P Reversed-phase

Martin, H.P., Reversed phase paper chromatography and detection of steroids of the cholesterol class, Biochim. Biophys. Acta, 25, 408, 1957. 

Lehtonen, P. Reversed-phase liquid chromatographic elution characteristics of substituted n-ethylbenzamides. J. Chromatogr., A 1983, 267, 277-284. 

Fig. 12. 5-HT and 5-HIAA measurements by reverse-phase separation. (A) Standards of 5-HT and 5-HIAA, 2 pmol injected onto the column (B) rat hippocampus (45 mg) sonicated in perchloric acid (5 vol wt), centrifuged, and 20 p,l supernatant injected onto the column. Chromatographic conditions column, Spherisorb 5 p, reverse phase, 25 cm x 4.6 mm mobile phase, sodium acetate, 0.1 M, citric acid, 0.1 M, pH 4.1, + methanol 10% flow rate, 0.8 ml/min electrode potential (carbon paste), +0.65 V volume injected, 20 p,l sensitivity, 2 nA/V (Gilbert et ai, 1981).

Recently (179), a macrocyclic antibiotic, Vancomycin, was used as a chiral mobile phase additive for TLC resolution of 6-aminoquinolyl-N-hydroxy succinimidyl carbamate (AQC) derivativatized amino acids and dansyl amino acids on chemically bonded diphenyl-P-reversed phase plates. Both the nature of stationary phase and the composition of the mobile phase have a strong influence on enantiomeric resolution typical results are given in Table 27. 

Lebiedzihska A, Marszall ML, Kuta J, Szefer P. Reversed-phase high-performance liquid chromatography method with coulometric electrochemical and ultraviolet detection for the quantification of vitamins Bi (thiamine), B (pyridoxamine, pyri-doxal and pyridoxine) and B12 in animal and plant foods. J Chromatogr A... 

Zhang T, Nguyen D, Franco P. Reversed-phase screening strategies for liquid chromatography on polysaccharide-derived chiral stationary phases. J. Chromatogr. A 2010 1217 1048-1055. 

SPE on Macherey-Nagel Chromabond HR-P reversed phase (wash with 0.01 M HCl, elute with 3 mL THE and 3 mL MeOH)... 

A useful guide when using the polarity index is that a change in its value of 2 units corresponds to an approximate tenfold change in a solute s capacity factor. Thus, if k is 22 for the reverse-phase separation of a solute when using a mobile phase of water (P = 10.2), then switching to a 60 40 water-methanol mobile phase (P = 8.2) will decrease k to approximately 2.2. Note that the capacity factor decreases because we are switching from a more polar to a less polar mobile phase in a reverse-phase separation. 

Another example is the purification of a P-lactam antibiotic, where process-scale reversed-phase separations began to be used around 1983 when suitable, high pressure process-scale equipment became available. A reversed-phase microparticulate (55—105 p.m particle size) C g siUca column, with a mobile phase of aqueous methanol having 0.1 Af ammonium phosphate at pH 5.3, was able to fractionate out impurities not readily removed by hquid—hquid extraction (37). Optimization of the separation resulted in recovery of product at 93% purity and 95% yield. This type of separation differs markedly from protein purification in feed concentration ( i 50 200 g/L for cefonicid vs 1 to 10 g/L for protein), molecular weight of impurities (<5000 compared to 10,000—100,000 for proteins), and throughputs ( i l-2 mg/(g stationary phasemin) compared to 0.01—0.1 mg/(gmin) for proteins). 

A liquid chromatography-mass spectrometry (LC-MS) method that can quantitatively analyze urinar y normal and modified nucleosides in less than 30 min with a good resolution and sufficient sensitivity has been developed. Nineteen kinds of normal and modified nucleosides were determined in urine samples from 10 healthy persons and 18 breast cancer patients. Compounds were separ ated on a reverse phase Kromasil C18 column (2.1 mm I.D.) by isocratic elution mode using 20 mg/1 ammonium acetate - acetonitrile (97 3 % v/v) at 200 p.l/min. A higher sensitivity was obtained in positive atmospheric pressure chemical ionization mode APCI(-i-). 

Obtained P values for the description of benzodiazepine s retention in reversed-phase HPLC are used. The equation... 

A.M. Krstulovic and P.R. Brown, Reversed Phase HPLC. Theory, Practical and Biomedical Applications, J. Wiley Sons, New York, 1982. ISBN 0471053694. 

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.]... 

E. A. Hogendoom and P. van Zoonen, Coupled-column reversed-phase liquid cliro-matogr-aphy in envuonmental analysis , J. Chromatogr. 703 149-166 (1995). 

In 1993, Jorgenson s group improved upon then earlier reverse phase HPLC-CZE system. Instead of the six-port valve, they used an eight-port electrically actuated valve that utilized two 10-p.L loops. While the effluent from the HPLC column filled one loop, the contents of the other loop were injected onto the CZE capillary. The entii e effluent from the HPLC column was collected and sampled by CZE, making this too a comprehensive technique, this time with enhanced resolving power. Having the two-loop valve made it possible to overlap the CZE runs. The total CZE run time was 15 s, with peaks occurring between 7.5 and 14.8 s. In order to save separation space, an injection was made into the CZE capillary every 7.5s,... 

G. P. Blanch, J. Villen and M. Heiraiz, Rapid analysis of free eiytlnodiol and uvaol in olive oils by coupled reversed phase liquid clnomatogi aphy-gas clnomatography , 7. Agric. Food Chem. 46 1027-1030 (1998). 

Figure 11.12 GC analysis of (a) urine sample spiked with opiates 3 p.g/ml) and (b) blank urine sample. Peak identification is as follows 1, dihydrocodeine 2, codeine 3, ethylmor-phine 4, moipliine 5, heroin. Reprinted from Journal of Chromatography, A 771, T. Hyotylainen et al., Determination of morphine and its analogues in urine by on-line coupled reversed-phase liquied cliromatography-gas clrromatography with on-line derivatization, pp. 360-365, copyright 1997, with permission from Elsevier Science.

Figure 12.11 Coupled SEC-RPLC separation of compound Chemigum mbber stock (a) SEC ti ace (b) RPLC trace of fraction 1, dibutylphthalate (c) RPLC trace of fraction 2, elemental sulfur. Coupled SEC conditions MicroPak TSK 3000H (50 cm) X 2000H (50 cm) X 1000 H (80 cm) columns (8 mm i.d.) eluent, THE at a flow rate of 1 mL/min UV detection at 215 nm (1.0 a.u.f.s.) injection volume, 200 p-L. RPLC conditions MicroPak MCH (25 cm X 2.2 mm i.d.) column flow rate, 0.5 mL/min injection volume, lOpL gradient, acetonitrile-water (20 80 v/v) to 100% acetonitrile at 3% acetonitrile/min UV detection at 254 nm (0.05 a.u.f.s.). Reprinted from Journal of Chromatography, 149, E. L. Jolmson et al., Coupled column cliromatography employing exclusion and a reversed phase. A potential general approach to sequential analysis , pp. 571-585, copyright 1978, with permission from Elsevier Science.

Figure 13.7 Selectivity effected by employing different step gradients in the coupled-column RPLC analysis of a surface water containing 0.40 p-g 1 bentazone, by using direct sample injection (2.00 ml). Clean-up volumes, (a), (c) and (d) 4.65 ml of M-1, and (b) 3.75 ml of M-1 transfer volumes, (a), (c) and (d), 0.50 ml of M-1, and (b), 0.40 ml of M-1. The displayed cliromatograms start after clean-up on the first column. Reprinted from Journal of Chromatography, A 644, E. A. Hogendoom et al, Coupled-column reversed-phase liquid chromatography-UV analyser for the determination of polar pesticides in water , pp. 307-314, copyright 1993, with permission from Elsevier Science.

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