Aqueous gradient elution

Dibenzyl-14-crown-4 (lithium ionophore VI 6,6-dibenzyl-l,4,8,ll-tetra-oxa-cyclo-tetradecane) [106868-21-7] M 384.5, m 102-103°. Dissolve in CHCI3, wash with saturated aqueous NaCl, dry with MgSOa, evaporate and purify by chromatography on silica gel and gradient elution with C6Hg-MeOH followed by preparative reverse phase HPLC on an octadecyl silanised silica (ODS) column and eluting with MeOH. It can be crystd from MeOH (v Br 120 cm , C-O-C). [7 Chem Soc Perkin Trans 1 1945 1986.]... 

The purified protein was subjected to reversed-phase HPLC analysis by using a 150 X 1 mm Cig column with gradient elution from 0.1% aqueous trifluo-roacetic acid (TEA) to 0.1% TEA in acetonitrile, over a period of 55 min, at a flow... 

The function of the post-column solvent addition was not discussed but is presumably to overcome, in the early stages of the gradient elution, the need to spray a largely aqueous mobile phase. 

Use of 10 pm LiChrosorb RP18 column and binary eluent of methanol and aqueous 0.1 M phosphate buffer (pH 4.0) according to suitable gradient elution program in less than 20-min run time with satisfactory precision sensitivity of spectrophotometric detection optimized, achieving for all additives considered detection limits ranging from 0.1 to 3.0 mg/1, below maximum permitted levels Simultaneous separation (20 min) of 14 synthetic colors using uncoated fused silica capillary column operated at 25 kV and elution with 18% acetonitrile and 82% 0.05 M sodium deoxycholate in borate-phosphate buffer (pH 7.8), recovery of all colors better than 82%... 

As the vast majority of LC separations are carried out by means of gradient-elution RPLC, solvent-elimination RPLC-FUR interfaces suitable for the elimination of aqueous eluent contents are of considerable use. RPLC-FTTR systems based on TSP, PB and ultrasonic nebulisa-tion can handle relatively high flows of aqueous eluents (0.3-1 ml.min 1) and allow the use of conventional-size LC. However, due to diffuse spray characteristics and poor efficiency of analyte transfer to the substrate, their applicability is limited, with moderate (100 ng) to unfavourable (l-10pg) identification limits (mass injected). Better results (0.5-5 ng injected) are obtained with pneumatic and electrospray nebulisers, especially in combination with ZnSe substrates. Pneumatic LC-FI1R interfaces combine rapid solvent elimination with a relatively narrow spray. This allows deposition of analytes in narrow spots, so that FUR transmission microscopy achieves mass sensitivities in the low- or even sub-ng range. The flow-rates that can be handled directly by these systems are 2-50 pLmin-1, which means that micro- or narrow-bore LC (i.d. 0.2-1 mm) has to be applied. 

Operation of the interface should be compatible with all chromatographic conditions which are likely to be encountered, including flow rates from around 20 nlmin-1 to around 2 mlmin-1, solvent systems from 100% organic phase to 100% aqueous phase, gradient elution, which is of particular importance in... 

RP-HPLC has also been used for the analysis of flavan-3-ols and theaflavins during the study of the oxidation of flavan-3-ols in an immobilized enzyme system. Powdered tea leaves (20Qmg) were extracted with 3 X 5 ml of 70 per cent aqueous methanol at 70°C for lQmin. The combined supernatants were filtered and used for HPLC analysis. Flavan-3-ols were separated in a phenyl hexyl column (250 X 4.6 mm i.d. particle size 5 /im) at 30°C. Solvents A and B were 2 per cent acetic acid in ACN and 2 per cent acetic acid in water, respectively. Gradient elution was 0-lQmin, 95 per cent B 10-4Qmin, to 82 per cent B to 40-5Qmin 82 per cent B. The flow rate was 1 ml/min. Theaflavins were determined in an ODS column (100 X 4.6 mm i.d. particle size 3pm) at 30°C. The flow rate was 1.8 ml/min and solvent B was the isocratic mobile phase. The data demonstrated that flavan-3-ols disappear during the oxidation process while the amount of theaflavins with different chemical structures increases [177],... 

RP-HPLC with gradient elution was employed for the study of the influence of theaflavins and thearubigins on the adsorption of black tea on calcium carbonate. Separation of tea constituents was performed in an ODS column (250 X 4.9mm i.d. particle size 5 im). Aqueous solvent was 1 per cent citric acid, pH adjusted to 2.8 with sodium hydroxide and the organic solvent was ACN. The gradient initiated at 8 per cent ACN, was increased to 31 per cent in 50min. Theaflavins and thearubigins were detected at 460 nm, while total polyphenolics were detected at 280 nm. The flow rate was 1.5 ml/min. The results demonstrated the involvement of theaflavins and thearubigins in the adsorption process [185],... 

Normal-phase HPLC has also found application in the analysis of pigments in marine sediments and water-column particulate matter. Sediments were extracted twice with methanol and twice with dichloromethane. The combined extracts were washed with water, concentrated under vacuum and redissolved in acetone. Nomal-phase separation was performed with gradient elution solvents A and B being hexane-N,N-disopropylethylamine (99.5 0.5, v/v) and hexane-2-propanol (60 40, v/v), respectively. Gradient conditions were 100 per cent A, in 0 min 50 per cent A, in 10 min 0 per cent A in 15 min isocratic, 20 min. Preparative RP-HPLC was carried out in an ODS column (100 X 4.6 mm i.d. particle size 3 jum). Solvent A was methanol-aqueous 0.5 N ammonium acetate (75 25, v/v), solvent B methanol-acetone (20 80, v/v). The gradient was as follows 0 min, 60 per cent A 40 per cent A over 2 min 0 per cent A over 28 min isocratic, 30 min. The same column and mobile phase components were applied for the analytical separation of solutes. The chemical structure and retention time of the major pigments are compiled in Table 2.96. 

The chemical structures of betanin and indicaxanthin found in the prickly pear are depicted in Fig. 2.150. Pigments were extracted by homogenizing fresh fruit flesh with methanol (1 5, w/v). The suspension was fdtered and the liquid phase was applied for spectrophotometry and RP-HPLC. Liquid chromatographic separation was performed in an ODS column (250 X 4.6 mm i.d. particle size 5 pan) at ambient temperature. Gradient elution started with 1 per cent aqueous acetic acid and changed to 12 per cent solvent B in... 

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