 3

Exaple: Calculae he adjused eenion ie and capaciy faco fo benzene and oluene in he GC expeien? Mehane (as a solven) peak is a 4 s; benzene a 5 s and oluene a 333 s. k Fo Benzene 5 4 09 s 5 4 5.

9 4 Reenion Tie and Paiion Coefficien The capaciy faco is equivalen o he ie he solue spends in he saionay phase ove he obile phase and can be elaed o he paiion coefficien: C V s s k C V Vs K V k

4 Exaple: Calculae he adjused eenion ie and capaciy faco fo benzene and oluene in he GC expeien? Mehane (as a solven) peak is a 4 s; benzene a 5 s and oluene a 333 s. k Fo Benzene s Fo Toluene k s Reenion Tie and Paiion Coefficien The capaciy faco is equivalen o he ie he solue spends in he saionay phase ove he obile phase and can be elaed o he paiion coefficien: C V s s k C V Vs K V k Relaive eenion can be elaed o eenion ie, capaciy faco, and/o paiion coefficien k K α k K Physical basis of choaogaphy: he geae he aio of paiion coefficiens beween obile and saionay phases, he geae he sepaaion beween wo coponens of a ixue V V V Exaple: If use he open ubula choaogaphy colun, whee ehane (as a solven) peak is a 4 s and benzene peak a 5 s. Calculae he paiion coefficien (K) fo benzene beween saionay and obile phases and he facion of he ie benzene spends in he obile phase. Coss - secional aea of colun V π π 4 (4) μ Coss - secional aea of coaing V s π hickness π(4.5) μ 5 4 V s k 5.0 K K 30 4 V s k k s Facion of ie in obile phase : 0.7 k k 5.0 s Scaling Up Analyical and pepaaive Keep colun lengh consan Coss-secional aea of colun ~ ass of analye ~ volue flow ae (if ainain consan linea flow ae) ~ saple volue applied o colun If change he colun lengh, hen he ass of saple can be inceased in popoion o he incease in lengh Scaling equaion: Mass Radius Mass Radius Diffusion One ain cause of band boadening is diffusion. Diffusion coefficien (D): easues he ae a which a subsance oves andoly fo a egion of high concenaion o a egion of lowe concenaion. Sd deviaion of diffusive band speading: σ D 4

5 Efficiency of Sepaaion Efficiency of Sepaaion Solues oving hough a colun can spead ino a Gaussian disibuion wih a sandad deviaion, σ (K is a consan). Resoluion of wo peaks: Δ ΔV 0.589Δ Resoluion w w w av av / av A esoluion of.5 gives an essenially coplee sepaaion of A and B. Δ ΔV 0.589Δ Resoluion w w w av av / av Colun Efficiency-Theoeical Plaes (N) Main & Synge (94): Teaed a choaogaphic colun as if i wee siila o a disillaion colun ade up of nueous discee bu coniguous naow layes (heoeical plaes). L Plae Heigh L N H Lx L Lx 6L N H σ w 6 N w σ σ H L Nube of Theoeical Plaes σ w/ w / Exaple: A solue wih a eenion ie of 407 s has a base widh of 3 s on a. colun. Find he plae heigh and nube of plaes. 6 N w (6)(407) 3 L. H N Facos Affecing Resoluion Facos Affecing Resoluion - Colun Lengh N Resoluion ( γ ) 4 γ :sepaaion faco Incease esoluion: Incease colun lengh (Squae oo of N) Change phase ineacion Incease capaciy faco (Incease facion of ie solue spends in saionay phase) Doubling he colun lengh inceases esoluion by () / 5

Colun Efficiency- van Deee Equaion van Deee (Duch, 956): B H A Cu u x x - H is plae heigh - u is he flow ae hough he colun - A, Muliple pahs (Eddy diffusion)

colun, analye can diffuse hough any diffeen pahs aound he saionay phase.

Is invesely popoional wih flow ae Equilibaion Tie Van Deee Plo fo Gas Choaogaphy Soe solue is suck in he saionay phase, which falls behind he solue in he

6 Colun Efficiency- van Deee Equaion van Deee (Duch, 956): B H A Cu u x x - H is plae heigh - u is he flow ae hough he colun - A, Muliple pahs (Eddy diffusion) -B/u, Longiudinal diffusion (olecula diffusion) -Cu, Equilibaion ie (esisance o ass ansfe) Muliple Pahs (Eddy Diffusion) Longiudinal Diffusion In a packed colun, analye can diffuse hough any diffeen pahs aound he saionay phase. Solue diffuses fo he high concenaion wihin he band o egions of lowe concenaion on he edges of he band. Is invesely popoional wih flow ae Equilibaion Tie Van Deee Plo fo Gas Choaogaphy Soe solue is suck in he saionay phase, which falls behind he solue in he oving fowad obile phase. Resuling in speading he oveall zone of solue Is popoional o flow ae A inial plae heigh of ~3 is obained wih flow ae of ~35 L/in. Because longiudinal diffusion in a gas is uch fase han diffusion in a liquid, he opiu linea flow ae in gas choaogaphy is highe han in liquid choaogaphy. 6

Asyeic Bandshapes Theoeically, he band coing off a colun should be Gaussian bu his is no always he case This usually occus when he paiion coefficien, K (C s /C ) changes duing he un K can becoe eihe

7 Asyeic Bandshapes Theoeically, he band coing off a colun should be Gaussian bu his is no always he case This usually occus when he paiion coefficien, K (C s /C ) changes duing he un K can becoe eihe bigge o salle K becoes bigge when oo uch solue has been pu ino he colun (oveloading)-so uch solue is dissolved ha he saionay phase acs like he solue K becoes salle due o ailing-his is when he solue binds songly o soe sies on he colun Asyey and K Isohe: a gaph of Cs vs C a a given epeaue Oveloading poduces a gadual ise and an abup fall of he choaogaphic peak (load less solue). A long ail occus when soe sies eain solue oe songly han ohe sies (silanizaion o block OH). Ch. 4 Gas Choaogaphy (GC) GC Pocess In gas choaogaphy, vapo-phase analye is swep hough he colun by a gaseous obile phase (caie gas) Gas-liquid cho (liquid saionay phase) Gas-solid cho (solid saionay phase) The obile phase is usually He, N, o H depending on he applicaion The analye is a volaile liquid o gas ha is injeced hough a sepu (ubbe disk) Scheaic Diaga of GC Open Tubula Coluns Gaseous analye is anspoed hough he colun by a gaseous obile phase. Fused silica (SiO ) coaed wih a polyiide ha can wihsand 350 C. Typically, inne diaees ae and lenghs ae Copaed o packed coluns: give highe esoluion, shoe analysis ie, geae sensiiviy, lowe saple capaciy 7

Effec of Inne Diaee on Resoluion Effec of Colun Lengh on

Tubula Coluns Wall-coaed: liquid saionay phase on inside

solid suppo aached o inside wall of colun Poous-laye: solid

handle lage saples) Saionay Phases Chosen based on he ule ha

Songly pola coluns ae bes fo songly pola solue.

8 Effec of Inne Diaee on Resoluion Effec of Colun Lengh on Resoluion Effec of Saionay Phase Thickness on Resoluion Open Tubula Coluns Wall-coaed: liquid saionay phase on inside wall of colun Suppoed-coaed: liquid saionay phase coaed on solid suppo aached o inside wall of colun Poous-laye: solid saionay phase on inside wall of colun (highe suface aea, handle lage saples) Saionay Phases Chosen based on he ule ha like dissolves like. The silica backbone and he polaiy. Songly pola coluns ae bes fo songly pola solue. As a colun ages, saionay phase bakes off and Si-OH goups becoe exposed (ailing peaks). 8

Pola saionay phase: songly eains he pola solues (alcohols ae songly eained).

9 Reenion Tie The Kovas Reenion Index (I) Reenion index elaes he eenion ie of a solue o he eenion ies of linea alkanes Fo a linea alkane, I 00 # of C aos (ex. fo ocane I800; fo nonane, I900) log (unknown) log ( n) I 00 n ( N n) log ( N) log ( n) Non-pola saionay phase: copounds elue osly based on boiling poin. Pola saionay phase: songly eains he pola solues (alcohols ae songly eained). N: # of cabon aos in lage alkane n: # of cabon aos in salle alkane Exaple: If eenion ies fo ehane, ocane, and nonane in a GC un ae 0.5, 4.3, and 8.5 inues especively, wha is he eenion index fo an unknown ha elues a 5.7 inues? log5. log3.8 I 00 8 (9 8) 836 log8.0 log3.8 Tepeaue Pogaing The epeaue of he colun is aised duing he sepaaion o incease solue vapo pessue Deceases eenion ie Shapens peaks Caie Gas Heliu is he os coon caie gas. The choice is osly dependen on he ype of deeco used. H povides he fases sepaaions and a bee esoluion, bu liied by i s eaciviy 9

Saple Injecion Saple Injecion Sandwich injecion echnique The ai bubble befoe he saple: pevening saple fo volailizing in he

% of he saple; ipuiies do no ge ono he colun in lage concenaions. Spliless injecion: ace analyses < 0.0% of he saple.

Deecos Mos coon: Theal conduciviy deeco (TCD) Flae ionizaion deeco (FID) Ohe deecos: Mass specoee (MSD) Infaed specoee (IRD)

will decease. Response o all analyes, bu sensiiviy is no vey good.

10 Saple Injecion Saple Injecion Sandwich injecion echnique The ai bubble befoe he saple: pevening saple fo volailizing in he injeco oven befoe you injec i. The ai bubble behind he saple: peven saple and solven fo ixing. Spli injecion: analyes ae > 0.% of he saple; ipuiies do no ge ono he colun in lage concenaions. Spliless injecion: ace analyses < 0.0% of he saple. On-colun injecion: go saigh ono he colun ahe han hough an injeco oven; fo saples ha heally decopose. Deecos Mos coon: Theal conduciviy deeco (TCD) Flae ionizaion deeco (FID) Ohe deecos: Mass specoee (MSD) Infaed specoee (IRD) Elecon capue (ECD) Niogen-phosphoous (NPD) Aoic eission (AED) Theal Conduciviy Deeco (TCD) Measues how uch a subsance can anspo hea fo a ho o cold egion. Heliu is he coonly used caie gas (has a nd highes heal conduciviy afe H ) When an analye eeges fo he colun wih i, conduciviy will decease. Response o all analyes, bu sensiiviy is no vey good. Flae Ionizaion Deeco (FID) The os coon deeco fo GC Response nealy all analyes (insensiive o nonhydocabons) Has geae sensiiviy han a TCD. Eluae is buned in a ixue of H and ai. Mos cabon aos (excep CO) poduce adicals ha poduce CHO in he flae: CH O CHO e Deeco Figues of Mei Measue he elecon cuen poduced, which is popoional o he nube of olecules pesen. 0

Ch23. Introduction to Analytical Separations

Ch23. Introduction to Analytical Separations Ch3. Inoducion o Analyical Sepaaions 3. Medical Issue : Measuing Silicones Leaking fo Beas Iplans High olecula ass poly(diehylsiloane), PDMS, [(CH 3 ) SiO] n : Used as GC saionay phase, gels in beas iplans