Elution monitoring

Lin, S. H. et al. Peak crossover in high-performance Uqnid chromatography elution monitored using whole-column detection. J. Chromatogr. A. 2008,1201 128-131. 

Polymer particles are suspended in water (25% MeCN) and then slurry-packed into stainless steel columns (250 mm x 4.6 mm i.d.) using an air-driven fluid pump (Haskel, Burbank, CA, USA) and water (25% MeCN) as the packing solvent. The packed columns are washed on-line on a Beckman HPLC system (comprising a solvent module 126 and diode array detector 168) using MeCN (20% acetic acid) to remove the print molecule until a stable base line is obtained. The mobile phase is then changed to a citrate buflfer (pH 3.0, 25 mM citrate) containing 10% MeCN (v/v) at a flow rate of 1 ml. min . For a test of chiral resolution, a racemic mixture of (+)- and ( )-isoproterenol (20 pL at 2 mM in the mobile phase) is injected, and the elution monitored at 280 nm. Acetone can be used as a void marker for the calculation of capacitor factor (k ) and separation factor (oc). 

In LC, the most common means for monitoring the eluant is to pass it through a cell connected into an ultraviolet spectrometer. As substances elute from the column, their ultraviolet absorption is measured and recorded. Alternatively, the refractive index of the eluant is monitored since it varies from the value for a pure solvent when it contains organics from the column. 

The reaction can be conveniently monitored by TLC using silica plates and eluting with 1 4 ethyl acetate-heptane. 

If unidentified peaks are detected the stability of the protein under the chromatographic conditions should be checked. In all analytical investigations of proteins on SEC columns it is desirable to be able to monitor the eluted peaks at a very high sensitivity of the ultraviolet detector. Therefore, very pure (analytical grade) salts and buffers should be used. 

The precision of SEC must be established before a comparison of columns and calibration standards can be made. Consistency in flow rate or elution time is the first requirement to obtain precision in SEC. Consistency in flow rate or elution time can be monitored by the elution time of the PEO standards, which are run before and after the samples. Elution time or flow rate can be considered consistent if the elution times of the PEO standards before and after the samples agree within 0.1 min. 

Flow markers are often chosen to be chemically pure small molecules that can fully permeate the GPC packing and elute as a sharp peak at the total permeation volume (Vp) of the column. Examples of a few common flow markers reported in the literature for nonaqueous GPC include xylene, dioctyl phthalate, ethylbenzene, and sulfur. The flow marker must in no way perturb the chromatography of the analyte, either by coeluting with the analyte peak of interest or by influencing the retention of the analyte. In all cases it is essential that the flow marker experience no adsorption on the stationary phase of the column. The variability that occurs in a flow marker when it experiences differences in how it adsorbs to a column is more than sufficient to obscure the flow rate deviations that one is trying to monitor and correct for. 

A method which uses supercritical fluid/solid phase extraction/supercritical fluid chromatography (SE/SPE/SEC) has been developed for the analysis of trace constituents in complex matrices (67). By using this technique, extraction and clean-up are accomplished in one step using unmodified SC CO2. This step is monitored by a photodiode-array detector which allows fractionation. Eigure 10.14 shows a schematic representation of the SE/SPE/SEC set-up. This system allowed selective retention of the sample matrices while eluting and depositing the analytes of interest in the cryogenic trap. Application to the analysis of pesticides from lipid sample matrices have been reported. In this case, the lipids were completely separated from the pesticides. 

Purification of Antibiotic 66-40 — Dissolve 28 g of crude Antibiotic 66-40 in 100 ml of distilled water and charge to an anion exchange adsorption column (Dowex 1 X2) in the hydroxyl form. Slurry 2,000 g of the resin in water in to a column 2yj in diameter and 36 "high. Elute the column with distilled water at a rate of about 23 ml/min collecting 100 ml fractions and monitor with a conductivity meter and by disc testing against Staphylococcus aureus. 

Stalcup aiid co-workers [14] adapted this method to a continuous elution mini-prep electrophoresis apparatus shown in Fig. 11-3. In this apparatus, the end of the electrophoretic gel is continuously washed with elution buffer. The eluent can then be monitored using an HPLC detector (Fig. 11-4) and sent to a fraction collector where the purified enantiomers, as well as the chiral additive, may be recovered. In this system, the gel configuration was approximately 100 mm x 7 mm, and was aircooled. The number of theoretical plates obtained for 0.5 mg of piperoxan with this gel was approximately 200. A larger, water-cooled gel was able to handle 15 mg of... 

High-pressure pumps operating at up to 6000 psi are required to force solvent through a tightly packed HPLC column, and electronic detectors are used to monitor the appearance of material eluting from the column. Alternatively, the column can be interfaced to a mass spectrometer to determine the mass spectrum of every substance as it elutes. Figure 12.18 shows the results of HPLC analysis of a mixture of 10 fat-soluble vitamins on 5 jam silica spheres with acetonitrile as solvent. 

A solution of the 2-azido ester or amide (ca. 2 g) in a mixture of MeOII (95 mL) and sodium-dried THF (95 mL) was photolyzed under N2 in a Hanovia photochemical reactor (110-W medium-pressure Hg lamp with a Pyrex filter). The reaction was monitored by observing the rate of disappearance of the absorption band (Nf) at 2140 cm 1 (irradiation times of 3-5 h were generally required). When the reaction was complete the solvent was removed in vacuo and the brown residual oil chromatographed on alumina [petroleum ether (bp 60-803C)/benzene 7 3]. Further elution with benzene followed by removal of the solvent gave the product (the esters as pale yellow oils, the amides as crystalline solids), which were further purified by vacuum distillation or by recrysiallization. 

The reddish yellow solution is diluted with 4-5 volumes of cold water containing 5 mM 2-mercaptoethanol to reduce the conductivity to 0.7 m 2 1 or less, and applied to a column of DEAE-cellulose (coarse grade 5 x 15 cm) equilibrated with 2mM potassium phosphate, pH 8.0, containing 5mM 2-mercaptoethanol. The column is first washed with the cold equilibration buffer, then luciferin is eluted with a linear increase of potassium phosphate from 2 mM to 0.3 M, monitoring the effluent by fluorescence and the absorption at 390 nm. The rest of the purification method described below is adapted from the... 

At this point, the solution containing the component to be measured (Ax) also contains any other compounds from the original matrix that are soluble in the solvent used in the analysis. For the analysis to be accurate, other components in the matrix cannot interfere by eluting at the same retention time as the components to be measured. For accurate MS analyses, the matrix component must not interfere with production of the ions being measured for either the internal standard or the component to be measured. In some cases, to eliminate interferences, it may be necessary to increase the resolution of the mass spectrometer by narrowing the mass window being monitored. Alternatively, MS/MS can be used to avoid chemical interference (see Chapter 1). 

The reaction can be monitored by TLC elution with 3 1 petroleum ether/ethyl acetate, Rf = 0.24 (O-tosylserine), Rf= 0.60 (iodoalanine). 

Monitoring of the reaction by TLC on silica gel showed complete consumption of the starting amine (elution with 20 1 CH2C12/CH30H, visualization by UV and by iodine dibenzylamine Rf = 0.4, nitrone Rf = 0.7). 

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