Liquid Chromatography - Instrumental Analytical Chemistry - Lecture Slides, Slides of Analytical Chemistry

Download Liquid Chromatography - Instrumental Analytical Chemistry - Lecture Slides and more Slides Analytical Chemistry in PDF only on Docsity! LIQUID CHROMATOGRAPHY Increasing polarity Water-insoluble \ Water-soluble Monpolar : Tonic re Nonionic polar a 10? 15 Molecular weight Ss 5 (Gel permeation) (Gel Filtration) 108 Figure 2-1 Applications of liquid chromatography. (Prov D. 1. Seumdory, in Chromatography, itd ad, E. Heftrmann, Hd, p. 81. New York: Van Nostrand Reino, 1575, Withe praredasdene.) . Docsity.com 20 Lo Particle Size and Plate Height 44,7 um 34,9 uu 22.6 jum __ 13.2 am ——G _. Md KA — =I Sa zr alan 0 1.0 2.0 hu 40 Linear velocity, cms Figure 28-2 Ecfect of particle size ot packing and flow rate upon plate height # in liquid chromatography. Column di- mirtisions: 30 crm & 2.4 mim. Solute: NW, N-dietliy)-n-amizia zohenzcne Mobile phase: mix- ture of hexane, methylene chdo- Tide, isapropylalcaho), (From &, E, ifajors, J. Chaematogr. Sci., 1973, 22, 82, WHA perercigs ton} Docsity.com ¢ Gradient Elution (a0 Gradiant élusion Prok identily 1. Benzene 2. Monochlorubenzens 3. Oxthuctichlorohantens 4. 1,3,3-trichiocabenzenc S$. 1 4,a-tachiorebensenz &. 1.2.4 -tthlervbensencs 7. 1,2,3,4-terach inropenzenc 8. 124.8 tetracklorcbonsiw 9. Penmechlocobonsene 10. Hoxachlorgbenzene th} Isveratic chatian 1 1 i 1 _t i a 1 15 20 25 a0 Retention time, min Tigers 2S Tacganovenaciit mm sepaiailon emaoncy py geavternt eluliow. Crahummn: lomo > 201 mom i... presision. bore stainless; packing: 19% Peruaphasc® (5. Sample: Sow of chlovinaved] Genes 11 Loprmopaucal. Latector UV photometer (254 um). Gondivions: temperanite, are, Secssure, [200 pa aren f. J. Kirkfarad, Wocer Practice oft aq. . ula CLummialuurMpily, 6 Sd. ew raex: intcractetce, 171. Reprinted LPOCSity.cOM pernissian of fof weifew fe Soom. Frc) Reciprocating Pump Colomn — | Pulse j Motor Seal A — Smee | Sey is cheek valves Reciprocating =—— Solvent oe 28-6 A reciprocating pump for HPLC, Docsity.com Sample Introduction Load sample Inject sample Figure 28-7 A sampling loop for liquid chromatography. (Courtesy of Beckman Instru- merits, Fullerton, CA,) With the valve handle as shown on the left, the loop is filled from the syringe, and the mobile phase flows from pump to column. When the valve is placed in the position on the right, the loop is inserted between the pump and the col- (By that the mobile phase sweeps the sample onto the column. Docsity.com LC Detectors TABLE 29-1 Performances GE LC Detectors Commercialty fame LoD Mas LOD LE Detectar Available (Commescial cetectorsy (state of the Bute Abtctbemce Yea" 100 pg-l ag I pe — Finoreicence Yest 1-10 pg Wie Hlectrochemical Yest 10 pg-t ng 100 f Reltactivn index Ves 100 og—f jug ling Conductivity ¥es 300 peo! og 300 pe Mass spectrometry Vea? 100 pe-l ag lpe FLIER Yer? lig Vl ag Light scattering? Wes 10 pz 500 ng Optkal activity Ne — lng Flement selective No — 102g Photolonizatton No _— 1 pe-t ag “Man LOTD is ogenl ated for injecuend amps tyes aia equ Bro fimes the orale ng ened wt of 0d grand, 10 WC Asjoctd for oneainaa wl pL informe Te miacboer Le. 2Same dcGnbion as 0, sbere, but Gr dgicced rolmme Ls generally wmaler, “Commariily available for mer cboes LC alsa, ally avedinble, yet comly, 5 kew-angip Hen peattcrme snd siephetomnedry, ALS. Yeung end R. H, Symavec, dal, Chess, 1PR6, 58, 1348 Wath pamiioions Docsity.com 3D Chromatogram Corticosterone Dexamethasone Cortisone Absorbance TSS Lend TITT ITTY I TTTY PUTT TTT TPT PETTITTE 220) 230 240 250 260 270 280 290 300 310 320 Wavelength, nm 28-10 Absorption spectra of the eluent from a mixture of three steroids taken econd intervals. (Courtesy of Hewiett-Packard Company, Palo Alto, CA.) Docsity.com Refractive Index Detector Lens Light Mask | source a Transducer L___ pal] Amplifier and Recorder Reference Optical power supply zero Zero adjust Figure 28-11 Schematic of a differential tefractive-index detector, (Courtesy of Waters Associates, inc., Milford, MA 91757} Docsity.com Normal Phase vs Reverse Phase Docsity.com Chain Length Effect Peak identification 1. Uracil 2, Phenol 3. Acetophenone 4. Nitrebenzene 5. Methyl benzoate Methyi 6 ’ Octy] 6. Toluene Octadecyl 23 17145 ] 2 4 Lotitil it Lotititititipy iy | Libitititititrbiti ti tity | 02468 0 2 4 6 8 10 12 14 1é O 2 4 6 8 1012 14 16 18 20 22 24 Time, min Time, min Time, min 28-15 Effect of chain length on performance of reversed-phase siloxane ns packed with 5-1m particles. Mobile phase: 50/50 methanol/water, Flow rate: 1.0 in. Docsity.com Mobile Phase Properties TABLE 28-2 Properties of Comman Chromategraphic Mabile Phases Refractive Viscosly, Boiling Puluity Eben Solvemt Intex" <P Paint, %C Inder, Strength: 29 Flaoroatkanes4 LZ? 1.29 04-25 HR meg 0.25 Cyclohenanc 1423 6.90 BI 0.04 —92 r-Heuane 1370 0.30 oy O1 an LChloroturane L400 (L432 7B 19 2h Carbon tempciloride 4.459 os 7 iG O.1N #Poopy] echer 1365 | O38 68 24 ous Toleny ape 0.55 119 ad oz Diethyl wither 1.330 1.4 35 28 0.38 Tetoslyare fara 1.405 046 66 AC 0.57 Chloroferm 1443 053 61 41 bag Bthenol 1asg 1.04 78 43 O88 Eby! acetate. 1am 943 F 44 ass Dicxane. Lamu 12 101 48 O56 Methanol Lae O54 Cif al 0.95 Apetonirike. LAL 034 $2 3a 0.65 Nitromethenc 1380 O61 [oi 6.0 fui Bihy.ene glycal 1431 165 182 69 Lit Water 1333 oa 130 102 Lage - oe “The came dir io a enon TEE cf vavosity, | unig, 1 CP = JN got "On Aly Mobipdewsen by U3 xives 2"on 4.0; “Tregration epend poo we olechler welche, Rang= ul rt afran. Docsity.com Bonded Phase Apps Peak indentification 1. Vitamin C 2. Saccharin 3. Caffeine 4. Sodium benzoate D2 4 6 8 10 e, min & (a) Peak indentification . Methy] parathion . Ciodrin Parathion Dyfonate . Diazinen EFN Ronnel . Trithion SA RMP wp Figure 28-17 Typical applice- tions of bonded-phase chro- matography. (a) Soft-drink addi- tives. Colymn: 4.4 * 250 mm paced with polar (nitrile) bonded- phase packings. [socratic solvent 6% HOAC/9496 HO, Flow rate: 1.0 cm3/min, (Courtesy of DuPont Instrument Systers, haratratngion, DE} (b) Organophesphate insecticides. Column: 4.5 % 250 mm paced with 5-pm, Cg, bonded-phase par Hcles. Gradient: 67% CHaOH/33% H20 to 80% OED ET SRI Rov Time, min . ments Fic, Danbury, CT) Both used (b) 254.nm UV detectors, | : Docsity.com Derivatives 48, Anzecing 18. Beammoduteric scld 20. [han inotobotrric acid 21, Tyrozina 22 a Aminobotpric acig | 23. Methionine 24. Valing 27) faeleu cing va 477 oxin Wigewe 28-08 | Choomatogvem of octhopbthalaldshyde sertvative | of 30 amino acids oF plyyst- Sfogicsl dpccbnce. Columno; § umn Cag, Revered-phasc. Schrent Az O05 4d Nag HIP, pea 7.4, 9612.2 CEO ATED). Fiaoretcenee deteckors ex citation 204 xu; enoission 425 miei, Gatepravitesd Wath pevemiratow flows R Pflefir et aly Aime, La, TPO, 13 OG Copyright 199 hy literesariewnt Sclenttfle Crt Hem iccabionas, Bre} Docsity.com Reversed Phase — lon Pair TABLE 28-4 Systems for Reversed-Phase Ion-Patr Chromatograms Sample Mobile Phase Counter-[on § “PeSetonyPie Amines 0.1 M HClOA,O/acetonitrile clo, Bps H,0/CH,0H/H,S0, C)oH,,50, RP Carboxylic acids pH7.4 (CqHy)gN* BP pH74 (CeHy)N* Le Sulfonic acids HyO/C3HOH (Ci sHay(CHa NT BP pH?4 (CoHy),Nt Lb pH3s Bis-{2-ethylhexyl)phosphate ic Dyes PH 2-4; HO/CHOH (Caf),Nt BP "Bonded-phase, bAdsorbed 1-pentanal. bed bis-(2-cthylhexyl}phogphoric acid/CHIC],. Docsity.com IC Suppressor RKegenerant Eluent flow, flow Open screen t | I | Regencrant screen Membrane Eluent sereen Membrane Regencrant Screen Regenerant flow Figure 28-22 A micromembrane suppressor. Eluent flows through a narrow channel that contains a plastic scresD. that reciuces the void volume and appears to increase mas+ transfer tates. The eluent is sepacated from the suppressor solution by 50-ym exchange resins. Regenerant flow is in the direction opposite to eluent flow. (Courtesy of Dionex Co” boration, Sunnyvale, CA.) Docsity.com Indirect Photometric Detection Time, min 0 3 6 9 CTT TT Absorbance ———- Figure 28-24 Indirect photometric detection of several anions by elution. Eluent; 10-3 M disodium phthalate, 10-3 M boric acid, pH 10, Flow rate: 5 mL/min. Sample volume: 0.02 mL. UV detector. Sample ions: (a) 18-1 car- bonate; (b) 1.4-ug chloride, (c) 3.8-~g phosphate; (d) 5-ug azide; (@) 10-ug nitrate. (Reprivted with permission from H. Sail, Anal. Chem., 1985, 55, 240A, Copyright 1983 American Chemical So- ciety) Docsity.com lon Exclusion ) so, Formic eo [™ + Time, min 4 Higere 28-26 An ion-exchusion chromatogram for a mix ture of six weak acids. Docsity.com Applying Size Exclusion CH, (CHy)nCOOH lution time, min {a) (b) Figure 28-28 Applications of size-exclusion chromatography. (a) Separation of fatty acids, Column: polystyrene based, 7.5 x 600 am, with exclusion Hmit of 1 x 108, Mobile phase: tetrahydrofuran. Flow rate: 1.2 mL/min. Detector: refractive index. (b) Analysis of a commercial epoxy resin (n = number monomeric units in the polymer). Column: porous silica 6.2 x 250 tim. Mobile phase: tetrahydrofuran, Flow rate: 1.3 mL/min. Detector: UV absorption. (Courtesy of DuPont Instrament Systems, Wilmington, DE,) Docsity.com  Sample 2 ~4 Sample 1 4 Wa “| Origin line ~ I I 1 1 ! I 1 l 2 i 5 | 2 4 & a a a B A Migration distance, cm (b) / Figure 28:33 _‘Thin-layer clirémadtograms. pe me Docsity.com  More TLC Solvent Sample front application f eee ® Mix e Theobromine e Theophylline 1 . Caffeine * 3Isobutyl-1-methyl xanthine ers e Mix Figure 28-32 Separation of xanthine derivatives on a C-18 reversed-phase plate. Mobile phase: methanol/0.1 M K,HPO, (55:45 v/v). Detection: Jodine va- por, Development time: 1 hr, Rp values: theobromine 0.68, theophylline 0.56, caffeine 0.44, 3-isobutyl-methyl xanthine 0.21. Docsity .com