Reversed-phase internal

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.]... 

However, complete hydrolysis of carotenoid esters sometimes is not achieved in 1 to 3 hr. The saponification degree can be verified easily by the presence of carotenol ester peaks eluting later than the peaks of P-carotene on reversed phase columns. Retinol palmitate, added as an internal standard to orange juice, also serves to indicate whether saponification is complete, since it is converted to retinol which elutes at lower retention time. The mixture is subsequently washed with water until free of alkali in a separatory funnel. Other more polar solvents such as CH2CI2 or EtOAc, and diethyl ether alone or mixtured with petroleum ether can be used to increase the recovery of polar xanthophylls from the water phase. 

Wheat samples are extracted with dilute ammonia on the ASE200. The extracts are amended with isotopically labeled internal standards. The extracts are purified by sequential octadecyl reversed-phase solid-phase extraction (Cig SPE) and ethylenediamine-iV-propyl anion exchange (PSA) SPE. The samples are analyzed by LC/MS/MS. This method determines crop residues of flucarbazone-sodium and A-desmethyl flucarbazone with a limit of quantitation (LOQ) of 0.01 mgkg for each analyte. 

Restricted access phases are another approach to exploiting the differences in characteristics of analytes. Large analytes are excluded from an internal surface on which an adsorptive stationary phase is present. A herbicide analysis for Metsulfuron methyl, Bentazone, Bromoxynil, methylchlorophenoxy acid, and Mecoprop in the presence of humic acid was performed on restricted access reversed phase media.52 The cytostatic compound epirubicin and its metabolites were separated from plasma using a Pinkerton GFF II column.53 Gradient separations of polymers on reversed phase and on normal phase represent an alternative to gel permeation chromatography. Polyesters of noncrystalline materials were separated on a variety of such phases.54... 

A review of the properties of silica as applied to reversed phase separations summarizes a number of issues that have been debated for many years.71 The review categorizes unmodified silanols as free, geminal, vicinal, and internal. The pK, values of silanols average about 7.1, but some silanols may have pK, values as low as 3. As reported in Chapter 4, heavy metal... 

Purification of double-stranded DNA on micropellicular anion exchange and reversed-phase materials has been reviewed.43 Micropellicular phases adsorb only at the surface and have no internal pores. For this reason, the surface area and hence the capacity of micropellicular phases tends to be low. Using small particles (1-3 p in diameter) increases the surface area but may be impractical for preparative work above the mg scale. 

A wide variety of methodologies have been employed for the analysis of antioxidants in polymers and some standard methods are available. For high-density polyethylene ASTM method D5524 (ASTM International) — Determination of phenolic antioxidants in high-density polyethylene, describes a method whereby the sample is ground to a small particle size and then extracted by refluxing with cyclohexane. The cyclohexane extract is then examined by reverse-phase HPLC with UV detection. 

Alhaique et al. [62] used a reversed phase high performance liquid chromatography method for the determination of miconazole in bulk or pharmaceuticals using bezafibrate as internal standard. 

Dean et al. [93] used a high performance liquid chromatographic method for the simultaneous determination of primaquine and carboxyprimaquine in plasma with electrochemical detection. After the addition of the internal standard, plasma was deproteinized by the addition of acetonitrile. Nitrogen-dried supernatants, resuspended in mobile phase were analyzed on a C8 reversed-phase column. Limits of detection for primaquine and carboxyprimaquine were 2 and 5 ng/mL with quantitation limits of 5 and 20 ng/mL, respectively. The assay sensitivity and specificity are sufficient to permit quantitation of the drug in plasma for pharmacokinetics following low dose (30 mg, base) oral administration of primaquine, typically used in the treatment of malaria and P. carinii pneumonia. 

Dobutamine hydrochloride may be determined in plasma levels, after extraction, on a C18 reversed-phase column eluted with 22% aceto-nitrile-78% 0.1 M phosphate buffer (pH 2.0) at 2 ml/minute. The drug and its metabolite are detected by a fluorescent detector with an excitation wavelength of 195 nm and a 330 nm emission cut off filter. The retention times of dobutamine and the 3-methoxy metabolite are 5.2 and 7.9 min., respectively. The lower limit of sensitivity is 10 ng/ml. Reproducibility is 5% over a 25-300 ng/ml range. Nylidrin is used as an internal standard (6). 

Reverse Phase High-Pressure Liquid Chromatography of Halcino-nide. Progesterone is used as internal standard. 

Many other types of solid phase adsorbents, including those based on conventional and specialty materials like restricted access media (RAM), can increase analysis speed and improve assay performance. These types of materials, also known as internal reversed-phase packings, are especially useful for assaying target compounds in biological samples such as serum and plasma. They are chemically modified porous silicas that have hydrophilic external surfaces and restricted-access hydrophobic internal surfaces. The ratio of interior to external surface areas is large. Macromolecules such as proteins cannot enter the pores of the RAM (they are excluded from the hydrophobic internal surface) and they elute quickly through the column. However, the smaller analyte molecules that can enter the pores are retained via interactions with the hydrophobic bonded phase within... 

Internal-surface reversed phase (ISRP) column, 6 447... 

A separation of myoglobin and lysozyme has been presented by Nicoud [44]. This purification was performed on SMB containing 8 columns using ACA 54 (Biosepra, France) as support. Very pure extracts (>98%) and raffinates (>98%) were obtained from a 50-50 mixture. An internal profile is given in Fig. 7. Another recently presented example is the separation of cyclosporine A from cyclic oligopeptide and other impurities in the reversed phase mode or by adsorption on silica gel presented by Schulte et al. [10]. 

Fig. 2.26. Reversed-phase HPLC separation of (a) Sobrasada sausage extract and (b) saponified Sobrasade sausage extact in an ODS column at maximum absorbances at each point in time. Peak identification 1 - 2, 4 - 6, 8, 12, 14-17 = unidentified free 3 = capsorubin 7 = violaxanthin 9 = capsanthin 10 = anteraxanthin 11 = cw-capsanthin 13 = lutein and zeaxanthin 18 = cantaxanthin, internal standard 19 = cryptoxanthin 20, 24, 25, 28 = unidentified monoester 21 = /J-cryptoxanthin 22 = capsorubin monoester 23, 26, 27, 29 = capsanthin monoester 30, 31 = lutein-zeaxanthin monoester 32 = /1-carotene 33 = cis-f)-carotene 34, 37, 39, 41, 43 = capsanthin diester 35 = capsorubin diester 36, 38, 40, 42, 44 = unidentified diester. Reprinted with permission from J. Oliver et al. [56],...

Fig. 2.55. Gradient reversed-phase HPLC analysis of flavonoids in white onions (a) and celery (b). ODS column of 150 X 3.9mm i.d particle size 5pm. Mobile phase 20min gradient of 15-35 per cent acetonitrile in water adjusted to pH 2.5 with TFA. Fowrate lml/min. Upper and lower traces represent samples before and after hydrolysis, respectively. Detection wavelength 365 nm. IS = internal standard Qc = quercetin Ap = apigenin Lt = luteolin. Reprinted with permission from A. Crozier et al. [159],...

To increase Vs, the chromatographer can increase the surface area of the stationary phase materials in normal-phase liquid chromatography, increase the stationary phase volume in reversed-phase or partition liquid chromatography, or increase the ion-exchange capacity in ion-exchange liquid chromatography. In general, if the internal diameter of a column is constant, the retention time... 

In HPLC, a sample is separated into its components based on the interaction and partitioning of the different components of the sample between the liquid mobile phase and the stationary phase. In reversed phase HPLC, water is the primary solvent and a variety of organic solvents and modifiers are employed to change the selectivity of the separation. For ionizable components pH can play an important role in the separation. In addition, column temperature can effect the separation of some compounds. Quantitation of the interested components is achieved via comparison with an internal or external reference standard. Other standardization methods (normalization or 100% standardization) are of less importance in pharmaceutical quality control. External standards are analyzed on separate chromatograms from that of the sample while internal standards are added to the sample and thus appear on the same chromatogram. 

Szoka FJ, Papahadjopoulos D. Procedure for preparation of liposomes with large internal aqueous space and high capture by reverse-phase evaporation. Proc Natl Acad Sci USA 1978 75 4194. 

Waste water Addition of internal standard elution effluents) from Sep-Pak C s reverse-phase (1,3-DNB column with methanol/water... 

Sample dried, sieved and ground extraction with ultrasonication in acetonitrile containing internal standard dilution with aqueous CaCl2 filtration from reverse-phase column with methanol/water... 

In comparison to their silica counterparts, organic polymer monoliths generally exhibit lower efficiencies in the reversed-phase HPLC separation of small molecules. This reduced performance is primarily due to the lack of mesopores (see Section 1.3.2.5) and the presence of micropores in the polymer matrix, which cause slow internal diffusion. However, there are some promising approaches trying to accomplish the chromatography of small molecules on organic polymer monoliths. 

A clean-up process-scale reverse-phase HPLC (RP-HPLC) step has been introduced into production of human insulin prb. The C8 or C18 RP-HPLC column used displays an internal volume of 801 or more, and up to 1200 g of insulin may be loaded during a single purification run (Figure 8.7). Separation is achieved using an acidic (often acetic acid-based) mobile phase (i.e. set at a pH value sufficiently below the insulin pi value of 5.3 in order to keep it fully in solution). The insulin is usually loaded in the water-rich acidic mobile phase, followed by gradient elution using acetonitrile (insulin typically elutes at 15-30% acetonitrile). 

---