Mobile-phase gradients

FIGURE l.l Hydrophobic interaction and reversed-phase chromatography (HIC-RPC). Two-dimensional separation of proteins and alkylbenzenes in consecutive HIC and RPC modes. Column 100 X 8 mm i.d. HIC mobile phase, gradient decreasing from 1.7 to 0 mol/liter ammonium sulfate in 0.02 mol/liter phosphate buffer solution (pH 7) in 15 min. RPC mobile phase, 0.02 mol/liter phosphate buffer solution (pH 7) acetonitrile (65 35 vol/vol) flow rate, I ml/min UV detection 254 nm. Peaks (I) cytochrome c, (2) ribonuclease A, (3) conalbumin, (4) lysozyme, (5) soybean trypsin inhibitor, (6) benzene, (7) toluene, (8) ethylbenzene, (9) propylbenzene, (10) butylbenzene, and (II) amylbenzene. [Reprinted from J. M. J. Frechet (1996). Pore-size specific modification as an approach to a separation media for single-column, two-dimensional HPLC, Am. Lab. 28, 18, p. 31. Copyright 1996 by International Scientific Communications, Inc.. Shelton, CT.]... 

Fig. 3-16. Chromatograms of mixed libraries of 16 L (a) and 16 D (b) seleetors using reeiproeal stationary phase CSP 13. Conditions eolumn 50 x 4.6 mm i.d. mobile phase gradient of 5-20 % 2-propanol in hexane flowrate, 1.2 mL min UV deteetion at 254 nm. (Reprinted with permission from ref. [92]. Copyright 1999, Ameriean Chemieal Soeiety.)...

The second is associated with the irreproducibility of the mobile phase gradients that may be formed. The latter is overcome by forming the gradient at a conventional flow rate and then splitting prior to the injector. 

Supercritical fluid chromatography (SEC) was first reported in 1962, and applications of the technique rapidly increased following the introduction of commercially available instrumentation in the early 1980s due to the ability to determine thermally labile compounds using detection systems more commonly employed with GC. However, few applications of SEC have been published with regard to the determination of triazines. Recently, a chemiluminescence nitrogen detector was used with packed-column SEC and a methanol-modified CO2 mobile phase for the determination of atrazine, simazine, and propazine. Pressure and mobile phase gradients were used to demonstrate the efficacy of fhe fechnique. 

For analysis, the HPLC mobile phase gradient started at 3 17 acetonitrile-0.15% acetic acid in water and ended at 9 1 acetonitrile-0.15% acetic acid in water (0-32min). The HPLC column was a Zorbax RX-C8, 2.1-mm i.d. x 150mm, 5-p.m particle size column with a flow rate of 0.2mLmin and a 100-p.L injection volume. 

Figure 7.13 Separation of a test eixture using automated multiple development with a universal mobile phase gradient from acetonitrile through dlchloromethane to carbon disulfide on a silica gel HPTLC plate. The chromatogram was scanned at different wavelengths to enhance the chromatographic information.

UV/VIS, F RI ECD, ELCD ELSD, MS UV-grade non UV-absorbing solvents No mobile phase gradients Conducting mobile phase Volatile solvents and volatile buffers... 

Instrumentation requirements for SEC are somewhat simpler than those of other modes of HPLC, since mobile phase gradients are not used however, adequate computer support for data acquisition and processing is essential. Method development involves finding a suitable solvent for the sample and choosing a mixed bed column or, more often, a set of columns in series to match the pore size of the column(s) with the size distribution of the sample. 

FIGURE 16.8 HPLC chromatogram of cytochrome c and myoglobin digest, using a 250 cm x 4.6 mm ODS C18 Vydac column and a linear mobile-phase gradient, 5-50% B, in 50 min. Buffer A was 0.1% TFA in water and buffer B was 0.1 TFA in acetonitrile. UV detection was carried out at 214 nm, at room temperature (reprinted with permission from Electrophoresis). 

FIGURE 17.9 Gradient LC separation of the FAE mixture, stationary phase M N Nucleosil C18, 25 x 0.46 cm i.d., mobile phase gradient of MeOH-H20, flow rate 1 mL/min (reprinted from Pasch et al., 2005, with permission of European Polymer Federation). 

Note 1 Best examined with a less aqueous, more organic mobile phase gradient, or isocratic condition. 

Fig.4 Reconstructed SRM chromatograms obtained from the LC-ESI-MS/MS analysis of a 100 ng mL-1 standard mixture of estrogens (in the NI mode) and progestogens (in the PI mode). Column Purospher STAR RP-18e (125x2 mm, 5 pm, Merck). Mobile phase gradient acetonitrile/water. Flow rate 0.2 mL min-1...

Zhou and Pietrzyk [41] found that increasing the mobile-phase ionic strength not only increases the retention of AS and AES on a reversed stationary phase, but also improves the resolution since the peak widths are significantly reduced. The authors achieved baseline separation of a multicomponent alkane sulfonate and alkyl sulfate mixture from C2 to Cis using a mobile-phase gradient whereby acetonitrile concentration increases and LiOH concentration decreases. 

Fig. 9. Reversed-phase separations of cytochrome c digests obtained with trypsin-modified beads (left) and trypsin-modified monolithic reactor (right) in a tandem with a chromatographic column (Reprinted with permission from [90]. Copyright 1996 Wiley-VCH). Conditions digestion (left curve) trypsin-modified beads reactor, 50 mm x 8 mm i.d., 0.2 mg of cytochrome c, digestion buffer, flow rate 0.2 ml/min, 25 °C, residence time, 15 min (right curve) trypsin immobilized onto molded monolith other conditions the same as with trypsin-modified beads. Reversed-phase chromatography column, Nova-Pak C18,150 mm x 3.9 mm i.d., mobile phase gradient 0-70% acetonitrile in 0.1% aqueous trifluoroacetic acid in 15 min, flow rate, 1 ml/min, injection volume 20 pi, UV detection at 254 nm...

Fig. 17. Rapid reversed-phase separation of proteins at a flow-rate of 10 ml/min (Reprinted with permission from [127]. Copyright 1999 Elsevier). Conditions Column, 50x4.6 mm i.d. poly(styrene-co-divinylbenzene) monolith,mobile phase gradient 42% to 90% acetonitrile in water with 0.15% trifluoroacetic acid in 0.35 min, UV detection at 280 nm. Peaks ribonucle-ase (1), cytochrome c (2), bovine serum albumin (3), carbonic anhydrase (4), chicken egg albumin (5)...

Fig. 20. Test of stability of weak cation exchange monolithic column (ISCO). Conditions column, 50 X4.6 mm i.d., mobile phase gradient of sodium chloride in 0.01 mol/1 sodium phosphate buffer (pH 7.6) from 0.1 to 0.5 mol/1 in 4.5 min and to 1 mol/1 in 6.5 min, overall gradient time 11 min, flow rate 10 ml/min. Peaks Ribonuclease (1), cytochrome c (2), lysozyme (3). The two separations shown in this figure were achieved 503 runs apart...

Fig. 21. Separation of cytochrome (peak 1), ribonuclease, (peak 2), carbonic anhydrase (peak 3), lysozyme (peak 4), and chymotrypsinogen (peak 5) by hydrophobic interaction chromatography on a molded poly(acrylamide-co-butylmethacrylate-co-N,AT,-methylenebisacry-lamide) monolithic column. (Reprinted with permission from [ 135]. Copyright 1998 Elsevier). Conditions column, 50 x8 mm i.d., 10% butyl methacrylate,mobile phase gradient from 1.5 to 0.1 mol/1 ammonium sulfate in 0.01 mol/l sodium phosphate buffer (pH 7) in 3 min, gradient time 3.3 min, flow rate 3 ml/min...

Mobile-phase Gradient elution-2 minutes, from 20% B to 37% B in 15 minutes, where, A =... 

Mobile-phase Gradient elution of A (o-Hcxanc) and B (Isopropanol) ... 

FIGURE 9.18 Separation of 1, methyl benzyl amine 2, tartaric acid 3, diastereomer 4, drug candidate BMS-X 5, oxidation product and 6, vehicle polymers. Using a ES-PFP column (250 x 4. 6 mm with 5 p.m particles) with a mobile phase gradient starting with 95% modifier/5%C02 held for 25 minutes and ramped to 15%C02 for 5 minutes at 1.5mL/min, 311 K and 18.0 MPa modifier 43% H20/56% methanol). (Unpublished data from S. L. Phillips et al., unpublished data. With permission.)... 

Figure 1.1 HPLC chromatograms of samples of fluoxetine hydrochloride from four different suppliers (A-D). Reproduced from [7], Reproduced by permission from the publisher and authors. (Column 250 X 4.6 mm i.d. 5 pm Zorbax SB-C8 mobile-phase gradient acetonitrile water triflu-oroacetic 20 80 0.07 for 5 min, to 85 15 0.07 over 25 min, maintain at 85 15 0.07 for 5 min, return to initial conditions over 5 min, re-equilibrate for 10 min flow rate 1 mEmin injection 10 pi of 10 mg/ml solution in the initial mobile phase detector UV 260 nm.)...

Figure 13 Purification of pravastatin sodium by preparative liquid chromatography. Reprinted from [12], copyright 2001, with permission from Elsevier. (Column 125 X 4.6 mm i.d. 3 pm Hypersil ODS mobile-phase gradient methanol water triethylamine acetic acid 45 54.8 0.1 0.1 for 13 min, to 99.8 0 0.1 0.1 over 9 min flow rate 1.2 ml/min detector UV 235 nm.)...

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