Gradient Operation in HPLC. Dr. Shulamit Levin

Transcription

1 ien Operaion in HPLC Dr. Shulami Levin

2 % Organic ien Operaion in HPLC Inroducion - Opimizing ien Separaions The following diagram illusraes he cycle ime parameers ha are used in a ypical grien Dr. Shulami Levin ien Injec Ouline Typical Problems Encounered in ien Chromaography Inroducion Sraegies for Higher Throughpu ien Separaions o Achieve Maximum Throughpu and Opimal Resoluion sysem soluions mehod soluions Non-reproducible reenion imes Difficulies o ransfer from analyical o narrowbore columns Long reequilibraion imes Long cycle imes (injecion o injecion) More efficien analyses desired

3 Inroducion - Opions o Improve Sample Throughpu Ouline Sysem Soluions: Reduce ien Delay Volume Decrease ime Reduce Injecion Cycle ime Modify Insrumen Use Muliple Parallel Columns Adjus Deecor Sampling Rae Mehod Soluions: Use Shorer iens Use Higher Flow Raes Use Shorer Columns Use a Smaller Paricle Size Decrease Time Increase Temperaure Inroducion Sraegies for Higher Throughpu ien Separaions o Achieve Maximum Throughpu and Opimal Resoluion sysemsoluions reduce grien delay volume decreasere-equilibraion ime reduce injecion cycle ime mehod soluions Tes Probe Srucures Iniial Separaion and Condiions S O N N N N OH -hydroxy-7-aza-benzoriazole H3C O C CH3 CH O HC NH mehyl 3-amino--hiophenecarboxylae O A B C D Condiions: Column: Symmery C, 5 µm,.6 X 50 mm Mobile phase: A=0.% TFA in waer, B=0.% TFA in aceonirile ien: 0-60% B in minues Column emperaure: 30.0 C Flow rae: ml/min. S O NH -mehylbenzene sulfonamide HN C -aminobenzophenone A - -hydroxy-7-aza-benzoriazole B - -mehylbenzene sulfonamide C - mehyl 3-amino--hiophenecarboxylae Deecor: 5 nm Injecion volume: µl H. Weller, Brisol-Myers Squibb Pharmaceuical Research Insiue D - -aminobenzophenone

4 B A C D Proporioning Valve Volumes in an HPLC Sysem Sysem Volume Waers 690 <650 µl Solven delivery Injecor Column Volume Column Deecor Deerminaion of Sysem Precolumn Volume Heigh Definiion: Delay volume is he volume of plumbing beween he poin he grien is formed and he inle of he column Measured by linear grien Heigh Measured by sep grien Exra Column Volume Effec of Precolumn Volume Reducing Delay Volume Volume (µl) Sysem componens affecing dwell volume: Pump ien Mixers Injecor ien Shape and Precolumn Volume Volume (µl) in heory real life n %B ie ien Theoreical ien No column,.00 ml/min 3.9x50 mm,.00 ml/min.x50 mm, 0.9 ml/min Injec Injec Delay Volume Time

5 Ouline Inroducion Sraegies for Higher Throughpu ien Separaions o Achieve Maximum Throughpu and Opimal Resoluion sysem soluions reduce grien delay volume decrease re-equilibraion ime reduce injecion cycle ime mehod soluions %B ien Shape and Delay Volume Time Theoreical ien No column,.00 ml/min 3.9x50mm,.00 ml/min.x50mm, 0.9 ml/min Calculaion ofien Equilibraion Volume is a necessary parof grien chromaography. Boh he HPLC sysem and he column mus be a iniial condiions a hebeginning of each run o ensure reproducible chromaographic separaions. The re-equilibraion volumecan be divided ino wo pars, he sysem washou and he column re-equilibraion. For good sysem/columnequilibraion rr = (3V T + 5Vc)/F where: rris he re-equilibraion imein minues, VTis he oalsysem volume, Vc ishe column volume in ml F is he flowrae in ml/min. column volume = 0.7(πrL/) sysem volume= µl 3.9 x 50 mm. x 50 mm.0 x 50 mm.0 ml/min 0.90 ml/min ml/min Column 0 6 Volume, ml Time, min 3xSysem 5xColumn

6 Reducion of Time Reducion of Time (Approach - Increase Flow Rae) Reduce ime, wo Approaches: ie Injec n. increase flow rae. reduce column volume ie Injec n Column: 3.9 X 50 mm G ra die Injec n ien Time Column: 3.9 X 50 mm G ra die Injec n ien Time ie Injec n Column volume (c.v.) =0.60 ml 5 minue ml/min insrumen delay volume (d.v.) = 650 µl grien volume = x g c.v. =3 Toal re-equilibraion ime, r = {3(0.65) + 5(0.7)(0.6)}/ =.0/ =.0 min. Column volume (c.v.) =0.60 ml 5 minue ml/min insrumen delay volume (d.v.) = 650 µl grien volume = x g c.v. =3 Toal re-equilibraion ime, r = {3(0.65) + 5(0.7)(0.6)}/ =.0/ =.0 min. Reducion of Time (Approach - Increase flow rae) re-equilibraion ime is reduced by 50% Reducion of Time (Approach - Reduce Column Volume) 0 6 Symmery C Alkylphenones 3.9 x 50 mm 0-00%B 3.0 min, 3 ml/min.5 min, ml/min 9.0 min, ml/min ien Injec Column:. X 50 mm Column volume (c.v.) = 0.70 ml 5 minue ml/min insrumen delay volume (d.v.) = 650 µl grien volume = g x c.v. = 0.5 Toal re-equilibraion ime, r = {3(0.65) + 5(0.7)(0.7)}/ =.5 min. ien Injec Column volume (c.v.) = 5 minue Column:. X 0 mm ml ml/min insrumen delay volume (d.v.) = 650 µl grien volume = g x c.v. = 0.35 Toal re-equilibraion ime, r = {3(0.65) + 5(0.7)(0.069)}/ =.0 min. re-equilibraion ime is reduced by 0% 5

7 Reducion of Time (Approach - Reduce Column Volume). x 0 mm. x 50 mm Condiions: Symmery C, 5 µm Mobile phase: A=0.% TFA in waer, B=0.% TFA in aceonirile ien: 0-60% B in 5 minues Column emperaure: 30.0 C Deecor: 5 nm Injecion volume: µl Flow rae: ml/min. Ouline Inroducion Sraegies for Higher Throughpu ien Separaions o Achieve Maximum Throughpu and Opimal Resoluion Increase hroughpu by approximaely 5% by reducing column volume from 0.70 (50 mm lengh) o (0 mm lengh). sysem soluions reduce grien delay volume decrease re-equilibraion ime reduce injecion cycle ime mehod soluions Reducing Reduce Cycle Times by: Programming a sysem purge in he mehod which occurs during he injecion of he sample or... Employing wo columns and performing column swiching. Faser Chromaography - Shorer Cycle Time Samples per day 30 +6% 76 Column: 3.9 X 50 mm 3 ml/min 3 min. grien 0 6

8 Higher Throughpu Through Column Swiching Column: Symmery, C, 5 µl, 9 X 50 mm Flow Rae: 0 ml/min. requires 5 column volumes = 50 ml = 7.5 min. period = unused ime Column swiching can reduce runimes by approx. 30% 0 Injec ien Hold Summary - Sysem Soluions Reducing ien Delay Volume Use 0. mm (5") i.d. ubing inse of 0.5 mm (9") o reduce sysem volume; Shoren all ubing lenghs; column columns Reduce he exra-column volume in he auo-injecor by employing a smaller loop 0 hrs Noe: a second pump mus be employed 0.5hrs 6.5hrs Remove grien mixers Summary - Sysem Soluions (con'd) Inroducion Ouline Achieve Faser ien Chromaography By... Reducing Time Reducecolumn volume Increase flow rae Reducing Cycle Time Program injeciono occur during re-equilibraion Implemencolumn swiching Sraegies for Higher Throughpu ien Separaions o achieve maximum hroughpu and maximize resoluion sysem soluions mehod soluions use shorer griens use higher flow raes use shorercolumns use smaller paricle sizes increase emperaure 7

9 Rs Opimizing Separaions The following diagram illusraes he cycle ime parameers ha were opimized o achieve high hroughpu goals. Injec ien Wha Facors Influence ien RP-HPLC Separaions... Facors Influencing Resoluion in ien RP-HPLC Separaions... Resoluion, Rs, beween wo closelyresolved analyesin grien RP-HPLCis a funcion of column efficiency N, seleciviy a, and he reenion facor: Rs = w N lnα Efficiency Seleciviy Reenion Bc0 + c = %B/minue = % %. 0 + Upon subsiuion of he acual variables ( %/g (grien ime)) for c, grien slope, one can see he relaionship beween grien ime and resoluion, and... Resoluion Dependance on and Flow Rae for a ien Mehod (Symmery C,.6X 50 mm, 5 µm) B. g g...furher derivaizaion of his erm shows he relaionship beween resoluion and flow rae, F, and column lengh, L, or column volume, πrl/. Rs = w lnα I is he effec of hese variables, F, g gand L, ha we will invesigae. B. g N g Efficiency Seleciviy Reenion B. %. 0+ %..πr.l/f + ε Flow [ml/min] 5 6 [min] Rs = w N ln α % B..ε.or.L/F + } g Efficiency Seleciviy Reenion. Effec of changing grien run ime, g. Effec of changing flow rae, F

10 Impac of Reducing (g) on Resoluion : minues : minues 5.50 Condiions: Column: Symmery C, 5 µm,.6 X 50 mm Mobile phase: A=0.% TFA in waer, B=0.% TFA in aceonirile ien: 0-60% B in noed grien ime Column emperaure: 30.0 C Flow rae: ml/min. Deecor: 5 nm Injecion volume: µl Summary - Impac of on Resoluion Resoluion increases as grien ime increases : minues : minue Longes grien ime provides bes resoluion -Shores grien ime maximizes hroughpu -Reducing jus grien ime sacrifices resoluion Throughpu decreases as grien ime increases.50 Impac of Flow Rae (F) on Resoluion Summary - Impac of Flow Rae on Resoluion Flow rae: ml/min. Flow rae: ml/min. Flow rae: 5 ml/min Condiions: Column: Symmery C, 5 µm,.6 X 50 mm Mobile phase: A=0.% TFA in waer, B=0.% TFA in aceonirile ien: 0-60% B in minues Column emperaure: 30.0 C Deecor: 5 nm Injecion volume: µl - Resoluion goes hrough an opimum due o he combinaion of grien expansion and decrease in plae coun Resoluion goes hrough an opimum due o he combinaion of grien expansion and decrease in plae coun Opimum resoluion is approximaely o ml/min for mos pracical separaion problems.50 9

11 Rs Resoluion Dependance on boh Flow Rae and for a ien Mehod (Symmery C,.6 X 50mm, 5 µm) Rs = N ln α w %. B. ε.or.l/f + g Efficiency Seleciviy Reenion.0.00 Reducion of Cycle Time Flow rae: 0.5 ml/min.. ime: 6 min. Flow rae:.0 ml/min. ien ime:.0 min..00 Condiions: Column: Symmery C, 5 µm,.6 X 50 mm Mobile phase: A=0.% TFA in waer, B=0.% TFA in aceonirile Column emperaure: 30.0 C Deecor: 5 nm Injecion volume: µl Flow [ml/min] 5 6 [min] 3. Effec of changing grien run ime, g, and flow rae, F Flow rae:.0 mlmin. ien ime:.0 min. Flow rae: 5.0 ml/min. ien ime:.0 min Flow rae increased proporional o grien ime decrease. Eluion paern is mainained as cycle ime is decreased resuling in an increase in hroughpu..00 Summary - Reducion of Cycle Time To obain he maximum sample hroughpu he grien ime mus be jused inversely proporionally o he flow rae. As shown in he previous slide he sample hroughpu was increased by 00% upon increasing he flow rae o 5 ml/min. and reducing he grien ime o minues. Inroducion Ouline Sraegies for Higher Throughpu ien Separaionso achievemaximum hroughpuand maximize resoluion sysem soluions mehodsoluions useshorer griens usehigher flowraes useshorer columns usesmaller paricle sizes increaseemperaure 0

12 Impac of Column Lengh on Resoluion How Shor is Too Shor? I is no he column lengh which influences he separaion in so much as he number of grien volumes moving across he column. The Number of Column Volumes per Minue Impacs Resoluion Approaches: Approach : ien volume in no proporion o he column volume (grien run ime consan while changing he column lengh). Approach : Scale grien volume in proporion o he column volume (change he grien run ime proporionally wih he column lengh).

13 Column Volume o ien Volume Relaionship (Approach ) -ien volume no scaledo column volume Impac of Column Lengh on Resoluion (Approach ) -ienvolume no scaled ocolumn volume 50 mm column Column volume = 0.5 ml 5 minue ml/min grien volume = g x f.r. = 5 Toal volume = g.v./c.v. = 0 column vols. 0 mm column Column volume = 0. ml 5 minue ml/min grien volume = gx f.r. = 5 Toal volume = g.v./c.v. = column vols ,. x 0 mm. x 30 mm. x 50 mm Condiions: Symmery C, 5 µm Mobile phase: A=0.% TFA in waer, B=0.% TFA in aceonirile ien: 0-60% B in 5 minues Column emperaure: 30.0 C Deecor: 5 nm Injecion volume: µl Flow rae: ml/min. Mainain resoluion by no scaling grien volume proporionally o column volume. However maximun reducion of analysis ime is no realized as when grien volume is scaled. 0.0 Column Volume o ien Volume Relaionship (Approach ) -ien volume scaled o column volume 50 mm column 0 mm column Wha Facors Influence ien RP-HPLC Separaions... Column volume = 0.5 ml 5 minue ml/min Column volume = 0. ml minue ml/min L (column lengh) is varied. ien volume is scaled in proporion o he column volume. grien volume = gx f.r. = 5 grien volume = gx f.r. = Toal volume = g.v./c.v. = 0 column vols. Toal volume = g.v./c.v. = 0 column vols. Rs = w N lnα Efficiency Seleciviy B. %. ε.πr.l/f + g Reenion Terms are consan

14 Impac of Column Lengh on Resoluion (Approach ) -ien volume scaled ocolumn volume Summary - Impac of Column Lengh on Resoluion x 0 mm ien ime = minue. x 0 mm ien ime = minues. x 30 mm ien ime = 3 minues. x 50 mm ien ime = 5 minues Condiions: Symmery C, 3.5 µm Mobile phase: A=0.% TFA in waer, B=0.% TFA in aceonirile ien: 0-60% B in noed ime Column emperaure: 30.0 C Deecor: 5 nm Injecion volume: µl Flow rae: ml/min. -Reduce analysis ime by >50%. -Tre-off: reducion in resoluion Maximum sample hroughpu is realized when he grien volume is scaled proporionally o he column volume ien Delay Time ien di en G ra Final Hold Impac of he Number of Column Volumes on Peak Shape Injec Column:.6 X 50 mm Column volume (c.v.) = 0.3 ml 5 minue ml/min insrumen delay volume (d.v.) = 650 µl grien volume = gx c.v. = 6 Toal sysem volume = 0.7(c.v.) + d.v. =. ml Injec Column:. X 50 mm Column volume (c.v.) = 0.7 ml 5 minue 0. ml/min insrumen delay volume (d.v.) = 650 µl grien volume = gx c.v. = 6 Toal sysem volume = 0.7(c.v.) + d.v. = 0.7 ml x 0 mm Peak Widh for Peak # = Condiions: Symmery C, 5 µm Mobile phase: A=0.% TFA in waer, B=0.% TFA in aceonirile ien: 0-60% B in minues Column emperaure: 30.0 C Deecor: 5 nm Injecion volume: µl Flow rae: ml/min. Column re-equilibraion ime = oal vol./f.r. Column re-equilibraion ime = oal vol./f.r. =.5/ = 0.67/0. =. min. = 3.6 min. grien is delayed by a facor of

15 Rs Rs Reducing he Effec of ien Delay Volume - Makegrien seeperby increasing he flowrae ordecreasing he grien ime Inroducion Ouline Increase flow rae ( o 3 ml/min.) peak widh for peak # = 0.0 peak widh for peak # = peak widh for peak # = Increase grien slope by 50% ( o min. gr) Sraegies for Higher Throughpu ien Separaions o achieve maximum hroughpu and maximize resoluion sysemsoluions mehod soluions use shorer griens use higher flow raes use shorer columns use smaller paricle sizes increase emperaure Impac of Paricle Size (dp) on Resoluion Comparison of Resoluion Dependence on Paricle Size.00 Symmery C, 3.5 µm,. x 50 mm 0.90 Rs (peak A and B)= Symmery C, 3.5 µm, Symmery C, 5 µm, 0.60 Rs (peak B and C)=.79.6 X 50 mm.6 X 50 mm 0.50 Rs (peak C and D)= Max. Max ml/min., 6 min. ml/min., 6 min grien =. grien = 6. A B Flow [ml/min] 5 6 ien Time [min] Flow [ml/min] 5 6 ien Time [min] Symmery C, 5 µm,. x 50 mm Rs (peak A and B)= 6.7 Rs (peak B and C)= 7.3 Rs (peak C and D)= 6.5 C A B C D D Condiions: Columns: Symmery C, 5 µm,.6, X 50 mm and Symmery C 3.5 µm,.6 X 50 mm Mobile phase: A=0.% TFA in waer, B=0.% TFA in aceonirile ien: 0-60% B in minues Column emperaure: 30.0 C Deecor: 5 nm Injecion volume: µl Flow rae: ml/min. -Achieve increased resoluion wih he smaller paricle size maerial in he same grien ime -Increase hroughpu and resoluion wih smaller paricle size if flow rae is increased

16 Summary - Impac of Paricle Size on Resoluion Inroducion Ouline Resoluion is increased as a resul of using a smaller paricle size. This is due o he increase in he number of heoreical plaes. If he flow rae is increased as well as he paricle size being decreased, an increase in sample hroughpu is realized wih increasing resoluion. Sraegies for Higher Throughpu ien Separaions o achieve maximum hroughpu and maximize resoluion sysem soluions mehod soluions use shorer griens use higher flow raes use shorer columns use smaller paricle sizes increase emperaure Impac of Temperaure C Faser Separaion Narrower Peaks Lower Backpressure Lower Viscosiy eaer Sensiiviy- Summary - Mehod Soluions To obain he fases hroughpu: increase flow rae decrease column volume decrease paricle size scale grien volume wih decrease in column volume increase emperaure o reduce viscosiy of mobile phase allowing increases in flow rae 5