Amino acids reversed-phase HPLC

The stability of the CBI derivative is sufficient for its isolation and complete characterization (11), an accomplishment that is not realized with most OPA adducts. Thus, the CBI derivatives of a number of representative amino acids and amines have been isolated and their fluorescent properties determined as a function of the media and other relevant parameters encountered in reverse-phase HPLC (RP-HPLC). 

Amino acid analysis, by reverse-phase HPLC, of acid-hydrolyzed uncross-linked recombinant resilin and cross-linked recombinant resilin clearly shows the presence of dityrosine in the cross-linked sample (Figure 9.3c). Further evidence of the presence of dityrosine was obtained by UV irradiation (Xmax,ex 315 nm Xmax,em 409 nm). Dityrosine endows natural resilin with pH-dependent blue fluorescence [38] on UV irradiation. The cross-linked recombinant resilin material was similarly fluorescent, strongly suggesting dityrosine cross-links. 

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. 

The most popular current techniques for amino acid analysis rely on liquid chromatography and there are two basic analytical methods. The first is based on ion-exchange chromatography with post-column derivatization. The second uses pre-column derivatization followed by reversed-phase HPLC. Derivatization is necessary because amino acids, with very few exceptions, do not absorb in the UV-visible region, nor do they possess natural fluorescence. 

Nucleotides, peptides, and amino acids also differ subtly in their polarities Some are more hydro-phobic than others. Thus, separation via reverse phase HPLC is possible. A reverse phase column, such as C18 or C8, has a low- to medium-polarity stationary phase. The more hydrophobic sample components interact to a greater degree with the stationary phase, and therefore elute more slowly than the more hydrophilic components. The sample elution order is from most hydrophilic to most hydrophobic. 

Reverse-phase HPLC can be used for the separation of peptides and proteins. Smaller peptides (less than 50 amino acid residues) may be satisfactorily separated on octadecylsilane (C-18) bonded phases whereas for adequate recovery of larger molecules, tetrylsilane (C-4) or octylsilane (C-8) is recommended. Porous column packing with gel permeation and reverse phase properties is usually required for proteins with relative molecular masses greater than 50 000. 

An example of a simple CZE method for peptide analysis and characterization is the one developed for protegrin IB-367.37 IB-367 is a peptide containing 17 amino acid residues that possess antimicrobial properties, and it is being developed for treatment of oral mucositis associated with aggressive cancer chemotherapy as well as other topical applications. This polycationic product was chemically synthesized using solid-phase and purified by preparative reversed-phase HPLC. IB-367 is rich in cysteine and arginine residues. 

CSPs and chiral mobile phase additives have also been used in the separation of amino acid enantiomers. Another technique that should be mentioned is an analysis system employing column-switching. D-and L- amino acids are first isolated as the racemic mixture by reverse-phase HPLC. The isolated fractions are introduced to a second column (a CSP or a mobile phase containing a chiral selector) for separation of enantiomers. Long et al. (2001) applied this technique to the determination of D- and L-Asp in cell culture medium, within cells and in rat blood. 

Purify the FMLPK-Fl on a Cjg reverse-phase HPLC column by isocratic elution with a solution of 30% acetonitrile and 0.2 M acetic acid. Monitor the effluent at 254 nm. The peak corresponding to FMLPK-Fl should be clearly resolved from unreacted FITC and its breakdown products. Confirm the identity of FMLPK-Fl by performing an amino acid analysis of an acid-hydrolyzed sample. 

As described in the section dealing with liquid chromatographic methods, the reaction of 0,0-di-substituted tartaric anhydrides with racemic amino alcohols in acidic solution leads exclusively to the corresponding diastereomeric monoesters, which are easily separated by reversed-phase HPLC. However, H- and sometimes 13C-NMR spectra of these compounds are in many cases also highly useful for determining the diastereomeric ratio and in many cases the absolute configuration. 

On reversed-phase HPLC, the sulfones usually appear in an intermediate position between the more hydrophobic t t[CH2-S] peptide and the more polar t t[CH2—SO] sulfoxides, much as seen in the amino acid analysis by oxidation of Met.162 Unlike sulfoxides, once formed, sulfones are resistant to reduction to sulfides or sulfoxides.1[5T Incorporation of the tp[CH2—S] element and its oxidized counterparts into litorin (positions 8-9), a bombesin-like peptide, gives rise to receptor antagonists that are more potent when in the sulfoxide forms than in the sulfone form. 

The use of nonpolar chemically bonded stationary phases with a polar mobile phase is referred to as reverse-phase HPLC. This technique separates sample components according to hydrophobicity. It is widely used for the separation of all types of biomolecules, including peptides, nucleotides, carbohydrates, and derivatives of amino acids. Typical solvent systems are water-methanol, water-acetonitrile, and water-tetrahydrofiiran mixtures. Figure 3.15 shows the results of protein separation on a silica-based reverse-phase column. 

B 4. List the order of FMOC-amino acids from a typical reversed-phase HPLC column. Explain the order in terms of the relative polarity of the amino acid derivatives. Hint See Chapter 3. 

Gas chromatography (GC) for amino acid analysis is the alternative to HPLC that has found the greatest acceptance. It requires the preseparation derivatization of the amino acids to render them volatile. For this purpose, amino acids are frequently converted into acylated esters. N-Trifluoroacetyl-n-butyl esters and /V-heptafluorobutyrylisobutyl esters are most commonly employed. There have been comparative studies (3,4) that document similar (if not equivalent) analytical results for GC and the classic ion-exchange chromatographic method applied to a variety of food samples. Comparison (5) of GC to the reversed-phase HPLC determination of amino acids (phenylisothiocyanate derivatized) also shows excellent agreement. 

Sulfosalicylic acid has most commonly been used to precipitate proteins prior to ion-exchange amino acid analysis (11). In this mode, SSA allows for a very simple sample preparation that requires only centrifugation of the precipitated sample and then direct injection of the resulting supernatant solution. The supernatant solution is already at an appropriate pH for direct injection. Also, the SSA does not interfere chromatographically since it elutes essentially in the void volume of the column. It has been noted that, if an excessive amount of SSA is employed, resolution of the serine/threonine critical pair can suffer (12). The use of SSA prior to reversed-phase HPLC can be more problematic, since its presence can interfere with precolumn deriva-tization. For example, Cohen and Strydom (13) recommend the separation of the amino acids from the SSA solution on a cation-exchange resin prior to derivatization with phenylisothiocya-nate (PITC). 

A problem with the chromatographic determination of cysteic acid is that there is almost no retention of cysteic acid. For both reversed-phase HPLC and ion-exchange amino acid analyzers (usually employing cation-exchange resins), cysteic acid is essentially eluted within or near the void volume of the column. This makes it more susceptible to unknown chromatographic interferences from various matrices. When cysteine is alkylated by 3-bromopropylamine, the product (S-3-aminopropylcysteine) looks very similar to lysine in structure. Hale et al. (90) show that this alkylated species affords excellent chromatographic separation on four different commercially available amino acid analysis systems and that, indeed, it does elute very near lysine in each case (see Fig. 4). 

Richfield-Fratz et al. (206) determined aniline, benzidine, 4-aminophenyl, and 4-amino-azobenzene in sunset yellow. The determination involved chloroform extraction followed by diazotization and coupling with the disodium salt of 3-hydroxy-2,7-naphthalenedisulfonic acid and analysis by reverse-phase HPLC. Using a similar system, Peiperl et al. (207) determined benzidine in sunset yellow. Dithionite was used to reduce any combined benzidine present. 

We were also able to use FAB mass spectrometry to determine the amino acid sequence around the active site serine in the acyl transference domain of rabbit mammary fatty acid synthase.6 The synthase was labelled in the acyl transferase domain(s) by the formation of O-ester intermediates after incubation with [" " C]-acetyl- or malonyl-CoA (Fig. 2A). The modified protein was then digested with elastase (Fig. 2B), and radioactive material isolated via successive purification steps on Sephadex G-50 and reverse phase HPLC. The isolated peptides were then sequenced by FAB MS. The data summarized in Table II established the sequences of both the acetyl and malonyl hexapeptides to be N-acyl-Ser-leu-Gly-Glu-Val-Ala. 

Tsanaclis et al. [33] described a reversed-phase HPLC method for determination of vigabatrin in plasma after derivatization with phthalal-dehyde. Serum was mixed with y-amino-y-phenylbutyric acid and methanol. Derivatization was carried out with phthaldehyde in borate buffer (pH 9.5) containing 2-mercaptoethanol. The resulting mixture was separated on a column (15 cm x 4.6 mm) of C18 Microsorb (5 /an) with 10 M H3P04-acetonitrile-methanol (6 3 1) as mobile phase with a flow rate of 2 ml/min and fluorimetric detection between 418 and 700 nm (excitation at 370 nm). The detection limit was 0.08 fig/ml of vigabatrin. The CVs were 9%, 5%, and 5% at 2.14, 20.1, and 83.61 fig/ml/min, respectively. Common anticonvulsant did not interfere. 

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