Dissolved in the mobile phase

The solvent used was 5 %v/v ethyl acetate in n-hexane at a flow rate of 0.5 ml/min. Each solute was dissolved in the mobile phase at a concentration appropriate to its extinction coefficient. Each determination was carried out in triplicate and, if any individual measurement differed by more than 3% from either or both replicates, then further replicate samples were injected. All peaks were symmetrical (i.e., the asymmetry ratio was less than 1.1). The efficiency of each solute peak was taken as four times the square of the ratio of the retention time in seconds to the peak width in seconds measured at 0.6065 of the peak height. The diffusivities obtained for 69 different solutes are included with other physical and chromatographic properties in table 1. The diffusivity values are included here as they can be useful in many theoretical studies and there is a dearth of such data available in the literature (particularly for the type of solutes and solvents commonly used in LC separations). 

Errors in the molecular weight data from HPSEC are usually due to improperly prepared samples, column dispersity, or flow rate variations. The sample to be analyzed should be completely dissolved in the mobile phase and filtered prior to injection onto the column. A plugged column inlet frit will invalidate results. In addition, do not load the column with excess sample. Column overloading affects the accuracy of data by broadening peaks, reducing resolution, and increasing elution volume. For best results, the concentration of the injected sample should be as low as possible while still providing adequate... 

The ideal HPLC detector should have the same characteristics as those required for GC detectors, i.e. rapid and reproducible response to solutes, a wide range of linear response, high sensitivity and stability of operation. No truly universal HPLC detector has yet been developed but the two most widely applicable types are those based on the absorption of UV or visible radiation by the solute species and those which monitor refractive index differences between solutes dissolved in the mobile phase and the pure mobile phase. Other detectors which are more selective in their response rely on such solute properties as fluorescence, electrical conductivity, diffusion currents (amperometric) and radioactivity. The characteristics of the various types of detector are summarized in Table 4.14. 

The solid state reference electrode has no internal Cl solution but uses Cl ions in the mobile phase to keep its potential constant. This means that a certain amount (typically 1 to 10 mM) KC1 must be dissolved in the mobile phase. 

It may be difficult to imagine a liquid mobile phase used with a liquid stationary phase. What experimental setup allows one liquid to move through another liquid (immiscible in the first) and how can one expect partitioning of the mixture components to occur The stationary phase actually consists of a thin liquid film chemically bonded to the surface of finely divided solid particles, as shown in Figure 11.8. It is often referred to as bonded phase chromatography (BPC). Such a stationary phase cannot be removed from the solid substrate by heat, reaction, or dissolving in the mobile phase. 

The HPLC pump draws the mobile phase from the reservoir via vacuum action. In the process, air dissolved in the mobile phase may withdraw from the liquid and form bubbles in the flow stream unless such air is removed from the liquid in advance. Air in the flow stream is undesirable because it can cause a wide variety of problems, such as poor pump performance or poor detector response. Removing air from the mobile phase, called degassing, in advance of the chromatography is a routine matter, however, and can be done in one of several ways 1) helium sparging, 2) ultrasonic agitation, 3) drawing a vacuum over the surface of the liquid, or 4) a combination of numbers 2 and 3. 

The efficiency of any chromatographic technique depends upon the number of sequential separations or equilibria that take place, which in the case of paper chromatography are due to the large number of compartments of cellulose-bound water. The test solutes are carried up the paper dissolved in the mobile phase and encounter successive compartments of water. At each one, rapid partition between the two phases occurs leaving the mobile phase to carry up the residual solute to the next water compartment and another partitioning effect. The solute, which is dissolved in the water and hence not carried up the paper, is now presented with fresh solvent rising up the paper and again is redistributed between the two phases. 

Ray used high performance liquid chromatography to estimate diloxanide furoate and tinidazole in single and combined dosage forms [39], Tablets were dissolved in the mobile phase, and 20 pL was injected on to a stainless steel column (30 cm x 3.9 mm) of p-Bondapak Cig. 8 3 methanol 0.05 M phosphoric acid (pH 3) was used as the mobile phase (flow rate of 2.5 mL/min), and detection was on the basis of the UV absorption at 254 nm. 

If a mobile phase, carrying a constant concentration of solute (X0), is fed continuously onto a chromatographic column and equilibrium is allowed to be established, the eluent from the column will also contain the solute at a concentration (X0). If a sample of the same solute, dissolved in the mobile phase at a concentration of (Xj), is now injected onto the column where either (X0Xj), then this will result in a perturbation on the concentration (X0) and, from the plate theory, this perturbation will pass through the column and be sensed by the detector at the end of the column. 

From Table 8 it is obvious that the resolution always increases with an increase of the number of benzene rings and that riboflavine is a more powerful selector than the nucleotides, but not as good as TAPA. An interesting experiment shows that it is not always necessary to have the selector coated or bound to the solid phase but that it can sometimes be used as well, dissolved in the mobile phase. The n-dodecyl ester of N-(2,4-dinitrophenyl)-L-alanine is able to discriminate between the enantiomers of l-aza-[6]-helicene, when used as a chiral dopant in the mobile phase in HPLC on a reversed phase column 93) (see Table 9). The usefulness of this dopant must be due to the known ability of a dinitrophenyl moiety to form CT-complexes with polycyclic aromatic hydrocarbons the presence of a chiral site near this group causes resolution of helicenes, because the steric interactions in diastereomeric complexes will be quite different. 

According to the modified procedure (602), milk is thoroughly mixed in its storage container immediately before transfer of the 1 ml aliquot in the extraction tube. This is necessary because approximately 50% of phenylbutazone in milk is associated with the cream. The sample is extracted with 2.4 ml diethyl ether and 2.4 ml petroleum ether in presence of 1 ml ethanol and 100 1 25% ammonia solution. The organic layer that contains the milk lipids is discarded. Five ml hexane-tetrahydro furan (4 1) is added to the aqueous layer, which is tiien acidified with hydrochloric acid and the layers are mixed. Under the acidic conditions, phenylbutazone partitions quantitatively into tlie organic layer, which is collected, evaporated, and dissolved in the mobile phase to be analyzed by liquid chromatography. Separation is performed on a reversed-phase column using an isocratic 0.02 M phosphate buffer/methanol mobile phase, and determination is by ultraviolet detection at 264 nm (Fig. 29.18.2). The limit of detection and limit of quantification were 3.0 and 5.4 ppb, respectively (Table 29.17). 

For studies on the relationship between log k and the percentage of strong solvent, olive oil was dissolved in the appropriate strong solvent at a concentration of 50 mg/ml. For time-normalization studies, olive oil was dissolved in the mobile-phase mixture at this same concentration level whenever possible. In cases where olive oil was not soluble in the mobile phase, it was dissolved in the strong solvent. The column void time was determined by measuring the av-... 

Perfetti et al. (131) described a method for the determination of ethoxyquin in milk. Milk solids were precipitated by adding acetonitrile, and the water-acetonitrile supernatant was washed with hexane to remove fat. The addition of NaCl caused the water-acetonitrile solution to separate into an aqueous phase and an acetonitrile phase, thus separating ethoxyquin from most water-soluble impurities. A large volume of water was then added to the acetonitrile layer, and ethoxyquin was partitioned into hexane and removed at reduced pressure. The residue was dissolved in the mobile phase and analyzed on a 250-mm X 4.6-mm-ID. Ultrasphere ODS column using fluorescence detection with excitation of 230 nm, and emission of 418 nm, respectively. A mixture of water and acetonitrile with a diethylamine-acetic acid buffer was the mobile phase. Recoveries from milk samples fortified at 1, 5, and 10 ng/g averaged 78%, with a coefficient of variation of 5.0%. Low concentrations (less than 1 ng/g) of apparent ethoxyquin were detected in commercial milk samples analyzed by this method. 

The determination of OXO in Japanese oyster was realized using reversed-phase HPLC. Samples were extracted with LLE and SPE recoveries were 88.3% (193). Oyster samples were homogenized with a phosphate buffer adjusted to pH 7. After centrifugation, supernatants were concentrated using an SPE C-18 cartridge. Before use, the cartridge was activated with MeOH and phosphate buffer. After the sample had been passed, the cartridge was flushed with water and the analytes were eluted with MeOH-orthophosphoric acid (9 1). The eluate was evaporated, and the residues were dissolved in the mobile phase. The method developed was validated and the study of OXO stability was performed. The limits of detection and determination were 10 and 40 ng/ml, respectively. 

Pharmacokinetic and residue analysis of ENRO and its metabolite CIPRO in chicken samples were performed using HPLC after extraction with dichloromethane and sodium phosphate buffer (pH 7.4) (202). After shaking and centrifuging, the organic phases were dried and the residues dissolved in the mobile phase. Extraction recovery was 87% for ENRO and its metabolite. 

The above method may also be used to determine Vitavax (5,6-dehydro-2-methyl-l,4-oxathion-3-carboxanilide) [196]. The procedure is the same with the exception that 10"47V sulfuric acid is used for the oxidation. The separations are made on columns (61 cm X 4.8 mm) containing Bondapak Ci8-Corasil using acetonitrile-water (1 4) as the mobile phase at a flow-rate of 1.05 ml/min. The sample is dissolved in the mobile phase for injection into the HPLC system. Concentrations as low as 20 ppb can be detected in natural waters. 

Assuming we have selected the proper mode of chromatography, will the mixture dissolve in the mobile phase Ion-exchange columns must be run in polar-charged solvents. Size separation columns are not, in theory, affected by solvent polarity, and size columns for use in both polar and nonpolar solvents are available. In partition chromatography, we have nonpolar columns that can be run in polar or aqueous solvents, and polar columns that are only run in anhydrous, nonpolar solvents. Intermediate columns such as cyanopropyl or diol can be run in either polar or nonpolar solvents, although often with differing specificity. An amino column (actually a propylamino) acts in methylene chloride/hexane like a less polar silica column but in acetonitrile/water... 

Amantea and Narang [58] used a reversed-phase HPLC method for the quantitation of omeprazole and its metabolites. Plasma was mixed with the internal standard (the 5-methyl analog of omeprazole), dichlor-omethane, hexane, and 0.1 M carbonate buffer (pH 9.8). After centrifugation, the organic phase was evaporated to dryness and the residue was dissolved in the mobile phase [methanol-acetonitrile-0.025 M phosphate buffer of pH 7.4 (10 2 13)] and subjected to HPLC at 25 °C on a column (15 cm x 4.6 mm) of Beckman Ultrasphere C8 (5 ym) with a guard column (7 cm x 2.2 mm) of Pell C8 (30-40 /im). The mobile phase flow-rate was 1.1 ml/min with detection at 302 nm. The calibration graphs are linear for <200 ng/ml, and the limits of detection were 5, 10, and 7.5 ng/ml for omeprazole, its sulfone, and its sulfide, respectively. The corresponding recoveries were 96.42% and 96% and the coefficients of variation (n = 5 or 6) were 3.0-13.9%. 

The separation of cimetidine and its metabolites is usually carried out by extraction of the biological medium with 1-octanol fran an aqueous alkaline pH solution followed by mixing, addition of an internal standard and centrifugation. The extraction with octanol is repeated and the combined extracts are re-extracted with dilute hydrochloric acid. The aqueous acid solution is then separated, ethanol is added and mixed. This is then followed by saturating the mixture with a large amount of potassium or sodium carbonate to "salt out" the ethanol layer which contains the cimetidine and its metabolite, the sulfoxide. Several different internal standards have been used Metiamide, 1-methyl-3-[2-[[(5-methyl-imidazole-4-yl) -methyl] thio]ethyl]-2-thiourea,19 31 39 (N-cyano-N1-methy1-N"-(3-(4-imidazolyl)-propyl)guanidine32, and 13-hydroxy-theophylline. 0 After extraction the samples are either evaporated to dryness and reconstituted with a known amount of ethanol, injected directly or dissolved in the mobile phase for the HPLC analysis. 

Fig. 10.17. Capillary electrochromatography of PTH-amino acids with gradient elution. Column, 207 (127) mm x 50 pm i.d. packed with 3.5 pm Zorbax ODS particles, 80 A pores. Starting eluent (A), 5 mM phosphate, pH 7.55, 30% acetonitrile gradient former (B), 5 mM phosphate, pH 7.55, 60% acetonitrile flow-rate (through inlet reservoir), 0.1 ml/min gradient, 0-100% B in 20 min voltage 10 kV current, 1 pA temperature, 25°C UV detection at 210 nm electrokinetic injection, 0.5 s, 1 kV. Peaks in order of elution formamide PTH-asparagine PTH-glutamine PTH-threonine PTH-glycine PTH-alanine PTH-tyrosine PTH-valine PTH-proline PTH-tryptophan PTH-phenyialanine PTH-isoleucine PTH-leucine. The concentration of the PTH-amino acids dissolved in the mobile phase was 30-60 pg/ml. Reprinted with permission from Huber et al. [68]. Copyright 1997 American Chemical Society.

A disadvantage of the operation of RPLC columns at elevated temperatures may be a more rapid detoriation of the column because the silica is more rapidly dissolved in the mobile phase. This effect may also lead to a reduced reproducibility of the system (peakwiths and capacity factors). 

The most common way to create an RP-IPC system is to use a genuine chemically bonded reversed phase column (e.g. C18 see section 3.2.2.1) and to use large pairing ions with a hydrophobic alkyl chain dissolved in the mobile phase. This technique was introduced by Knox and Laird, who named it soap chromatography [380]. Because of the usually long alkyl chains of the pairing ions, the use of Cl 8 phases is to be recommended in order to avoid effects that are related to the critical chain length (see section 3.2.2.1). 

The sample should be dissolved in the mobile phase. If it is not very soluble, other solvents can be used, but they should be as similar to the mobile phase as possible, and blanks should be run to see the effect of this new solvent on the column. A better alternative is to use larger sample volumes. The upper limit is about one-thirtieth of VM, which would be about 100 pL for a conventional analytical column. 

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