Columns separation, isocratic

Many industrial laboratories conducting significant amounts of additive analyses have developed a universal HPLC method which may be used to separate most of the additives of interest. Thomas [417] has reported a method that can separate over 20 common primary and secondary stabilisers. Verdurmen et al. [197] employ a gradient ranging from 60 % acetonitrile/40 % water to 100% acetonitrile subsequently, all components are eluted off the column in isocratic mode. Irganox 1063 is used as a suitable internal standard since this compound is not frequently encountered in commercial polymers, elutes without overlap to other additives and shows good UV absorbency. In order... 

The decolourization of the azo dye amaranth was also investigated using atomic hydrogen permeating through a Pt-modified palladized Pd sheet electrode. The decolouration products were separated by RP-HPLC in an ODS column. The isocratic mobile phase was 0.1 M aqueous orthophosphoric acid. The flow rate was 1.2 X 10 2 cm3/s and decomposition products were detected at 236 nm. The RP-HPLC system separated two analytes with retention times of 3.4 and 4.5 min, as demonstrated in Fig. 3.47. The peaks were... 

The glycoalkaloids and their hydrolysis products, with the exception of the aglycone, are ana-lyzable in a single isocratic run. Most analyses were done on reverse phase columns. Separation... 

Fig. 19. Efficiency of RPC in protein separation. Isocratic RPC separation of murine epidermal growth factor (after elution as a single peak from a diethylaminoethyl cellulose column). Column RP 18 (300x7.8 mm dP = 10 pm) 40 °C eluent 0.04 M triethylamine acetate in water — acetonitrile (74 26), pH 5.6 flow rate 0.8 ml/min UV detection at 254 nm, 0.05 AUFS injection 100 pg in 100 pi. The a peak was due to the polypeptide of 53 amino acid residues (M =6040 g/mol), the p component did not contain the asparagine residue in position 1. (From Ref.77) with permission)...

Reversed-phase chromatography also separates, isocratically, vitamin D2 or D3 from their respective previtamins and inactive isomers (207), but, unlike normal-phase chromatography, it can separate vitamin D2 from D3 using nonendcapped stationary phases (198). The 25-hydroxylated metabolites of vitamins D2 and D3 can be separated from one another using a Vydac 201 TP column (37). The separation of vitamin D2 from vitamin D3, and 25-hydroxyvitamin D2 from 25-hydroxy vitamin D3, allows the D2 form of the vitamin or its metabolite to be used as an internal standard for quantifying the corresponding D3 form. 

A sensitive and selective liquid chromatography-tandem mass spectrometry (LC-MS-MS) assay for the simultaneous determination of donepezil (D) and its pharmacologically active metabolite, 6-O-desmethyl donepezil (6-ODD) in human plasma is developed using galantamine as IS [30]. The analytes and IS were extracted from 500 /A aliquots of human plasma via solid-phase extraction (SPE) on Waters Oasis HLB cartridges. Chromatographic separation was achieved in a rim time of 6.0 min on a Waters Novapak Ci8 (150 mm x 3.9 mm, 4 /an) column under isocratic conditions. Detection of analytes and IS was done by tandem mass... 

Figure 4.2 Analysis of Ado, AMP, ADP, and ATP by HPLC. Separations were carded out on reversed-phase Cig columns, eluted isocratically. The detection was at 254 nm. (A) The mobile phase was composed of 10 mM potassium phosphate (pH 5.5) and 20% methanol as the mobile phase. (B) The mobile phase contained 65 mM potassium phosphate (pH 3.7), 5% methanol, and 1 mM n-tetrabutyl ammonium phosphate.

The separation of reactant from product was carried out on a reversed-phase column eluted isocratically with an elution buffer of 0.1 Af potassium phosphate containing 10% methanol at pH 3.2. N-Methyldopamine (N-M-DA) was added to each reaction mixture as an internal standard. The eluent was monitored with an electrochemical detector. The separation of these three compounds is shown in Figure 9.2A. 

The separation and identification of natural dyes from wool fibers using reverse-phase high-performance liquid chromotog-raphy (HPLC) were performed on a C-18 column. Two isocratic four-solvent systems were developed on the basis of the Snyder solvent-selectivity triangle concept (1) 10% acetonitrile, 4% alcohol, and 2% tetrahydrofuran in 0.01 M acetic acid and (2)7% acetonitrile, 8% alcohol, and 5% tetrahydrofuran in 0.01 M acetic acid. Samples were also eluted in 30% acetonitrile. Spot tests and thin-layer chromatography were performed on all samples to confirm HPLC results. The systems also were found to be potentially useful in the identification of early synthetic dyes. A system of sample preparation that minimizes the reaction of samples was discussed. The application of this HPLC separation technique to samples from 20th century Caucasian rugs and American samples unearthed from the foundation of Mission San Jose was examined. 

Figure 4. HPLC separation of phenolic components of an aqueous leachate from the surface of corn leaves infected with Colletotrichum graminicola. a) untreated leachate b) base hydrolyzed leachate c) acid hydrolyzed leachate. Compounds 1 and 2 are isomers of p-coumaric acid. Compounds separated isocratically on a reversed phase C-18 column with a 70 % to 30 % mixture of absolute methanol and 1 % acetic acid. Reproduced with permission from Ref. 69. Copyright 1989 Academic Press, Inc.

Radhofer-Welte and Dittrich [35] described a rapid and sensitive HPLC method for the determination of lornoxicam in plasma samples of humans and laboratory animals. After addition of the internal standard, tenoxicam, the plasma was acidified and extracted by dichloro-methane via Extrelut columns or by solid-phase extraction using Cis columns. After evaporation of fhe solvenf, fhe separation is performed on a Ci8 column in isocratic mode wifh a mobile phase consisting of 0.1 M phosphafe buffer (pH 6)-methanol, and defection at 372 nm. The limit of deferminafion was set to 10 ng/ml using 0.5 ml of sample but can be extended down to 2 ng/ml plasma. Using solid-phase extraction with Cis columns both lornoxicam and its main metabolite 5 -hydroxylornoxicam can be determined while extraction via Extrelut was used in studies where only lornoxicam was to be determined. The method was used in several thousand samples of pharmacokinetics and bioavailability studies in animals and humans. 

An example for the separation of ecdysteroids with HP-RPC systems was published by Louden et al.119 and related to the separation of an ecdysteroids extract from the plant Lychnis flos-cuculi (Caryopyllaceae). These authors used an analytical 4.6 mm x 100 mm 5 pm C18 silica column with isocratic elution (eluent ACN and DzO 99.8% isotopic purity 20 80 v/v) at 1.0 ml min 1 and DAD between 190 and 360nm. This study also explored the application of HPLC—NMR spectroscopy and HPLC—NMR spectroscopy—MS to these ecdys-teroid-containing plant extracts, showing the advantages and limitations of the use of complex multiply hyphenated detection systems, which incorporate detectors of differing sensitivities. 

An example of the separation of coumarins with comprehensive normal-phase x reversed-phase LC systems has been published by Dugo etal for coumarins and psoralens in cold-pressed lemon oil. For the separation in the first dimension, the authors used a capillary normal-phase 1.0 mm X 300 mm 5 pm silica column with isocratic elution (eluent -hexane ACN (75 25)) at 20 pi min-1. In the second dimension, a monolithic 4.6 mm X 25 mm C18 silica column (including a 4.6 mm X 5 mm guard column) was employed with linear gradient elution (eluent A water and eluent B ACN) at 4 ml min-1. The interface between the first and the second dimension was a 10-port, 2-position valve equipped with two storage loops. The incompatibility of the solvents that were used in the two dimensions (NPC and RPC) and its effects in the separation were overcome by using a combination of a capillary column in the first dimension and an analytical monolithic column in the second dimension. With this NPC X RPC system, 11 heterocyclic compounds were analyzed and depicted in 2D contour plots. 

Binder, S.R. Regalia, M. Biaggi-McEachern, M. Mazhar, M. Automated liquid chromatographic analysis of drugs in urine by on-line sample cleanup and isocratic multi-column separation. 

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