Reversible acetonitrile

Reversed-phase chromatography is widely used as an analytical tool for protein chromatography, but it is not as commonly found on a process scale for protein purification because the solvents which make up the mobile phase, ie, acetonitrile, isopropanol, methanol, and ethanol, reversibly or irreversibly denature proteins. Hydrophobic interaction chromatography appears to be the least common process chromatography tool, possibly owing to the relatively high costs of the salts used to make up the mobile phases. 

Fig. 12. Tryptic map of it-PA (mol wt = 66,000) showing peptides formed from hydrolysis of reduced, alkylated rt-PA. Separation by reversed-phase octadecyl (C g) column using aqueous acetonitrile with an added acidic agent to the mobile phase. Arrows show the difference between A, normal, and B, mutant rt-PA where the glutamic acid residue, D, has replaced the normal arginine residue, C, at position 275.

This reversed-phase chromatography method was successfully used in a production-scale system to purify recombinant insulin. The insulin purified by reversed-phase chromatography has a biological potency equal to that obtained from a conventional system employing ion-exchange and size-exclusion chromatographies (14). The reversed-phase separation was, however, followed by a size-exclusion step to remove the acetonitrile eluent from the final product (12,14). 

Reversed-phase hplc has been used to separate PPG into its components using evaporative light scattering and uv detection of their 3,5-dinitroben2oyl derivatives. Acetonitrile—water or methanol—water mixtures effected the separation (175). Polymer glycols in PUR elastomers have been identified (176) by pyrolysis-gc. The pyrolysis was carried out at 600°C and produced a small amount of ethane, CO2, propane, and mostiy propylene, CO, and CH4. The species responsible for a musty odor present in some PUR foam was separated and identified by gc (Supelco SP-2100 capillary column)... 

Sugar analysis by hplc has advanced greatly as a result of the development of columns specifically designed for carbohydrate separation. These columns fall into several categories. (/) Aminopropyl-bonded siHca used in reverse-phase mode with acetonitrile—water as the eluent. (2) Ion-moderated cation-exchange resins using water as the eluent. Efficiency of these columns is enhanced at elevated temperature, ca 80—90°C. Calcium is the usual counterion for carbohydrate analysis, but lead, silver, hydrogen, sodium, and potassium are used to confer specific selectivities for mono-, di-, and... 

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-). 

Silica gel, per se, is not so frequently used in LC as the reversed phases or the bonded phases, because silica separates substances largely by polar interactions with the silanol groups on the silica surface. In contrast, the reversed and bonded phases separate material largely by interactions with the dispersive components of the solute. As the dispersive character of substances, in general, vary more subtly than does their polar character, the reversed and bonded phases are usually preferred. In addition, silica has a significant solubility in many solvents, particularly aqueous solvents and, thus, silica columns can be less stable than those packed with bonded phases. The analytical procedure can be a little more complex and costly with silica gel columns as, in general, a wider variety of more expensive solvents are required. Reversed and bonded phases utilize blended solvents such as hexane/ethanol, methanol/water or acetonitrile/water mixtures as the mobile phase and, consequently, are considerably more economical. Nevertheless, silica gel has certain areas of application for which it is particularly useful and is very effective for separating polarizable substances such as the polynuclear aromatic hydrocarbons and substances... 

The dispersions of 12 solutes were then measured in a packed reverse phase column using the same mobile phase as that used in the determination of their diffusivities acetonitrile/water). The column dispersion (H) was then plotted against l/Dm... 

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.]... 

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 13.9 Coupled-column RPLC-UV (215 nm) analysis of 100 p.1 of an extract of a spiked soil sample (fenpropimoiph, 0.052 mg Kg ). LC conditions C-1, 5 p.m Hypersil SAS (60 m X 4.6 mm i.d.) C-2, 5 p.m Hypersil ODS (150 m X 4.6 mm i.d.) M-1, acetonitrile-0.5 % ammonia in water (50 50, v/v) M-2, acetonitrile-0.5 % ammonia in water (90 10, v/v) flow-rate, 1 ml min clean-up volume, 5.9 ml transfer volume, 0.45 ml. The dashed line represents the cliromatogram obtained when using the two columns connected in series without column switcliing. Reprinted from Journal of Chromatography A, 703, E. A. Hogendoom and R van Zoonen, Coupled-column reversed-phase liquid cliromatography in envir onmental analysis , pp. 149-166, copyright 1995, with permission from Elsevier Science.

Reverse-phase Squalane Zipax-HCP Cyanoethylsilicone Water and alcohol-water mixtures acetonitrile and acetonitrile-water mixtures... 

A similar irradiation of imide 9 in acetonitrile gives a mixture of ethyl 4,5-bis(ethoxy-carbonylamino)-l//-l,2-diazepine-l-carboxylate (10) and the isomeric ethyl 5,6-bis(ethoxycar-bonylamino)-l/f-l,2-diazepine-l-carboxylate (11) in the ratio 6 1. In contrast, when the irradiation is carried out in dichloromethane, the ratio is reversed to 1 2.5.81... 

HPLC on a PRP-1 column (0.7 x 30.5 cm, Hamilton), with 65% acetonitrile containing 0.05% acetic acid, at a flow rate of 4 ml/min. To reverse the possible hydration of the molecules, the material obtained in step 3 is first evaporated to dryness, and redissolved in chloroform. Immediately before injection onto the HPLC column, an... 

Purification of the activation products (PMs). The methylamine activation product dissolved in methanol is purified by chromatography, first on a column of silica gel using a mixed solvent of chloroform/ethanol, followed by reversed-phase HPLC on a column of divinylbenzene resin (such as Jordi Reversed-Phase and Hamilton PRP-1) using various solvent systems suitable for the target substance (for example, acetonitrile/water containing 0.15% acetic acid). 

The method is very useful for the synthesis of physiologically interesting a-mcthylamino acids, e.g., methyl dopa from the 3,4-dimethoxybenzyl derivative. The excellent stereoselection achieved in the process, however, is caused by the preferential crystallization of one pure diastereomerfrom the equilibrium mixture formed in the reversible Strecker reaction. Thus, the pure diastcrcomers with benzyl substituents, dissolved in chloroform or acetonitrile, give equilibrium mixtures of both diastereomers in a ratio of about 7 347. This effect has also been found for other s-methylamino nitriles of quite different structure49. If the amino nitrile (R1 = Bn) is synthesized in acetonitrile solution, the diastereomers do not crystallize while immediate hydrolysis indicates a ratio of the diastereomeric amino nitriles (S)I(R) of 86 1447. 

In this case also the silicon is four-coordinated as a result of the supplementary complexation by acetonitrile. With a distance of 2.328(2) A the RuSi bond can be considered as short. It is interesting to note that 12 is the only case in which a reversible coordination of the donor molecule at the silicon has been found [92]. From these results a high reactivity of 12 should be inferred however, no details are known so far [37]. 

Naphthalenedisulfonate-acetonitrile as the only mobile phase with a silica column coated with a crosslinked aminofluorocarbon polymer has proven to be an effective combination for the separation of aliphatic anionic surfactants. Indirect conductivity and photometric detection modes are used to monitor these analytes. The retention of these surfactants is found to depend on both the ionic strength and the organic solvent content of the mobile phase. The mechanism of retention is considered to be a combination of both reverse phase and ion exchange processes. Selective separation of both alkanesulfonates and... 

The alternative technique for analyzing 1,4-dioxane is HPLC. Scalia proposed a method by solid-phase extraction using octadecyl-bonded silica cartridges and analyzed directly on a reverse phase column with UV detection at 200 nm and acetonitrile-water as eluent [328-330]. 

A method offering the possibility for the separation, identification, and determination of alkyl- and alkylphenol ether carboxylates, even in mixtures with other nonionic and amphoteric substances, is carried out by HPLC using a reverse phase RP18 column and a mixture of methanol, water, and acetonitrile with the addition of an ion-pairing reagent as mobile phase working under isocratic conditions [242]. 

To retain solutes selectively by dispersive interactions, the stationary phase must contain no polar or ionic substances, but only hydrocarbon-type materials such as the reverse-bonded phases, now so popular in LC. Reiterating the previous argument, to ensure that dispersive selectivity dominates in the stationary phase, and dispersive interactions in the mobile phase are minimized, the mobile phase must now be strongly polar. Hence the use of methanol-water and acetonitrile-water mixtures as mobile phases in reverse-phase chromatography systems. An example of the separation of some antimicrobial agents on Partisil ODS 3, particle diameter 5p is shown in figure 5. 

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