Proteins normal phase HPLC

Chiral separations result from the formation of transient diastereomeric complexes between stationary phases, analytes, and mobile phases. Therefore, a column is the heart of chiral chromatography as in other forms of chromatography. Most chiral stationary phases designed for normal phase HPLC are also suitable for packed column SFC with the exception of protein-based chiral stationary phases. It was estimated that over 200 chiral stationary phases are commercially available [72]. Typical chiral stationary phases used in SFC include Pirkle-type, polysaccharide-based, inclusion-type, and cross-linked polymer-based phases. 

Compared to refined vegetable oils, the compositions of crude vegetable oils and oil and fat products are more complicated. These samples contain proteins, carbohydrates, and minerals that interfere with HPLC separation and reduce the lifetime of the HPLC column. These compounds need to be largely eliminated from the extract before HPLC analysis. Saponification and heating are used to weaken sample matrices to allow the solvent to fully access all tocopherols and tocotrienols of the sample. Liquid/liquid extraction is used to remove these polar compounds from the organic solvent layer that contains tocopherols and tocotrienols. The normal-phase HPLC method is usually used for crude vegetable oils and vegetable oil products reversed-phase HPLC can be used for animal fat products. 

Meyer et al. analyzed plasma retinol plus endogenous aW-trans and i-cis retinoic acid isocratically by normal-phase HPLC, using hexane 2-propanol acetic acid as mobile phase (90). Extraction of the retinoic acids required acidification of the sample however, too much acid can result in dehydration of retinol to anhydroretinol, and hydrolysis of endogenous retinoyl P-glucuronide (90). A synthetic retinoid sulfonic acid was used as internal standard. By using absorbance at 350 nm, limits of detection were 1.7 nM (0.5 (ig/L) for retinoic acid isomers, 35 nM (10 Xg/L) for retinol. 13-Demethyl retinoic acid has also been used as internal standard (142). Lanvers et al. used normal-phase HPLC with gradient elution (hexane 2-propanol glacial acetic acid) to analyze 13-c/x retinoic acid, 9-cis retinoic acid, aW-trans retinoic acid, retinol, and the 4-oxo metabolites of the retinoic acid isomers (143). Plasma samples were treated with ethanol to denature proteins, and then were extracted with hexane after addition of saturated ammonium sulfate solution (pH 5). 

Reverse-phase HPLC (RP-HPLC) separates proteins on the basis of differences in their surface hydophobicity. The stationary phase in the HPLC column normally consists of silica or a polymeric support to which hydrophobic arms (usually alkyl chains, such as butyl, octyl or octadecyl groups) have been attached. Reverse-phase systems have proven themselves to be a particularly powerful analytical technique, capable of separating very similar molecules displaying only minor differences in hydrophobicity. In some instances a single amino acid substitution or the removal of a single amino acid from the end of a polypeptide chain can be detected by RP-HPLC. In most instances, modifications such as deamidation will also cause peak shifts. Such systems, therefore, may be used to detect impurities, be they related or unrelated to the protein product. RP-HPLC finds extensive application in, for example, the analysis of insulin preparations. Modified forms, or insulin polymers, are easily distinguishable from native insulin on reverse-phase columns. 

Reverse-phase HPLC (RP-HPLC) separates proteins on the basis of differences in their surface hydrophobicity. The stationary phase in the HPLC column normally consists of silica or a polymeric support to which hydrophobic arms (usually alkyl chains such as butyl, octyl or... 

The decision about which HPLC column to choose is really controlled by the separation you are trying to make and how much material you are trying to separate and/or recover. I did a rather informal survey of the literature and my customers 15 years ago to see which columns they used. I found 80% of all separations were done on some type of reverse-phase column (80% of those were done on C18), 10% were size separation runs (most of these on polymers and proteins), 8% were ion-exchange separations, and 2% were normal-phase separation on silica and other unmodified media, such as zirconium and alumina. The percentage of size- and ion-exchange separations has increased recently because of the importance of protein purification in pro-teomics laboratories and the growing use in industry of ion exchange on pressure-resistant polymeric and zirconium supports. 

Normal aqueous micellar media can also be employed to extract and purify components from solid matrices. Proteins have been extracted from wheat kernals using aqueous NaLS (399). This same surfactant system has been employed in an improved method for the extraction of filth from cheese (417). In another application, aqueous solutions of Brij-35 micelles have been employed to extract components (i.e. vanillin and ethylvanillin) from smoking tobacco (106). In a similar manner, various phenolic compounds have been extracted from herbal/plant leaves using nonionic Triton X-100, Brij-35, or octyl glucoside (0G) (393). In both of these latter examples, the indicated compounds could be identified and quantitated by reversed phase HPLC using as mobile phase the same micellar solutions (refer... 

Chiral separations can be considered as a special subset of HPLC. The FDA suggests that for drugs developed as a single enantiomer, the stereoisomeric composition should be evaluated in terms of identity and purity [6]. The undesired enantiomer should be treated as a structurally related impurity, and its level should be assessed by an enantioselective means. The interpretation is that methods should be in place that resolve the drug substance from its enantiomer and should have the ability to quantitate the enantiomer at the 0.1% level. Chiral separations can be performed in reversed phase, normal phase, and polar organic phase modes. Chiral stationary phases (CSP) range from small bonded synthetic selectors to large biopolymers. The classes of CSP that are most commonly utilized in the pharmaceutical industry include Pirkle type, crown ether, protein, polysaccharide, and antibiotic phases [7]. 

Loro, K. A., Orlan, R., Zhang, R, Usherwood, P. N. R., and Nakanishi, K. (1993). Anal. Biochem. 215, 38-44. Separation of the sticky pqjtides from membrane proteins by HPLC in a normal-phase system. Author s note. We were unable to solubilize either DGK3M or any of the polyamino acids in the solvent systems reported in their work, suggesting their method may not be completely general. 

Most chiral HPLC analyses are performed on CSPs. General classification of CSPs and rules for which columns may be most appropriate for a given separation, based on solute structure, have been described in detail elsewhere. Nominally, CSPs fall into four primary categories (there are additional lesser used approaches) donor-acceptor (Pirkle) type, polymer-based carbohydrates, inclusion complexation type, and protein based. Examples of each CSP type, along with the proposed chiral recognition mechanism, analyte requirement(s), and mode of operation, are given in Table 3. Normal-phase operation indicates that solute elution is promoted by the addition of polar solvent, whereas in reversed-phase operation elution is promoted by a decrease in mobile-phase polarity. 

Figure 10.13 shows the excellent performance of reversed-phase HPLC in biotechnological research. It represents the separation of the tryptic hydrolysate of the normal form and of a mutant of tissue-type plasminogen activator. This protein is built up of 527 amino acids and has a mass of approximately 67 000 Da. The mutant differs in a single amino acid, which leads to a deviating retention time of this specific fragment. 

Vitamin K HPLC has provided the first assay of the phylloquinones and menaquinones that constitute vitamin K in plasma. Phylloquinone circulates bound to lipoproteins from which it can be extracted with hexane after ethanol protein precipitation. Removal of co-eluted lipids can be achieved with normal-phase cartridge columns. Reversed-phase HPLC is almost universally used for vitamin K measurement. Either UV (270 nm) or electrochemical detection is suitable. Electrochemical detection uses the reductive mode ( —1.3 V) to convert the quinone moiety to hydroquinone the main disadvantage being the need to remove oxygen from the mobile phase. 

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