Gradient peaks

Figure 4.36 Example of an optimum linear-solvent-strength gradient. Peak Identification 1 benzyl alcohol 2=2-pbenylethanol 3 = o-cresol 4 nitrobenzene 5 diethyl o-phthalate 6 - benzophenone 7 = naphthalene 8 = biphenyl and 9 = anthracene. (Reproduced with permission fr< ref. 557. Copyright Elsevier Scientific P d)lishlng Co.)...

The second solvent is too weak, so that peaks continue to appear after the end of the gradient (peaks 7 and 8 are eluted isocratically and are highly dispersed). 

The gradient peak capacity is significantly higher than the isocratic peak capacity, P , within the same time limits, as illnstrated in Figure 5.10. 

The gradient pattern is highly reproducible. We have also demonstrated that UDP-galactose GM2 galactosyltransferase is similarly located in this gradient peak (48). The peak galacto-syltransferase activities also corresponded to peaks of UMP-ase ( -ribonucleotide phosphohydrolase, EC 3.1.3.5) and UDP-phospho-hydrolase (nucleosidediphosphate phosphohydrolase, EC 3.6.16) activities which are marker enzymes for the endoplasmic reticulum. 

Fig. 7. Fractionation by anion exchange chromatography of 1 ml portions of a 20% aqueous solution of ampicillin sodium (initial pH 8.5) kept at 22 °C for the specified periods. The DEAE-Sephadex A-25 column was eluted with 0.05 M phosphate (pH 7.4), with a linear sodium chloride gradient. Peak identities A, ampicillin B, a-aminobenzylpenicilloic acid C, dimer D, mixture of a trimer having a closed -lactam ring and a dimer having an open j -lactam ring E, tetramer /% mixture of a tetramer having an open j -lactam ring and a pen-tamer with an intact j -lactam ring G, hexamer H, octamer. (Bundgaard and Larsen 1977)...

FIGURE 21.11 Simulated sample-to-sample chromatographic peak shifts and elution order for a typical reverse-phase gradient. Peak retention shifts are random inside the indicated area and therefore not predictable. 

FIGURE 5.5.1 Thermoresponsive chromatography system, (a) Mechanism of elution control of solutes by changes in hydrophobic interaction with the PNIPAAm-grafted surfaces as a function of temperattire. (b) Chromatograms of a mixture of four steroids and benzene by temperature gradient (peaks 1, benzene 2, cortisone 3, prednisolone 4, hydrocortisone acetate and 5, testosterone) [34]. 

Methoxymelamine resins, namely CYREZ 963 and CYREZ 350 corrunercial resins, were analyzed using a C,g column (A = 225 nm) and a 50-min 25/75 -> 100/0 (at 35 min hold 15 min) methanol/water gradient. Peak shapes were excellent [250]. Distinct and characteristic profiles for these polymers were obtained. A series of 10 pL injections of 0.05% solutions was made in these analyses. 

Four crocins (1, 2, 4, and 4), crocetin, and cw-crocin were extracted from saffron and analyzed on a Cig column (A = 420 nm) using a complex 30-min 60/40 - 10/ 90 water (1% acetic acid)/methanol gradient. Peak shapes were excellent and all peaks were baseline resolved [384]. The last analyte eluted at 23 min (the internal standard eluted at 28 min). Linear ranges were reported as 5-50pg/mL to 30-300pg/mL, whereas detection limits were in the 0.2-3 pg/mL range (analyte dependent). 

Nicotinic acid, nicotinamide, inosine monophosphate, adenosine 5 -monopho-sphate (AMP), adenosine 5 -diphosphate (ADP) and adenosine 5 -triphosphate (ATP) were extracted from red blood cells and baseline resolved on a C g column (A = 280 nm or 254 nm) using a complex 22-min 4/96 -> 30/70 methanol/water (100 mM KH2PO4 with 8mM tributylamine at pH 5.5) gradient Peak shapes were excellent [448]. 

Benzothiazole, 2,2 -(dithiobis)benzothiazole, and 2-mercapto-, 2-(methylthio)-and 2-(thiocyanomethylthio)benzothiazoie were baseline resolved on 3 Ci8 column (2 = 250 nm, 380 nm, or 325 nm) using a complex 20-min 63/37 90/10 acet-onitrile/water (4mM NaH2P04 buffer at pH 4.5) gradient. Peak shapes were excellent. Extraction from industrial wastewater samples gave detection limits in the low (0.1-10) ng injected range [908]. 

Low-density polyethylene samples were analyzed for their additive content. Five additives (butylated hydroxytoluene, butylhydroxyethylbenzene, Isonox 129, Irga-nox 1076, Irganox 1010) were separated on a Cjg column (X = 200nm) using a 15-min 75/25 100/0 acetonitrile/water gradient. Peak were resolved and elution was complete in 11 min [1014], Levels of 70-1000 ppm were found. 

Dicarboxylic acids (Cg-Cig, even) were analyzed as their mono-coenzyme A, mono-camitine, and 4-nitrobenzyl esters [1063]. The mono-coenzyme A esters were baseline resolved on a C g column (photodiode array detector, A = 200-300 nm) using a complex 45-min 5/95 - 50/50 acetonitrile/water (50 mM KH2PO4 buffer at pH 5.3) gradient. Peak shsqjes were excellent. The mono-camitine esters were analyzed as their 4-bromophenacyl derivatives on a Cg column (A = 260 nm) using a complex 40-min 60/38/2 -> 95/0/5 acetonitrile/water/water (0.15 M triethylamine phosphate buffer at pH 5.6) gradient. The 4-nitrobenzyl derivatives were resolved on... 

Seven ftavanones and xanthones (euchrestaflavanone C and B, osajaxanthone, toxyloxanthone C, macluraxanthone, alvaxanthone, 8-prenylxanthone) were isolated from the root bark of osage orange trees and resolved on a C,g column (A = 280 nm) using a 42-min 60/40-> 40/60 (981.5/18.5/0.005 water/acetonitrile/TFA)/ (0/100/0.005 water/acetonitrile/TFA) gradient. Peaks were well resolved from one another and from other extracted compounds. Positive-ion APCI was used for peak identifrcation [1145]. 

The production of three peptides, neokyrotiopin, VV-hemorphin-4, and brady-kinin-potentiating peptide, was monitored throughout the peptic hydrolysis of bovine hemoglobin using a 65-min 100/0- 33/67 (at 30 min)-> 23/87 wato- (0.1% TFA)/(60/40/0.1 acetonitrile/water/TFA) gradient. Peaks of interest were resolved and eluted in 30 min however, the elution of large peaks was seen imtil 60 min [1295],... 

Several V-terminally truncated C-type natriuretic peptides were isolated from the venom of the habu snake [1297]. Separation of these components was achieved on a C 8 column (A = 214nm) using a 30-min 100/0 ->40/60 water (0.1% TEA)/ acetonitrile gradient. Peak shapes were excellent. 

Figure 12 Separation of 14 common anions under isocratic and capacity gradient conditions (a) with 20 mM LiOH as eluent (isocratic) (b) with 20 mM NaOH as eluent (isocratic) (c) with a 20 mM NaOH to 20 mM LiOH gradient. Peaks 1 = F, 2 = CH3C02-, 3 = C1-,4 = N02 , 5 = Br, 6 = N03 , 7 = S042-,8 = C2042-,9 = Cr04-, 10 = r, 11=P043-,...

Figure 17 (a) Structure of cyclenbowl (b) Preconcentration of perrhenate on cyclenbowl column by step gradient. Peaks 1 = F, ... 

FIGURE 10.15 Separation of 17-component amino acid hydrolyzate. Conditions Column, Dionex AS-8 gradient. Peaks (25 nmol each, except 12.5 nmol for cystine) a, arginine b, lysine c, threonine d, alanine e, glycine f, serine g, valine h, proline i, isoleucine j, leucine k, methionine 1, histidine m, phenylalanine n, glutamic acid o, aspartic acid p, cystine and q, tyrosine. (Reprinted from Welch, L.E., LaCourse, W.R., Mead, D.A., Jr., and Johnson, D.C., Ana/. Chem., 61, 555, 1989.)... 

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