Isocratic with HPLC

RP-PLC separation of bilirubin derivatives was performed in an ODS column (250 X 4.6 mm i.d. particle size 5 pm) at 34°C. Analytes were eluted isocratically with 0.1 M di-n-octylamine acetate in 8 per cent aqueous methanol. The flow rate was 0.75 ml/min and bilirubins were detected at 454 nm. Typical chromatograms are depicted in Fig. 2.161. The chromatograms demonstrate that RP-HPLC is a valuable tool for the study of the synthetic process of new bilirubin derivatives [333],... 

Nelis and De Leenheer (180) used isocratic NARP-HPLC with Zorbax ODS (a monomeric ODS stationary phase with a 20% carbon loading) and a mobile phase of acetonitrile/dichlorometh-ane/methanol (70 20 10) to separate nine carotenoids spanning a wide polarity range. This classic separation was achieved by virtue of the fact that the Zorbax ODS material supplied at that time was nonendcapped. The carotenes were retained by hydrophobic interaction with the ODS... 

For coumarins in orange fruits (115), the HPLC used a Zorbax Rx C8 (250-mm X 4.6-mm ID, 5 fim) column maintained at 25°C, and analysis was performed by binary-gradient elution using 0.1% HOAc in acetonitrile (eluent A) and 0.1% HOAc in HzO (eluent B). In the author s lab, standard coumarins could be separated by isocratic elution on Zorbax Rx C8 column with acetonitrile-0.1% HOAc in water (35 65) at 1.0 ml/min, as presented in previous work (1). The eluate from the column was passed to a UV detector (UV 330 nm) and then into a fluorescence detector (excitation at 340 nm, emission at 425 nm). As for the specificity, some of the coumarins do not have native fluorescence. Nine coumarins are separated under UV 330 nm, and three coumarins could not be detected with fluorescence detection. Detailed conditions for coumarin analysis in foods and absorption spectra of coumarins obtained by online diode array detector with HPLC were presented by Lee and Widmer (1). Since coumarins exhibit strong absorption in the ultraviolet region, absorption at approximately 313 nm has been used to estimate the dilution of cold-pressed lemon oil with distilled oil (12). Analysis of umbelliferone (7-hydroxy-coumarin) and scopoletin (6-methoxy-7-hydroxycoumarin) in citrus fruits was performed using... 

Figure 2.19 Separation of several nucleosides on ion-exchange HPLC was carried out on an ion-exchange column (AX-100) eluted isocratically with a mobile phase of 0.1 M sodium phosphate buffer (pH 7.3) containing 0.8 M sodium acetate. The column was monitored at 254 nm. A standard solution containing approximately 2 nmol each of adenosine, AMP, cAMP, ADP, and ATP was loaded onto the column.

In this assay, the substrate, Dopa, and the reaction product were separated by reversed-phase HPLC and eluted isocratically with 0.1 M potassium phosphate buffer at pH 3.0. The eluent was monitored with an electrochemical detector. The separation obtained with this procedure is shown in Figure 9.4B, together with results obtained after incubation of L-Dopa with the enzyme from rat cerebral cortex for 20 minutes at 37°C (Fig. 9.44). Using a calibration curve of the type shown in Figure 9.5, it was possible to show that 1.55 nmol... 

The assay developed for this activity involves the reaction of the P5C with o-aminobenzaldehyde (OAB) to form the reaction product dihydroquinozoli-nium (DHQ). The DHQ and unreacted OAB were separated by reversed-phase HPLC (LiChrosorb C18). The column was eluted isocratically with a mobile phase of 1 part methanol to 2 parts water, and the separation shown in Figure 9.37 obtained. The reaction mixture contained L-omithine (35 mAf), a-ketoglutarate, potassium phosphate (pH 7.4), and pryidoxyl phosphate in a total volume of 2 mL. The reaction was started by the addition of the homogenate and terminated by the addition of 1 mL of 3 N HO containing the OAB. Precipitated protein was removed by centrifugation (3000 rpm), and samples of the supernatant solution (10 nL) were injected for analysis. 

In this study, the reactants ATP, N MN, and ADP were separated by reversed-phase HPLC (/uBondapak C,8) eluted isocratically with a mobile phase of 25 mAf (NH PC (pH 8.0). Figure 9.86 illustrates the separation obtained under these conditions. 

The separation of the substrate and the product was accomplished by reversed-phase HPLC on an ODS column. The column was eluted isocratically with a mobile phase of methanol-water (33 67 v/v). The compounds were detected at 254 nm. The separations obtained are shown in Figure 9.121. Radiolabeled substrates were also used, and the eluent was assayed for radioactivity on fractions collected during the elution. 

Figure 9.140 HPLC elution profile of a sample taken from a discontinuous assay of crude mouse spleen extract, using iV,-fluorenylmethoxycarbonyl-EEY(P)AA [Fmoc-EEY(P)AA] as substrate. Chromatography was performed on a C g Novapak column (10 cm X 8 mm) eluted isocratically with 36% acetonitrile-water (0.1% TFA) (v/v) at a flow rate of 2 mL/min. Peaks are due to (A) methanol and methanol-soluble compounds derived from the sample of crude homogenate, (B) Fmoc-EEY(P)AA (1238 pmol), and (C) Fmoc-EEYAA (195 pmol). Appropriate controls showed that no interfering compounds eluted in the position of the peptides. Inset Fluorescence monitoring of HPLC of Fmoc-EEYAA (75 fmol) eluted isocratically with 36% acetonitrile-water (0.1% TFA). Excitation and emission wavelengths, 268 and 307 nm, respectively, with gain X 100 and 10 mV chart scale. (From Nash et al., 1993.)...

The identification of the reaction conjugates was carried out by reversed-phase HPLC on a Ct8 (/xBondapak) column eluted isocratically with a mobile phase of methanol-glacial acetic acid-water (20 1 79 v/v). The wavelength for detection was 254 nm. 

The HPLC enzyme assay method provides an alternative procedure with which to approach this problem. Clearly, to be able to assay these activities by the HPLC method, it is necessary to separate ATP, ADP, and AMP. This separation can be easily accomplished by ion-exchange HPLC eluted isocratically with a mobile phase containing a phosphate buffer and sufficient concentration of salt to elute the ATP. Under these conditions, the order of elution of the compounds would be AMP first, ADP next, and ATP last. 

ECD Characteristics. Compound-specific destructive concentration or mass-flow detector depending on operation compound sensitivities differ over a wide range useable primarily with isocratic RP-HPLC including some buffer salts, pre- or postcolumn derivatization can be used to increase number of measurable compounds. 

The vast majority of separations described for the leukotrienes have used isocratic reversed phase conditions with an ODS stationary phase, although a single report exists which used ion-pair reversed phase chromatography with gradient conditions. Examples of these separations will not be discussed, together with some of the problems associated with HPLC of the leukotrienes. 

It is universal for aU anions or cations in solution (i.e., a bulk property of mobile-phase detector), nondestructive, and a concentration detector compound sensitivities differ over an order of magnitude range, useable primarily with isocratic RP-HPLC without buffer salts (unless subsequent suppression column is used) and it is a premier detector for ion chromatography (IC). 

A solventless technique of solid-phase microextraction (SPME) has been also employed for HPLC determination of MC in a natural Microcystins sp. bloom in a freshwater, where three dominant MC variants MC-LR, -YR, and -RR were quantified. For this purpose a measuring system with commercial SPME-HPLC interface was employed. Microcystins were sorbed from acidified solutions using SPME fibers with carbowax/templated resin and polydi-methylsiloxane/divinylbenzene coating and desorbed at dynamic mode with HPLC eluent, which was used in isocratic elution mode and consisted of water and methanol with 0.05% TFA. For each toxin partition equilibrium was achieved within 60 min and example response obtained in SPME-HPLC system is shown in Fig. 6. The detection limits for all examined MC for 5 ml samples were reported at about 7 ppb. 

The instability of EC detectors towards changes in eluent ionic strength implies that isocratic elution is to be preferred, especially for applications requiring high sensitivity. Gradient elution can, however, be used in conjunction with HPLC-ED if the stability of the baseline and of the response is acceptable, as exemplified by some of the reports discussed in Chapter Either step or continuous gradients... 

The above HPLC methods are used for the analysis of limonin and/or nomilin in citrus juices. Herman et al. (1989) used a reverse phase HPLC method to quantify limonoids in fruits, seeds, leaves, stems and seedlings of Citrus ichangensis. The extraction procedure of Hasegawa et al. (1984) as described in Section 2.1.1 was followed. The dried extracts were dissolved in 50% methanol prior to injection. A reverse phase C-18 column (4.6 X 250 mm) was used. The column was eluted isocratically with water-methanol-acetonitrile (49 41 10) at a flow rate of 1 ml/min. Limonoids were detected by UV absorption at 210 nm. This... 

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