Eluent gradient elution

Purify of the crude residue by flash chromatography on silica gel using hexane ethyl acetate as eluent (gradient elution from 98 2 to 90 10). Pure 6-(dimethylphenylsilyl)-2,2-dimethyl-4-(2-phenylethyl)-5-hexyn-3-one (33) (0.141 g, 65%) as a clear oil. Characterize the product by 1H NMR, IR, MS spectroscopy, and elemental analysis. 

Eluent. Gradient elution with a mixture of acetonitrile and 0.05M acetic acid, commencing with 10% of acetonitrile and increasing linearly to 90% acetonitrile in 15 minutes. 

There is one mobile phase, consisting of the elements of the eluent. Gradient elution can also be performed by the use of several mobile phases. 

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%... 

The effectiveness of the elution step can be tailored by using a single eluent, pulses of different eluents, or eluent gradients. These systems are generally characterized by mild desorption conditions. If the eluting agent is bound to the protein, it can be dissociated by desalting on a gel filtration column or by diafiltration. 

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. 

Using the Tomtec Quadra 96 workstation, 0.1 mL of the ethyl acetate layer was transferred to a 96-well collection plate containing 0.4 mL of acetonitrile in each sample well. The solution was mixed 10 times by aspiration and dispersion on the Tomtec. The plate was then covered with a sealing mat and stored at 2 to 8°C until LC/MS/MS analysis. The HILIC-MS/MS system consisted of a Shimadzu 10ADVP HPLC system and Perkin Elmer Sciex API 3000 and 4000 tandem mass spectrometers operating in the positive ESI mode. The analytical column was Betasil silica (5 fim, 50 x 3 mm) and a mobile phase of acetonitrile water formic acid with a linear gradient elution from 95 5 0.1 to 73.5 26.5 0.1 was used for 2 min. The flow rate was 1.0 mL/min for the API 3000 and 1.5 mL/min for the API 4000 without any eluent split. The injection volume was 10 jjL and a run time of 2.75 min was employed. 

Fig. 2.5.11. (a) APCI-LC-MS(+), (b) ESI-LC-MS(-t-), (c) ESI-LC-MS(+), (d) ESI-LC-MS(+), (e) APCI-LC-MS(—) and (f) ESI-LC-MS(—) reconstructed ion chromatograms (RIC) of methanolic solution of the household detergent mixture as in Fig. 2.5.2. Chromatographic conditions (a), (b), (e), and (f) RP-Cig, methanol/water gradient elution (c) ion-pairing RP-Cla using trifluoro acetic acid (TFA) (5 mmol), methanol/water gradient elution (d) isocratic elution performed on PLRP-column, eluent methanol/water methane...

The traditional HPLC instrument is composed of two different parts the first part separates the components of the sample and the other part accomplishes the detection of the components separated. The part of the HPLC carrying out the separation contains a column, an injection device and the eluent delivery system (pump with filters, degasser and transfer tubing, eventually a mixer for gradient elution). One or more detectors, a signal output device coupled with appropriate software, are responsible for detection and primary data evaluation. Pumps deliver the eluent or the different components of the eluent into the column with a precise, constant and reproducible flow rate. 

GRADIENT ELUTION FOR THE REVERSED-PHASE HIGH-PERFORMANCE LIQUID CHROMATOGRAPHIC SEPARATION OF COLOUR PIGMENTS IN THE EXTRACTS OF PAPRIKA (<CAPSICUM ANNUUM). ELUENT A, METHANOL-ACETONITRILE (80 20, V/V) ELUENT B, BIDISTILLED WATER... 

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