Peptides normal phase HPLC

Chiral stationary phases for the separation of enantiomers (optically active isomers) are becoming increasingly important. Among the first types to be synthesized were chiral amino acids ionically or covalently bound to amino-propyl silica and named Pirkle phases after their originator. The ionic form is susceptable to hydrolysis and can be used only in normal phase HPLC whereas the more stable covalent type can be used in reverse phase separations but is less stereoselective. Polymeric phases based on chiral peptides such as bovine serum albumin or a -acid glycoproteins bonded to... 

The advantage of normal phase over the reversed-phase mode of liquid chromatography is that the column load can be 10-fold higher in the former. The main limitation of normal phase is that only those peptides that are soluble in organic solvents, such as chloroform and methanol or ethanol mixtures, will yield some separation. As a result, RP-HPLC is preferred for the separation of unprotected peptides as well as of protected or partially protected derivatives. 

The oviducts of the cockroaches L. maderae (31) and P. americana (32) also contain proctolin. In both instances, quantitative estimates of proctolin-like bioactivity were made following the separation of extracts on reverse-phase HPLC. After depolarization of the tissue in high potassium saline, the proctolin-like substance in . americana was released from oviducts in a calcium dependent fashion. Oviducts in L. maderae (31) showed some responsiveness to proctolin in a calcium-free medium and the peptide also appeared to facilitate the re-entry of calcium into muscle after depleted preparations were returned to normal levels of external calcium. 

In this section peptide separation by gel permeation, normal-phase adsorption, ion-exchange, and liquid-solid adsorption HPLC will be considered. Compared to reversed-phase HPLC, these elution modes have attracted considerably less interest for peptide separations. This is due to a number of factors arising from the diverse characteristics of the solutes... 

B. Gel Permeation and Normal-Phase Liquid-Liquid HPLC of Peptides... 

EL-4 cells (3 x 10 ) were lysed with N,N-dimethyl-N-(3-sulfopropyl)-3-[[(3a,5 P,7a, 12a)-3,7,12-trihydroxy-24-oxocholan-24-y 1]-amino]-l-propanaminium hydroxide (CHAPS). The nuclei and membranes were pelleted and the supematent lysate filtered to remove lipids. The lysate was sequentially passed over sepharose columns containing a) normal mouse serum b) Y-3 which is an anti-K monoclonal antibody. Both columns were washed with 45 coliunn volumes of progressively lower molarity salt solutions. The beads were then treated with acetic acid to release antigen-antibody complexes and the complex was denatured by boiling in 10% acetic acid. The mixture was filtered through a 3 kDa pore-size membrane and the filtrate containing MHC class I peptides subjected to reversed phase HPLC. 

Normal-phase LC is a less potent alternative [132,133] and has been recommended only when impurities in the crude were derived from the loss of side-chain protecting groups. If the crude product exhibits a significant amount of deletion peptides, then purification by reversed-phase HPLC techniques is required [131]. In this case, Cg or even C4 columns are the most suitable for peptides of —10 residues. For longer peptides the less polar diphenyl-based phase is recommended. 

Peptide mapping has also been used to characterize abnormal variants in human hemoglobin.Table 4.3 lists the amino acid sequences, calculated masses, and masses observed using PDMS for the tryptic peptides from the a-chain of normal human hemoglobin. In this case, peptides were purified by reversed-phase HPLC prior to mass-spectral analysis, and a human a-chain variant (Hb Bobbi) had been identified in which the HPLC peak corresponding to aT6 eluted earlier than expected. A comparison of the PDMS spectra shown in Figure 4.14 for the normal and variant aT6 revealed a mass difference of 22 Da, which corresponds to an Asp - His substitution, or a single substitution (G -> C) in the codon. 

The amino acid composition of peptides is generally assayed by carrying out acid hydrolysis with 6 M hydrochloric acid, followed by determination of the individual amino acids by HPLC. Various types of HPLC have been employed to separate amino acids, but reversed-phase chromatography on Cigcoiumns is the most commonly system used. Detection of amino acids normally involves derivatization, since their maximum absorbance is at 214 nm in which many other compounds also can absorb. The most frequently used derivatizing agents are dansyl chloride, phenyl isothiocyanate and o-phthaldialdehyde. 

Since peptides for research purposes are usually required in only mg to g amounts, the time-saving solid-phase peptide synthesis method148-501 can be used. The strategy is in principle similar to that in solution, with the difference that there is no need for isolation of the intermediate products. As the growing peptide chain is synthesized on a suitable resin the whole procedure lends itself to automation. The drawback is that every reaction step at the resin has to be forced to give an almost 100% yield. In practice, this cannot be accomplished, with the consequence that the desired product must be isolated from a mixture of side-products by the final, normally HPLC, purification procedure which is sometimes difficult to perform and also expensive. Peptides of up to 50 amino acid residues are now readily accessible using stepwise solid-phase procedures[501. 

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