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In this study, a chiral CE method was developed based on the partial filling technique with two chiral plugs for the simultaneous enantioseparation of some racemic drugs, including baclofen, carvedilol, cetirizine, chlorpheniramine, citalopram, fluoxetine, hydroxyzine, propranolol, tramadol, trihexyphenidyl. This method of capillary filling involves the application of two adjacent chiral plugs containing the same BGE, but with different chiral selectors in the plugs for the enantioseparation of a mixture of drugs which cannot be separated with single or mixed chiral selectors. By using this method, each plug can separate the enantiomers independently (same as a single chiral selector modified CE) and the possible interactions between the chiral selectors would be inhibited. The best results were obtained using a fused silica capillary (60 cm × 50 μm id (50 cm effective length)) with phosphate buffer (100 mM, pH 3.0) containing 10 mM hydroxypropyl-α/β-cyclodextrin and 10% (w/v) maltodextrin and detected by UV at 214 nm. The influence of the length and order of the chiral plugs on the enantioresolution was also studied and optimized. The proposed method was compared with a mixed chiral selector-CE system with a combination of hydroxypropyl-α-cyclodextrin/hydroxypropyl-β-cyclodextrin and maltodextrin in the BGE. According to the results, the modified partial filling method is a simple and efficient method for the simultaneous chiral separation and offers appropriate migration times and resolutions compared to the results obtained from mixed chiral selector CE.

The aim of this study was to investigate the feasibility of using multitime-regenerated immunoaffinity column (IAC) for aflatoxins B1, B2, G1 and G2 in peanut confection. After each use, the IAC was washed immediately with phosphate-buffered saline and stored for >12h prior to reuse. The evaluation procedure consisted of using extracts of naturallycontaminated peanut confection (4 replicates), aflatoxin-free peanut confection (duplicates), and aflatoxin-free peanut confection sample spiked with the 4 aflatoxins (AFT) at 3 levels in 4 replicates. Each day, 18 test extracts were analyzed using 18 designated IACs. After each use, the IACs were regenerated and reused for corresponding test extracts on the following day. This procedure was repeated daily over the course of 9days. Analytical steps included passing the test extracts through the IACs, washing the columns with water, and eluting AFT with methanol. The eluates were diluted with water and were subjected to reversed phase LC separation, post-column photochemical derivatization and fluorescence detection. After eluting AFT, IACs were immediately regenerated by washing with phosphate buffer solution and storing overnight at 8°C for re-use the following day. Results were analyzed using ANOVA and Tukey tests. The numbers of reuse varied for each AF: For AFB1 AFB2, AFG1and AFG2 could be reused for 9, 6, 6 and 0 times, respectively. According to AOAC method performance criteria, recoveries ranging from 70% to 125% are considered acceptable at the spiking levels used in this study.

Ethoxynonafluorobutane (ENFB) has been used as a component of new biphasic solvent mixtures. The suitability of several mixtures as solvent systems in countercurrent chromatography was tested. The applicability of the ENFB/2-PrOH/H(2)O mixture to the separation of enantiomers, in combination with a fluorinated chiral selector (CS), was evaluated. N-Perfluoroundecanoyl-l-proline-3,5-dimethylanilide (2), analogous to the previously used N-dodecyl-l-proline-3,5-dimethylanilide (1), was synthesized for this purpose. The capacity of the new solvent system to retain the fluorinated CS in the fluorinated phase used as stationary was examined. Chiral selector 1 was applied in analogous conditions for comparative purposes. Additionally, MTBE/phosphate buffer solvent system was also used with the two CSs. The ENFB/2-PrOH/H(2)O (25:35:40) mixture was found to be adequate in the enantioseparation of DNB-Leu and DNB-Leu-tBu. Enantioselectivity was maintained in the fluorinated solvent system without compromising eluting time. The complete separation of DNB-Leu-tBu was achieved and no leaks of CS to the mobile phase were detected.

Capillary zone electrophoresis (CZE) has been employed to characterize nanometer-sized thiolated alpha-cyclodextrin-capped gold nanoparticles (alpha-CD-S-AuNPs). The addition of tetrabutylammonium (Bu(4)N(+)) ions to the run buffer greatly narrows the migration peak of alpha-CD-S-AuNP. The optimal run buffer was determined to be 10mM Bu(4)N(+) in 30 mM phosphate buffer at pH 12 and an applied voltage of 15 kV. The effect of various tetraalkylammonium ions on the peak width and electrophoretic mobility (mu(e)) of alpha-CD-S-AuNP was studied in detail. Bu(4)N(+) ions assist in inter-linking the alpha-CD-S-AuNPs and narrowing the migration peak in CZE. This observation can be explained by the fact that each Bu(4)N(+) ion can simultaneously interact with several hydrophobic cavities of the surface-attached alpha-CDs on AuNPs. The TEM images show that alpha-CD-S-AuNPs with Bu(4)N(+) are linked together but in the absence of Bu(4)N(+), they are more dispersed. The migration mechanism in CZE is based on the formation of inclusion complexes between Bu(4)N(+) and alpha-CD-S-AuNPs which induces changes in the charge-to-size ratio of alpha-CD-S-AuNPs and mu(e). An inverse linear relationship (r(2)>0.998) exists between the mu(e) and size of alpha-CD-S-AuNPs in the core range 1.4-4.1 nm. The CZE analyses are rapid with migration time less than 4 min. A few nanoliters of each of the alpha-CD-S-AuNP samples were injected hydrodynamically at 0.5 psi for 5s. Our work confirms that CZE is an efficient tool for characterizing the sizes of alpha-CD-S-AuNPs using Bu(4)N(+) ions.

A RP-HPLC method for the analysis of oxalic, citric, formic, succinic, orotic, uric, pyruvic, acetic, propionic, lactic and butyric acids in dairy products with a simple treatment of the sample has been developed. A gradient programme pumping phosphate buffer at pH 2.20 and acetonitrile was used to separate the compounds on a C18 column. Various parameters affecting analysis have been optimised to take18 min with an excellent linearity (R0.999). The precision was good (R.S.D.5%) and the recovery found close to 100%. Its application to analyse the quality of some dairy products has been investigated.

A capillary electrophoresis (CE) assay has been developed for the quantitation and determination of the impurity profile of the potassium channel blockers 3,4-diaminopyridine and 4-aminopyridine. The compounds were separated from related substances using a capillary of 30 cm effective length, a 50 mM phosphate buffer, pH 2.5 and an applied voltage of 25 kV. The assay was validated with respect to specificity, linearity, range, limits of quantitation and detection, precision and robustness. The method allows the detection and quantitation of impurities at the 0.05% level. The feasibility of the assay was demonstrated by analyzing a commercial sample of 3,4-diaminopyridine. All known related substances could be detected in this sample with the present CE method.

Chiral resolution of native DL-lactic acid was performed by capillary electrophoresis using 2-hydroxypropyl-beta-cyclodextrin as a chiral selector. Various factors affecting chiral resolution, migration time, and peak area of lactic acid were studied. The running conditions for optimum separation of lactic acid were found to be 90 mM phosphate buffer (pH 6.0) containing 240 mM 2-hydroxypropyl-beta-cyclodextrin with an effective voltage of -30 kV at 16 degrees C, using direct detection at 200 nm. In order to enhance the sensitivity, sample injection was done under a pressure of 50 mbar for 200 s. On-line sample concentration was accomplished by sample stacking. With this system, D- and L-lactic acids in food products were analyzed successfully.

Capillary electrophoresis (CE) was applied as a fast method of siderophore separation. Siderophores are iron binding and regulating cell products, which facilitate iron transport into cells. A fast and efficient method of siderophore analysis is important for better understanding of the iron pathways in a sea environment or marine organisms. The best results of CE analysis were obtained using free zone CE in 25 m M phosphate buffer at basic pH using a constant voltage of 20 kV. Under these conditions it was possible to detect the presence of siderophores in seawater.

A capillary electrophoresis method for the enantioresolution of labetalol was developed using CDs as chiral selectors and uncoated capillaries. Various native (α-, β- and γ-CD), alkylated (hydroxy propyl-β- and γ-CD, methyl-β-CD) and anionic (sulfated-β-CD and sulfobutylether-γ-CD) cyclodextrins were tested and operational parameters such as buffer pH, concentration of CD were investigated. Propranolol was also studied as a model compound. Uncharged γ-CDs were more effective than β-CDs to separate the enantiomers of labetalol but no complete resolution of the four isomers was obtained. The use of charged cyclodextrins led to a combination of hydrophobic inclusion and ion-pairing interaction in the chiral recognition mechanism. Thus, a complete resolution of the four enantiomers of the labetalol was attained using 7.7 g/l sulfated-β-CD in a 30 mM phosphate buffer, pH 6.5.

The separation of alcohol-soluble proteins of tobacco seeds by free-solution capillary zone electrophoresis (CZE) has been described. The entire separation was achieved in less than 12 min using on-column UV detection at 214 nm. As buffer modifier, 20% acetonitrile was tested for use with 100 mM phosphate buffer (pH 2.5) containing 0.05% hydroxypropylmethylcellulose. This buffer provided high resolution and reproducibility of alcohol-soluble proteins of tobacco seed. Among four tested tobacco cultivars (Virginia tobaccos speight G-28, Yunyan-317, Hongda and Yunyan-85), each cultivar was found to possess a unique protein CZE peak pattern and this pattern could be regarded as a genetic marker to identify tobacco cultivar.

Meleme, one of thermal decomposition products of melamine, ws analyzed satisfactorily by high-performance cation-exchange chromatography using 20 m M phosphate buffer as eluent. The elution behavior of meleme and melamine at various pH values (pH 2.0–6.0) was examined and the determination was accomplishes simultaneously using photodiode array UV-Vis detection.

Capillary electrophoresis (CE) was applied as a method for monitoring of the separation process of systemin, an endogenous peptide from tomato plants. Systemin, an 18-amino-acid polypeptide, was isolated from tomato leaves and purified. A systemin peak was identified using synthetic systemin as a marker, and traced by CE and reversed-phase high-performance liquid chromatography (RP-HPLC) during all chromatographic steps. The best results of CE analysis were obtained in 25 mM phosphate buffer at acidic pH using a constant operating voltage of 30 kV.

Heparin-oligosaccharides, low-molecular-mass heparins (LMMHs) and heparins were determined using high-resolution capillary electrophoresis (HPCE) and polycrylamide gel electrophoresis (PAGE). The conditions for HPCE were 20 m M phosphate buffer (pH3.5) and 20°C. The method was equivalent to that used previously with a borate-sodium dodecyl sulfate buffer (pH8.8). Six LMMHs were determined using PAGE. The method showed a standard deviation of the average molecular mass from 6.4 to 19.8% and of their polydispersity from 0.7 to 10.2%. HPCE and PAGE revealed different important structural and compositional differences of heparins.

Conditions were established for the determination of hydroxy-substituted polynuclear aromatic hydrocarbons (hydroxy-PAHs) using high-performance liquid chromatography with electrochemical detection. Reversed-phase chromatography with methanol-aqueous phosphate buffer (pH 3.0) (50:50) was adopted for the separation of the hydroxy-PAHs. The figures of merit were calculated; the detection limits (signal-to-noise ratio = 2:1) ranged from 20 to 200 pg. The method was applied to the determination of these compounds in an aerosol sample and 5-hydroxyindane, 2-hydroxy-9-fluorenone and 2-nitro-1-naphthol were tentatively identified at the pg/m3 level. The presence of 2-nitro-1-naphthol (0.2 ng/m3) was confirmed by gas chromatography-mass spectrometry.

The direct separation of the two racemates and four diastereomers of nadolol by high-performance liquid chromatography (HPLC) is described. The racemates were well separated on non-chiral ODS columns using mixtures of phosphate buffer and methanol. The diastereomers could be separated on commercial chiral columns, using organic and aqueous mobile phases. The analytical HPLC method using an organic mobile phase was applied to semi-preparative HPLC for diastereomers.

A reversed-phase high-performance liquid chromatographic (HPLC) procedure for the determination of chlordiazepoxide, its four metabolites and three unstable photoproducts (oxaziridines) generated by UV irradiation is reported. The influence of pH and acetonitrile (CH 3 CN) and tetrahydrofuran (THF) percentages in the mobile phase was investigated. The optimum method uses a Nucleosil C 18 column (5 μm) with a mobile phase consisting of CH 3 CN-THF-0.06 M phosphate buffer (pH 5.8) (22:2:76). Eight drugs covering a wide range of lipophilicity and polarity were separated in 50 min. The absolute detection and quantification limits of benzodiazepines were less than 1 and 5 ng, respectively. The HPLC method alone or coupled with the UV irradiation procedure showed good precision (R.S.D. 1.7–4.0%) and excellent linearity in the range of 1.5–43 μg/ml (r⪖0.999).

A method for the determination of tartaric, malic, shikimic, lactic, acetic, citric, succinic, citramalic and fumaric acid in musts, wines and vinegars is proposed, based on high-performance liquid chromatography using two 3-μm C18 columns (250 × 4.6 mm I.D.) in series, providing an efficiency between 50 000 and 80 000 theoretical plates/m. The mobile phase is phosphate buffer (pH 2.35) to which is added a small amount of methanol (3%) as polar modifier. The relative standard deviations were

A sensitive high-performance liquid chromatographic (HPLC) analytical procedure was developed for the quantitative determination of sulphadiazine (SDZ) and trimethoprim (TMP) in Chinook salmon muscle tissue. SDZ and TMP were extracted from salmon muscle tissue using solid-phase extraction, and the extract was subsequently subjected to separate HPLC assay for each of the drugs. An Ultrasphere octadecylsilyl ion-pair column (250 × 4.6 mm I.D.) was used for both assays. A mobile phase of methanol−0.05 M phosphate buffer pH 2.5 (17:83), with ultraviolet detection at 280 nm was used for the SDZ assay. A mobile phase of acetonitrile−0.05 M phosphate buffer pH 2.5 (5:95), with ultraviolet detection at 224 nm was used for the TMP assay. The calibration curves for both assays in Chinook salmon tissue were linear over the concentration range of 0.1 to 10 pg/g for SDZ (r2 = 0.9990) and 0.1–15 μ/g for TMP (r2 = 0.9996). The minimum detectable quantities in Chinook salmon muscle tissue for both SDZ and TMP were 0.1 μ/g at signal-to-noise ratios of 10:1. The average recoveries for the drugs from Chinook salmon muscle tissue were 63% for SDZ and 42% for TMP.

A high-performance liquid chromatographic method was developed for the determinaton of 5-fluorouracil(5-Fu) in vitreous gel and liquid. After addition of an internal standard (5-fluorocytosine), the vitreous humour samples were extracted with n-propanol-diethyl ether (16:84, v/y) with average recoveries of 95%. The extract was evaporated to dryness and the residue was reconstituted in 1 ml of mobile phase (0.05 M phosphate buffer, pH 3.5) and chromatographed isocratically on a C18 reversed-phase column coupled to an ultraviolet detector set at 254 nm. This method was applied to the pharmacokinetic study of 5-Fu in the vitreous gel and liquid of rabbit eyes after the injection of the drug.

High-performance liquid chromatographic methods for the characterization of dirithromycin, a macrolide antibiotic derived from erythromycin, are described. Chromatography is performed on a Hypersil ODS column using a mobile phase consiting of acetonitrile-methanol-50 m M potassium phosphate buffer, pH 7.5 (44:19:37) with ultraviolet detection at 205 nm. The strength of the phosphate buffer can be used to control the selectivity of the separation of dirithromycin and related substances, especially the separation of erythromycylamine. The addition of methanol to the mobile phase improves peak shapes for the compounds of interest. Validation data for purity and related substances methods are described.

Cefixime (CX), an oral cephalosporin antibiotic, and its metabolites in human digestive organs were separated by various modes of high-performance capillary electrophoresis. The zone electrophoresis mode in phosphate buffer (pH 6.8) containing 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulphonate gave the best separation, permitting the complete resolution of CX and all of five metabolites. On the other hand, the plain zone electrophoresis mode in phosphate buffer (pH 6.8) offered a simple procedure for the direct determination of urinary CX concentration using intact urine samples.

Motilin is a gut hormone, which is involved in gastrointestinal motility. Capillary electrophoresis studies were made on 24 peptides that are N-terminal, C-terminal or internal fragments of motilin. The isoelectric point, total charge and hydrophobicity were calculated for all of the peptides. The effects of buffers and pH on migration time and resolution were studied. These included citrate buffer, pH 2.5; phosphate buffer, pH 7.0 and borate buffer, pH 10.0. A capillary zone electrophoresis method was developed to resolve 14 of the motilin peptides. Secondary structure predictions were made using the Chou-Fasman method. Circular dichroism spectra were collected to confirm presence of α-helix in several fragments. Effects of charge, hydrophobicity, secondary structure and length of the motilin fragments on migration time were studied.

In order to overcome problems related to the determination of free tryptophan in biological fluids using conventional methods, we have developed an accurate and reliable procedure based on a specific pretreatment of samples followed by a very rapid and sensitive reversed-phase high-performance liquid chromatographic analysis. The pretreatment consists of adding to the sample of a very low amount of 3 M phosphate buffer to maintain pH in the physiological range followed by ultrafiltration. The precision, reproducibility and sensitivity of our method were also evaluated. The recovery of each amino acid was greater than 92%. The use of a microbore column allows the detection of up to 0.2 pmol/μl of amino acid. The method has been applied to the analysis of samples obtained from 25 normal and 10 phenylketonuric subjects.

Peptides were separated by gel filtration high-performance liquid chromatography with gradient elution. Two gradient elution systems were applied: (1) 0 to 20% acetonitrile in 30 mM phosphate buffer, pH 8.0 and (2) 30 mM phosphate buffer, pH 8.0 to water; with the polymer-type gel filtration column we used, excellent separation of tryptic peptides from hen egg-white lysozyme was achieved within 50 min. System 2 was applied to a separation of V8 protease peptide from yam tuber chitinase and yielded two to three times higher amounts of peptides than reversed-phase high-performance liquid chromatography.

A gradient reversed-phase high-performance liquid chromatographic method was developed for the quantitative analysis of salmon calcitonin. A 0.054-M potassium monobasic phosphate buffer was modified with an acetonitrile—methanol (4:1) mixture as the mobile phase. Several gradient slopes are used during the separation and analysis so that potential peptidal component impurities in salmon calcitonin samples can be separated without wasting analysis time. The method is applicable to the determination of salmon calcitonin in the peptide portion of the sample as well as in the total sample. The analytical results are expressed as percent chromatographic purity instead of activitiy units.

Free phenolic acids in Italian wines and a sherry were identified by high-performance liquid chromatography. The samples were concentrated and passed through a Sep-Pak C18 cartridge and the acids were recovered by elution with 2 ml of tetrahydrofuran. The separation was carried out by gradient elution on a reversed-phase column with methanol—water and phosphate buffer (pH 2.7). Detection was carried out 280 and 230 nm.

Several species of calcitonin, namely, porcine, human, and salmon, are of clinical importance. Previous studies have shown that reversed-phase high-performance liquid chromatography (HPLC) can be used for the analysis of synthetic calcitonin. To confirm the homogeneity of synthetic HPLC purified salmon calcitonin, an analytical method, based on cation-exchange HPLC with a linear sodium chloride gradient in phosphate buffer was developed. The method is capable of selectively analyzing salmon calcitonin in complex synthetic matrices, separating it from synthetic by-products and degradation products, and provides a rapid and precise chemical assay for determining calcitonin content and quality.

High-performance liquid chromatography (HPLC) on newly developed spherical beads of hydroxyapatite was applied to the analysis of purified E. coli tRNAs (Val, Met, Tyr and Phe). tRNAs were eluted from the column separately with appreciable differences in retention time by a 45-min gradient of phosphate buffer (pH 6.8) of concentration from 64 to 123 mM; both the retention times and peak areas of respective tRNAs were highly reproducible. Total tRNA (tRNA(Total)) preparations obtained from E. coli and B. subtilis were also analysed on the column. It is possible even to elute tRNA(Total) which may, in general, contain 60 or more tRNA species with a relatively shallow gradient such as 75-132 mM. The recovery of tRNA from the column was as high as 90%. Owing to the complicated composition, the elution profile of tRNA(Total) had a wide spread irregular shape but, with a 1-h gradient more than ten peaks were easily detected. When the amino acid-accepting activity of tRNA in the eluate of tRNA(Total) was determined, for ten specific tRNAs, each activity peak was eluted sharply from the column. In addition, several tRNA activities were eluted in different fractions. This indicates that isoacceptors were separated by the column. The results show that HPLC on hydroxyapatite beads is useful for the purification and characterization of tRNA.

Relationship between the structure and retention time in a group of dipyridylsulphides have been investigated. The retention time has been determined by high-performance liquid chromatography on a chemically bonded octadecylsilica column with methanol-13 mM phosphate buffer (pH 7) as the mobile phase, using UV detection. From the values of the logarithm of the capacity factor the contributions of the half-molecular fragments have been calculated. The contributions of the fragments can be used to calculate the capacity factors of other dipyridylsulphides. One example of the study of the structure-antituberculous activity relationship from the carbothioamides series is presented.

A reversed-phase displacement chromatographic method has been developed for the purification of bovine and porcine insulin samples. Up to 500 mg of raw insulin could be purified on a Nucleosil C8 analytical column using a methanol-containing phosphate buffer carrier and a cetrimide-containing displacer. A proinsulin contamination level as low as 100 ppm in the collected fractions could be achieved.

The use of preparative liquid chromatography (LC) for the effective purification of potential X-ray contrast agents or intermediates is described. Pure products in grams to kilogram quantities have been obtained by reversed-phase preparative LC of highly water-soluble non-ionic compounds, a weak aliphatic carboxylic acid and an isophthalic acid derivative. The mobile phases used were mixtures of methanol-water alone or with the addition of ammonium acetate buffers, tetrabutylammonium chloride and phosphate buffer.

m- and p-Hydroxyphenylacetic acids have been identified and quantitated in rat brain by gas chromatography of their pentafluoropropionyl hexafluoroisopropyl diesters. The procedure differs from those employed in earlier studies on the catecholic acids in that a support-coated open-tubular column was used, a preliminary purification of the acids by thin-layer chromatography was carried out, and excess derivatizing reagent was removed by extraction with a phosphate buffer.

The elution behaviour of sodium dialkyldithiocarbamates (R2NCS2Na: RCH3, C2H5, n-C3H7 and n-C4H9) was examined by high-performance liquid chromatography on C1-, C8- and C18-bonded silica columns in the presence of tetraalkylammonium salts. The best separation of the carbamates was obtained from the C8-bonded column, with a mobile phase of methanol—1/15 M phosphate buffer (60:40) at pH 6.8 containing tetra-n-butylammonium bromide (0.025 M). This method was applied to the analyses of commercial R2NCS2Na. The results are in good agreement with those obtained by potentiometric titration.

The antilipolytic compound 5-fluoro-3-pyridinecarboxylic acid and 5-fluoro-3-hydroxymethylpyridine have been determined quantitatively in plasma (Srel = 5-7% in the concentration range 1-20 microgram/ml) by liquid chromatography on LiChrosorb RP-8 (5 micrometer) with phosphate buffer pH 3-4 as mobile phase, after precipitation of proteins and direct injection of the supernatant. Detection limits were 0.1-0.2 microgram/ml. The chromatographic retention is explained by adsorption of the uncharged compounds on to the support complemented by ion-pair adsorption with buffer components at extreme pH values.

A method is reported for the determination of alkaloids in cell cultures of Tabernaemontana species. The alkaloids were isolated by means of C 8 pre-concentration columns. After recovery they were analysed on a Phenyl type of column using phosphate buffer (pH 3.9)-acetonitrile-2-methoxyethanol (80:15:5) as the mobile phase. Detection was effected at 275 and 313 nm and peak-height ratios were used for identification.

A new procedure using high-performance liquid chromatography for the rapid separation of cellulase proteins is described. The cellulase components of Trichoderma reesei are fractionated on a DEAE anion-exchange column using a phosphate buffer at pH 6.2. Activities of the individual components obtained from T. reesei QM6a, a wild strain, and several mutant strains have been determined. Each system examined contained beta-glucosidase, at least two exo-beta-1,4 glucanases and five endo-beta-1,4 glucanases with the endo-beta-1,4 glucanases accounting for 20--36% and the exo-beta-1,4 glucanases for 64--80% of the soluble protein.

The retention of homologous n-alcohols in dilute phosphate buffer solution was determined on pentyl-, octyl- and dodecyl-agarose as a function of the degree of substitution of the alkyl-agaroses. The occurrence of two retention determining processes, i.e., liquid—liquid distribution and bimolecular association, is revealed by the retention data. The standard thermodynamic functions for the interaction were calculated and are discussed.

All of the casein components could be adsorbed onto Octyl-Sepharose CL-4B from a phosphate buffer solution and separated chormatographically using a gradient of 0–6 M urea. β-Casein, despite its higher average hydrophobicity, was eluted first. The large peptides α s1 -I and β-I (derived from α s1 − and β-casein by chymosin action) were readily separated from their precurson proteins using this technique, and the rare A variant of α s1 -casein was well resolved from the commoner B variant.

High-performance liquid chromatography of penimocycline Penimocycline is an antibiotic obtained by Mannich reaction between tetracycline and ampicilline. Separation of penimocycline from tetracyclines and other impurities has been studied by high-performance liquid chromatography. The most effective method is liquid-liquid partition on a Micropak CH column (non-polar hydrocarbon bonded on porous silica microparticles) and gradient elution with water-methanol, 0.02 M phosphate buffer (pH 7.6) and 1 m M EDTA. Some results on hydrolysis of penimocycline are given.

A new form of ion-pair chromatography is established and characterised, in which a zwitterion-pairing agent (11-amino undecanoic acid, C11AA, at up to 2 m M concentration in eluent) is used to enhance the retention of nucleotides in reversed-phase high-performance liquid chromatography. Excellent separations of these compounds (giving up to 5000 plates for a 100-mm column) are obtained within the pH range 4 to 6 (maintained by 75 m M phosphate buffer), with retention at pH 4 being up to twenty times that at pH 6. At fixed pH retention increases directly with the amount of C11AA adsorbed by the stationary phase. It is proposed that the enhancement of retention by addition of C11AA and its pH dependence arises from the formation of quadrupolar ion pairs between C11AA and the nucleotides. The order of elution, in general, is monophosphates (least retained), diphosphates, triphosphates. However, these groups can either be separated entirely from each other, or made to interleave by suitable adjustment of pH and C11AA concentration. Deoxy- and cyclic-nucleotides are more strongly retained than their parent compounds. The method is flexible and has great potential for further development.

A procedure for the optimum separation and determination of indole derivatives was developed, based on a generally applicable algorithm for computer processing of experimental data. Experiments were performed on a Separon SGX C 18 column with a mobile phase of citrate phosphate buffer (pH 3.1), containing various amounts of ethanol. A tandem combination of an UV-photometric detector at 267 nm and an electrochemical detector with a wall-jett cell was used. The detection limit was 10 −10 g for certain indole derivatives. The optimization by means of the computer program consists of the following steps: display of the dependence of the capacity factors on the ethanol content in the mobile phase, display of the course of the optimization function and construction of separation maps and simulation of chromatograms.

The retention factors of several acetyl-indandiones as functions of the methanol concentration, pH and tetrabutylammonium ion concentration of the eluent have been determined in an octadecyl silica-methanol-aqueous phosphate buffer reversed-phase system. Results have been interpreted by assuming that the acetyl-indandiones behave as monoprotic acids, and the apparent pKa values have been calculated. These indicate that acetyl indandiones are stronger acids than the simple aromatic carboxylic acids.

The retention behaviour of amines was investigated in systems with silica gel (LiChrosorb Si 60) as the support and an aqueous phosphate buffer containing organic modifiers as the mobile phase. N,N-Dimethyloctylamine and N,N,N-trimethyloctylamine were used as cationic modifiers, with 3,5-dimethylcyclohexylsulphate as the ion-pair forming counter ion. Adsorption isotherms were determined for the amines used as modifiers. At low concentrations of modifier a monolayer is formed on the solid phase and the retention times of the solutes decrease with increasing modifier concentration. Higher concentrations give rise to the formation of a multilayer or bulk phase of modifier, leading to rapidly increased retention of the most hydrophobic solutes and a change in retention order. They hydrogen-bonding properties of the solute seem to be of major importance for the retention on unmodified silica, while the hydrophobicity of the solute determines the retention to the adsorbed phase of the modifier.

Isocratic high-performance liquid chromatography on Hamilton PRP-1 column (25 x 0.46 cm I.D.) at 60°C allows the separation of oxytetracycline, 4,6-epidoxycycline, 4-epidoxycycline, methacycline, 6-epidoxycycline and doxycycline. The mobile phase is tetrahydrofuran—0.2 M phosphate buffer (pH 8.0)—0.2 M tetrabutylammonium hydrogen sulphate solution (pH 8.0)—0.1 M sodium edetate solution (pH 8.0)—water (6:10:5:1:78). The flow-rate is 1.0 ml/min, 40-μg samples are injected and detection is at 254 nm. The column lifetime exceeds 6 months. Results for a number of bulk samples and specialities are reported.

The retention behavior of three naphthaleneacetic acid derivatives on two hydrophobic supports, LiChrosorb RP 8 and Spherisorb ODS, has been studied with a mixture of phosphate buffer (pH 7) and methanol or acethonitrile as the mobile phase. The addition of quaternary ammonium compounds to the mobile phase increases the capacity factors, indicating a mixed retention mechanism that includes ion-pair partition. Constants for ion-pair extraction and ion-pair formation in the mobile phase have been derived. By gas chromatographic measurements, it was found that LiChrosorb RP 8 adsorbs about 0.18 ml of methanol per gram after equilibration wiht phosphate buffer (pH 7)-methanol (60:40, v/v) as the mobie phase. The effects of the support and the composition of the mobile phase on efficiencies, asymetry and selectivity factors are discussed.

A high-performance liquid chromatographic assay for the analysis of josamycin in human serum and urine is presented. The assay involves a simple solid-phase extraction procedure coupled with a phase separation step, separation on a reversed-phase C 18 column with UV detection by a multi-wavelength programmable detector. The mobile phase was acetonitrile-0.015 M phosphate buffer, pH 6.0 (5:2) at a flow-rate of 1.2 ml/min. The column temperature was maintained at 35°C. Linear calibration curves over the concentration ranges 0.1–2.0 mg/l (serum) and 0.5–5 mg/l (urine) were obtained with correlation coefficients of 0.9983 and 1.0000, respectively. The relative standard deviations of five replicate samples at the upper and lower limits of each calibration curve were below 7%. The recoveries at the upper and lower ends of the calibration range for serum were 77% and 70%, respectively, and those for urine were 76% and 80%, respectively.