Detectability comparisons, HPLC columns

Fig. 6. A comparison of the absorbance of fractions eluting off an HPLC column (UV/VIS) with the total molecular ion intensities of the fractions (TIC). Separation C-18 reverse phase colrnnn with 90 10 v/v water/acetonitrile (both containing 0.1% HAC) to 10 90 v/v over 20 min. Detection UV absorbance at 254 nm total ion coimts (TIC) with ESI source, m/z scanned 350-2000 in positive ion mode (Esquire LC/MS Ion Trap Mass Spectrometer/ Bruker)...

Stewart et. al.(21] have shown the usefulness of FI on-line ion-pair extraction in the determination of carboxylic acid drugs, using salicylic acid, valproic acid, and ibuprofen as model drugs. After a comparison of different chromophoric and fluorophoric cationic dyes in chloroform extractant. Gentian Violet was recommended as counterion for spec-trophotometric determination and Acridine Orange was recommended for fluorimetric determination. The system was used for post column detection in HPLC. 

Figure 16 Comparison of L-phenylalanine anilide imprinted polymer particles in HPLC (1) and CEC (2). Particles sizes for HPLC 20-25 pm, for CEC 2-10 pm. Mobile phase 90 5 5 AcN/HAc/H20 (v/v/v). HPLC column 15cm x 2.1 mm ID, flow rate 0.2mL min Detection 254 nm. CEC Capillary 40 cm x 75 pm ID, effective length 20 cm. Electroinjection 5 s, 300Vcm Separation 350Vcm Detection 254nm. (From Ref 26.)...

Figure 18 Comparison of dansyl-L-phenylalanine imprinted polymer particles in CEC (a) and HPLC (b). CEC capillary 25 cm x 75 pm ID, effective length 17 cm. Electroinjection 10 s, 10 kV. Separation 30 kV. Detection 280 nm. Mobile phase AcN/(100mmolL HAc) (80 20, v/v) (pH 3.0). HPLC Column 15 cm x 4.6mm ID, flow rate 1.0mLmin Detection 280 nm. Mobile phase AcN/HAc (98 2, v/v). Sample volume 30 pL. (From Ref. 22.)...

The comparison of analytical characteristics HPLC methods of determination of phenols with application amperometric and photometric detectors was caiiy out in this work. Experiment was executed with use liquid chromatograph Zvet-Yauza and 100 mm-3mm 150mm-3mm column with Silasorb C18 (5 10 p.m). With amperometric detector phenols were detected in oxidizing regime on glass-cai bon electrodes. With photometric detector phenols were detected at 254 nm. 

Nishiyama and Kuninori [65] described a combination method of assay for penicillamine using HPLC and postcolumn reaction with 6,6 -dithiodi(nicotinic acid). Thiols were separated by HPLC on a reversed-phase column (25 cm x 4.6 mm) packed with Fine Sil 08-10, with 33 mM KH2PO4 (adjusted to pH 2.2 with H3PO4) or 33 mM sodium phosphate (pH 6.8) as the mobile phase. Detection was by postcolumn derivatization with 6,6 -dithiodi(nicotinic acid), and measurement of the absorbance of the released 6-mercaptonicotinic acid was made at 344 nm. The detection limit for penicillamine was 0.1 nmol. A comparison was made with a... 

The experiments were performed with different concentration of p-coumaric acid. While in most cases, 500 ppm solutions were employed, saturated p-coumaric acid solutions (700ppm) were more suitable for detecting minor intermediates, p-coumaric acid and intermediates compounds evolution was monitored by HPLC equipped with a Yarian OmniSpher 5 Cig column. Identification and quantitations were achieved by comparison with standards. TOC analyses were carried out using a Shimadzu mod 5000 A apparatus. 

Ultra-performance HPLC (UPLC) utilizes sub-2-pm porous particles inside packed microbore columns up to 150 mm long. Significant improvements in terms of resolution, analysis time, and detection sensitivity have been reported. A side-by-side comparison of HPLC and UPLC was made to determine concentrations of alprazolam in rat plasma.10 UPLC provided a four-fold reduction in terms of LC/MS/MS cycle time that translated into higher sample throughput. Another important... 

Fig. 2.25. (a-c) Comparison of HPLC separation of carotenoid isomers with a C30 column. Column temperature 25°C, flow rate lml/min, detection 450 nm. Mobile phase was acetone and water mixtures. Reprinted with permission from T. Glaser et al. [52]. 

Fig. 3.107. Comparison of micro-HPLC separations of aromatic sulphonic acids in different mobile phases (a) 0.005 M tetrabutylammonium hydrogensulphate (TBAS) in 15 per cent (v/v) methanol in water (1) Laurent acid, (2) amino-F-acid, (3) Cleve-1,6- and Peri acids, (4) unidentified impurity, (5) Cleve-1,7-acid and (6) unidentified impurity, (b) 0.005 M tetrabutylammonium hydrogensulphate (TBAS) in 15 per cent (v/v) methanol in water with 0.01 M /Lcyclodextrin (CD) (1) Laurent acid, (2) amino-F-acid, (3) Cleve-1,6-acid, (4) Peri acids, (5) unidentified impurity, (6) Cleve-1,7-acid and (7) unidentified impurity. Column, Biosphere Si C18, 162 X 0.32 mm i.d. flow rate 5 pl/min, column temperature ambient, detection, UV, 220-230 nm. Reprinted with permission from P. Jandera et al. [164].

For comparison of impurity levels quoted as % area/area, the normalized PA [area divided by the respective MTs, often stated as corrected PA (Ac)] must be used in CE to compensate for the residence time difference of the species in the detector. In HPLC, the separation takes place on the column. After the column, all analytes travel through the detector at the same speed (that of the mobile phase) and hence have the same residence time in the detector cell. However, in CE, the electrical field also takes effect in the detection cell. Therefore, the residence time of the species that have a higher apparent mobility (as shorter Jm) will give a lower response than species with a lower mobility, for species with the same absorptivity and concentration. ... 

Hi. Lysine. Gamma radiolysis of aerated aqueous solution of lysine (94) has been shown, as inferred from iodometric measurements, to give rise to hydroperoxides in a similar yield to that observed for valine and leucine. However, attempts to isolate by HPLC the peroxidic derivatives using the post-column derivatization chemiluminescence detection approach were unsuccessful. This was assumed to be due to the instability of the lysine hydroperoxides under the conditions of HPLC analysis. Indirect evidence for the OH-mediated formation of hydroperoxides was provided by the isolation of four hydroxylated derivatives of lysine as 9-fluoromethyl chloroformate (FMOC) derivatives . Interestingly, NaBILj reduction of the irradiated lysine solutions before FMOC derivatization is accompanied by a notable increase in the yields of hydroxylysine isomers. Among the latter oxidized compounds, 3-hydroxy lysine was characterized by extensive H NMR and ESI-MS measurements whereas one diastereomer of 4-hydroxylysine and the two isomeric forms of 5-hydroxylysine were identified by comparison of their HPLC features as FMOC derivatives with those of authentic samples prepared by chemical synthesis. A reasonable mechanism for the formation of the four different hydroxylysines and, therefore, of related hydroperoxides 98-100, involves initial OH-mediated hydrogen abstraction followed by O2 addition to the carbon-centered radicals 95-97 thus formed and subsequent reduction of the resulting peroxyl radicals (equation 55). 

The anthocyanins exist in solution as various structural forms in equilibrium, depending on the pH and temperature. In order to obtain reproducible results in HPLC, it is essential to control the pH of the mobile phase and to work with thermostatically controlled columns. For the best resolution, anthocyanin equilibria have to be displaced toward their flavylium forms — peak tailing is thus minimized and peak sharpness improved. Flavylium cations are colored and can be selectively detected in the visible region at about 520 nm, avoiding the interference of other phenolics and flavonoids that may be present in the same extracts. Typically, the pH of elution should be lower than 2. A comparison of reversed-phase columns (Ci8, Ci2, and phenyl-bonded) for the separation of 20 wine anthocyanins, including mono-glucosides, diglucosides, and acylated derivatives was made by Berente et al. It was found that the best results were obtained with a C12 4 p,m column, with acetonitrile-phosphate buffer as mobile phase, at pH 1.6 and 50°C. 

The HPLC was equipped with a UV-Vis detector (VWD = 210 nm), a water symmetry Cig column, 150 mm x 3.9 mm, 5 p,m. The mobile phase was 25% acetonitrile in 75% 20 mM phosphate buffer (pH 2.8). The flow rate was set at 1.5 mL/min. Equal volumes (25 p,L) of standard and sample solutions were injected into the chromatographic system. Quantification of polysorbate 80 is based on a comparison of the response of oleic acid in sample and that of oleic acid in standard solution [6]. When polysorbate 80 was quantified by GC, the released oleic acid can be detected without derivatization and prepared according to the HPLC method. 

The solvent elimination problem became less of a problem with the commercialization of microbore columns. Hayes et al. (54) studied gradient HPLC-MS using microbore columns and a moving-belt interface. The heart of the system was the spray deposition device designed to be compatible with microbore-column flow rates. Nebulization of the eluent was found to be applicable to a variety of mobile-phase compositions and thus was readily compatible with gradient elution. Figure 13 shows a comparison of UV detection with that obtained with the HPLC-MS system. Applications of this system were demonstrated on water from coal gasification processes. 

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