Ch25-Note

Ch25-Note - Ch. 25 High-Performance(-Pressure) Liquid...

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Unformatted text preview: Ch. 25 High-Performance(-Pressure) Liquid Chromatography (HPLC) Two pumps for Injection port gradient eluticn Column Ultraviolet Computer for control and display detector 2. Small particles give high efficiency but require high pressure <22: ~=~ <2? | 62> ~=~ c l I i CHINE absorbence li'-]E-'|3'.ICIFI I I Deccmacsl cn Wattle. l‘ _ _ 1— I | | 30 . . i 45 T5 {II 15 Time Is] :b} Time (s; 45- 60 A20 ccmpcunds run on {a} 10—pm—diameter and [bi 5—pm—diameter silica particles. fli- fl h a Plate height as a function of flow rate fer stationary-phase particle size of 10. 5, and 3 pm. N ~ 3 500 Licm} N: number of plates dp {mm} L = cclumn length at, = particle diameter Plate height {pm} N a 2 smallest particle size has the least sensitivity tc flew 3. I’erforlltttlti'c as :t I'llrtt'tim: ul' ptlrlicie riiullluter Particle size Retention time Piste numher Required pressure If“ ism] {mini (N) (hart 5111 Si} 25 OCH] 1‘?! 31‘: Ill 42 [lth E? L5 9‘ 33 Hill} Till Ll} Er 125 flflfl 2 Jill] Theoretical performance of Siam-diameter X 25-Eltt-Iflnp capillary Fur rttiuimum plate height i't'c' solute with commit}: factor A" = 2 and diffusion ctr-efficient = h? X in ' "3 mil-'3 in water-acetnnitrilc elucnl. t-LUURL'H J. i:'. Mach-lair. K. L1 Patel. and .I. W. Jorgensoo. "Litlahigh Pressure Reversed Phase Capillary Liquid Chromatography with I.“ urn Particles." duet. (flit-m. [999. F}. Tilti. fill a column Iivith some particulate matter gravity.r drives solvent through column and allows the separation takes longer and longer for solvent to come through smaller particle size the shorter the plate the more theoretical plates per column the higher the pressure needed The Column Porous titanium trits Guard column removes dust. particles. irreversiva adsorbed impurities. snodlzed alumin um jacket Porous frits distribute liquid evenly Mam mlumn across the area of the column. {plastic} guts of the machine half a centimeter across business end of the machine precolumn on it Porous titanium frit morphologically ho mogeneous ‘ must be packed veryr carefullyr % flow wanted to be as even down as possible carrier liquid coming on to lhe column and comings on EKlt otl the path sophisticated tech in constructing 5- The Stationary Phase Most common is spherical, microporous silica particles. Hydngpn sanded 1|: not sum gmps Schematic oi silica gel. Has up to El pmol of site not groups per square meter. SiDH pit; range is broad. centered around 5. Example of peak "tailing," in this case of alkaloids {organic amines) in chromatogram (all. Less acidic silica with fewer SiDH groups is used for chromatogram its]. dipole pond to polar constituents large range at pita for the hydroxyl groups pretty acidic groups silica particle tor a normal gravity driven chromatogram normal stationary phase highly polar bond tightly to polar groups need a polar solvent to break up analyte different amounts 1lrl'hat allow separaion Scanning electron micrographs of silica particles. [at aggregate of spherical particles with 50% porosity and surface area of 1510 map. to} Spongelilre structure with 70% porosity and sec mzt‘g. Nominal pore sizes are ~1lIJ nm, but distribution is greater in b, which also dissolves more readily in base. Type a Silica Korean-DDS IDimn’ll'ryl-S'lfll Type B Elton Eel-hast Flat-CH3 (Dimefl'Ier1fl-J 'I DDIGE'IIII'I H.125 up 2 DHipmrninn Elfifipg 3 Amrptyllne DEE pg 4 Trimiorarnine I125 up Ushectorreepa1==_.. 12 1-1- 20 2'2 Tin-re (min; 1'] Elfi- EHI 2-! 5— Normal-Phase and Reversed-Phase Chromatography Normal Phase -Plain silica or -Polar stationary phase R = {CH233'NH2 {CH2}3-CN -More polar solvent has higher eluent strength DH Reversed Phase {usual mode} H20 -Nonpolar stationary phase '7' @/ Fl = {CHEJWCHE “El—5‘3“? (CHzerHa __ (GE; __ (CHEhCEHE Silica SIIDCH -Less polar solvent has higher eluent strength Bulky isohutyl groups sterioally protest the silosane bonds {Si-D-Si} from hydrolysis at low pH. homogenized and determined the nature of the polar groups bonded stationary phase most common are the reversed phase ohains very long hydroearons slightly more polar erganie environment normal phase head of silica or if u havoe the pound polar phases interact most stron Iy with polar groups oause things to elute less polar analyte t s more organic the more it will dissolve on the nonpolar stationary phase polar solvents will have lliltle eifeots eluling it nonpolar solvents tend to elule much more efficiently 7. Monolithic Silica Columns {a} {a} Single porous silica rods polymerized in shape from liquid precursors. {b} ~2 pm pores. {c} Enlarged vievv; ~13 nm pores in the solid are not visible. UM1M35 At a given pressure, flow rates are 9 X greater than with 3.5 pm spherical particles and the resolution is the same. silo strvclLJres can be made in a varietyr of wave can be assembled taking silicic acid in a solutions and neutralizing it and forming the lattice inside the solution vessel has the shape can make a single piece ofhighlv porous silica in the column hibrnogeneiljl.r in the lattice so LI can have the greatesl number of plales possible for We space 3- Elution Hon-uni n c: u '3‘ Pcntsni: Hermite Heptune Triehlorotrifluorocthono Toluene ' 'Il ' 'Ilu' ' :”_I Chluml'urrn m 5' ' agreed II"? E cclr'n' Dichloromotl'mne mm Ill EEJE'W”\|:| {‘1' n : Diethyl other FJ! I13] aerial: 33/ Mothyt .r-bulyl other .. Dilnill'lt' Awttmitrih: Acetone Tetrahydrofumn 2-ansutn] Methanol laj UEI I'IIL' lllllil'HLllL'l I;I.I1I.II| let wall: l> [‘II IIIII. Fur :ttlsorliliun t'ItrurImlngrrlplu rm \IIiL'H Elum‘lt strength {5"} i't.l'it'i flfll flfll 0.!sz 1122 1126 0.30 0.4-3 11.43 0.413 11.5 I L152 U53 1153 lift“ 0.?!) [TBIJIE 25-2' I'Tluolrlipit' writ“. :IIIII Illlrun'nIL-i L'tltiil'l' 1.1. Ll'lil'll'llljdlll'i 11F Hoist-II!» Ultraviolet cutoff (urn) I'Jfl W’S 20H '23] 2154 245 235i 215 25E: 2 it} 2 t5 £90 33L} 2 t2 1&5 2E5 ull'w. LN |_ R Snyder. in: Hl'xh-Fr'rfimlmm'r .i'J'e‘Im'rI f'l'rnmlm’ug-mpfiy I'E'. HI1rt-Li.1h.-et5.l.‘~"l1l. 3- [NL-u' ‘r'erk: t'tmjemie Pores. WEE]; mrrtfirt 6E 1m tram .S'rli'n'rtr Cir-Mr. 3rd or] [Mllikrp-‘Ifi. Ml: Hllrdirlc S: Jackson Laboratories. t'flut. Resell. eluent strength inoresses with polarity for normal-phase chromatography; most usually, one uses reverse-phase. so the order of eluent strength is reversed 9- Hardware: Pumps and Injectors Smog-loaded nulllel what: l'. :_L'I|I]I'"I. Syringe I'Itnhe 1I|Ler IUD-uL-mme Diana” reserwtir pislnri Sam.th Load position In|ect FIDSIIJEI‘i “Ii 10. Hardware: Detectors E .l'd'J.‘ ou: Uv Detector Lrgm SUUTUH Detector - - ... ': _':rtr.- =|| Fletractive Index Detector Path taken when TREE Reference out rclrac‘li'fl index Err-h - I 1-. HR F'hctooetl _ “5. Beecher plea: Fle'erenoe In th taken with pure solver“ in bO'II‘l momma-15 ’ Rh :1?” “a / “Sample out mm X Sample In 11- Hardware: Detectors Photodiode arrav ultraviolet detector Concave holographic Pholodiode arrave Devleriom lamp [3? Nebulizer preeeure relied Nllrogefi gee Nohflizer Hemed drill Mm Senate Evaporative Light- Soattering Detector Electrochemical Detector *Working electrode +Stainleee-eteel auxiliary Electrode 'High impedance reference electrode Mass Spectrometer Abeorhance [3.2 fig anthracene Aoeorhanoe 5 Time {min} Fir ItillaCerIe epectr om 245 295 Wavelength {nrnl icl 12. {Table 25-3 Comparison of" rontntereie] HPLC detectors Approximate limit of Idlel;ll;eel:iott‘I USeful with Detector (ng) gradient? Ultravioiet CI. 1~| Yes Refractive index IU-fl—I 000 No Eveporative light—scattering U. 1—I Yes Electrochemical 0.01—1 No Fluorescence I 0.00] —[}.{]I Yes Nitrogen (N NO N03 —:> hv] 0.3 Yes Conductivity (1.5—1 No Mass spectrometry 0.1—1 Yes Fourier transform infrared l 000 Yes a. Most detection limits from E. W. Yeung and R. E. Synosree, “Detectors for Liquid Chromatography” Anni. [Thea-rt. 1936. 58. IZSTA. EMBE- Isoeratic and Gradient Elution This series is carried out with different but oonstant ratios of aqueous buffer and GchN. Note that 35% aoetonitrile is a peer compromise {too much time}. fibflfl'bfllm El 233 r'll'f'l —- TIII'IE- |rTIiI'-'I - Tr'l'e [Ir'rr'll - 'l'n'mtL |r'l'|iI'-'| - TI"II|:' Hart} - E x E I s E E 1 4 5 D IE! 3'. I. I.I-.n. -..I .- .4 J.J .I I I I..I....I-.- .1.LI.I I I.I I I..|-.I..-. .1 . I. I..| I. I.I I. |.. |-.- -.J.I.L I..I.. {l‘ 5 11} I5 3'] 25- 31 35 in 45 g TIn1e1n'Inl—- H E l I:- 5 1U 15 2D 2'5 E 'I—Il'T'Elimil'lfi— E l 3 E E 2 ms 3 E i} 5 11} 15 2‘3 25 33 35 |1|:| 45 3 Timelmnt—- § a- El E a A .D a; . . '1} E! 1U 15 ED 3’5 3': 35 $13} $5 7‘1} Til-nu (minl —u— sin le pump that will pump one solvent mixture through if u ave more than one pump u can have as many solvent mixtures as pumps that leads to a gradient elution Iir'lcl optimum melhod of separaling certain mixture one pomp tr}.r diffemnt mixtures on Clifforsnt samples 14. Gradient Elution Gradient elution is analogous to temperature programming in gas ohromatography. The compounds are the same as on the last slide. 30% oH3oN o 5 ' 1o 15 a) 25 3o 35 Time [mint I- Abeomanoe at 22th nrn _~. II} 5 1D 15 21] 25 30 35 Tune tmint —- 15. Molecular m5 < eooo ii- 5! Method Development — Standard operating Eothlmn Sahbln'm Mancini: DI' Procedure (SOP) '"tflr’stmt mama": n ihnmnucncig {cl-lo:=| l1: GHQ-DH} flamed EI'JLIJIB II'I JIEITID'. WWW mum-phi“ Still-It'll il'l CHISEL acclonilrlc. or normal-phase lmlllems‘flm U'm ottoman-gapin- Iain-,1 acutan chromatin-grow E mam“ Adequate separation in reasonable time Robust method Determine the goal of the analysis Select method of sample preparation Select detector Selection of column, soluentls], gradientls}, traversed-pram cl'lron'alo-grapl'ly C "a. [33. CI. or phalhl I.‘II' Delilah? MEI" 16. r” . . . . Table 25-4 Starting contlltlous tor rerersetlaphase ch rnmatography' Stationaryr phase: Cm or C3 on Satin-diameter spherical silica particles. Less acidic Type B silica {Figure 25—?) is preferred. For operation above 50°C, stericall'}r protected silica (Figure 25—8) is preferred. Column: 0.46 X 15 cm column for 5—p:m particles“ 0.46 X 7".5 cm column for 15—pin particles (shorter run, same resolution) Flow rate: 2.0 mUmin Mobile phase: CHBCNIHED for neutral analytes CHJCNtaqueous bufleri’ for ionic analytes 5 so] 9t: CHECN in H20 to 100% CH3CN for gradient elution Temperature: 35°4U”C it' temperature control is available Sample size: 25—50 p:L containing ~25—50 pg of each analyte a. A [1.30 X I5 cm column reduces solvent consumptitm to {UNI-1.1.413]: = 4334- of the volume required for [JAE—cm diameter: reducing the flow to [0.433(10 mL-"minjl = 0.36 ml..-'min_ h. Buffer is 25—50 mM phosphatefpil 2—3 made by treating1 Elfilr’flL with KUI i. K” is more soluble than Na” in organic solvents and leads to less tailing. Add 0.2 g sodium axide per liter as a preservative if the buffer will not be used quickly. Flmu rate _ I115 mLfmin 5 ‘IEI 15 20 Column length {cm} 35- 13. UMEUBBADEGEDTDBDQQWD AcetnnitriIEfl-i ED i—I—I—l—i—l—i—i—I—I—I [I 20 40 50 SD 1GB MethanaIfl-IED 1—|—|—f—H—|—|—i—|—1—l D 10 20; 3G 46 5!} 65 7’0 3C! 96 100 Tetra Fwd mfu ranfl-IED i—i—fi—i—i—+—+—l—l—+—1 High 0x93 High T “faB —- Low “EB Low T WWW m3 3 SUE-Em»: 32-5273 3 I over 4‘3 mm over 20 min E I C D a E E E 0 IN a E 42 § 3 E E a E 0 5 m 15 2+) 25 30 35 4c: “‘ Time {min} —r 313% E In 32% E 5 over 40 min 150 E 30 G fl . 50 g a] D 3? g 40 .0 E 4: 20 c: ‘ I—I—I—I—I—l—I—I—I—I—l—hI—hI—l—hH—I—l—H—I—l—I—I—I—I—l—I—I—I—I—L CI 5 1C! 15 2'3 25 30 35- ,3 Time {min} —-.- 0 1D 20 30 4D 51] Time from injection {min} ...
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Ch25-Note - Ch. 25 High-Performance(-Pressure) Liquid...

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