Mobile Phase and pH

The previous chapters have dealt mainly with LC/MS analysis involving short run times, many samples, and relatively small numbers of compounds in samples. What about samples containing very complex compound mixtures, for example, natural products, samples from biomarker discovery, protein digests, and QA/QC method development or metabolite identification samples requiring detection of every component Such workflows often require several analysis steps with different columns and different mobile phases and pH values to increase the separation probability by changing the selectivities of individual runs. 

As mentioned earlier, CEC couples the separating power of HPLC and the high efficiency of CE. The packed capillary can be considered the heart of the CEC system because it acts as a pump and provides chromatographic selectivity. The design of the stationary phase related parameters such as mobile phase and pH and instrumental parameters such as pressurization, injection modes, temperature, and voltage polarity play an important role in this technique. 

The concentrations of benzoic acid, aspartame, caffeine, and saccharin in a variety of beverages are determined in this experiment. A Gig column and a mobile phase of 80% v/v acetic acid (pH = 4.2) and 20% v/v methanol are used to effect the separation. A UV detector set to 254 nm is used to measure the eluent s absorbance. The ability to adjust retention times by changing the mobile phase s pH is also explored. 

This experiment focuses on developing an HPLG separation capable of distinguishing acetylsalicylic acid, paracetamol, salicylamide, caffeine, and phenacetin. A Gjg column and UV detection are used to obtain chromatograms. Solvent parameters used to optimize the separation include the pH of the buffered aqueous mobile phase, the %v/v methanol added to the aqueous mobile phase, and the use of tetrabutylammonium phosphate as an ion-pairing reagent. 

An hplc assay was developed suitable for the analysis of enantiomers of ketoprofen (KT), a 2-arylpropionic acid nonsteroidal antiinflammatory dmg (NSAID), in plasma and urine (59). Following the addition of racemic fenprofen as internal standard (IS), plasma containing the KT enantiomers and IS was extracted by Hquid-Hquid extraction at an acidic pH. After evaporation of the organic layer, the dmg and IS were reconstituted in the mobile phase and injected onto the hplc column. The enantiomers were separated at ambient temperature on a commercially available 250 x 4.6 mm amylose carbamate-packed chiral column (chiral AD) with hexane—isopropyl alcohol—trifluoroacetic acid (80 19.9 0.1) as the mobile phase pumped at 1.0 mL/min. The enantiomers of KT were quantified by uv detection with the wavelength set at 254 nm. The assay allows direct quantitation of KT enantiomers in clinical studies in human plasma and urine after adrninistration of therapeutic doses. 

In the course of mixture separation, the composition and properties of both mobile phase (MP) and stationary phase (SP) are purposefully altered by means of introduction of some active components into the MP, which are absorbed by it and then sorbed by the SP (e.g. on a silica gel layer). This procedure enables a new principle of control over chromatographic process to be implemented, which enhances the selectivity of separation. As a possible way of controlling the chromatographic system s properties in TLC, the pH of the mobile phase and sorbent surface may be changed by means of partial air replacement by ammonia (a basic gaseous component) or carbon dioxide (an acidic one). 

The compatibility of the matrix with acids and bases is quite good. Ultrahydrogel columns can be used with mobile phases from pH 2 to pH 12 for extended periods of time. 

Scientific (Northbrook, IL) contain a silica support with a -y-glycidoxypropylsi-lane-bonded phase to minimize interaction with anionic and neutral polymers. The columns come in five different pore sizes ranging from 100 to 4000 A. The packing material has a diameter from 5 to 10 /cm and yields in excess of 10,000 plate counts. With a rigid silica packing material, the columns can withstand high pressure (maximum of 3000 psi) and can be used under a variety of salt and/or buffered conditions. A mobile phase above pH 8, however, will dissolve the silica support of the column (21). A summary of the experimental conditions used for Synchropak columns is described in Table 20.8. 

Today, the use of CHIRBASE as a tool in aiding the chemist in the identification of appropriate CSPs has produced impressive and valuable results. Although recent developments diminish the need for domain expertise, today the user must possess a certain level of knowledge of analytical chemistry and chiral chromatography. Nevertheless, further refinements will notably reduce this required level of expertise. Part of this effort will include the design of an expert system which will provide rule sets for each CSP in a given sample search context. The expert system will also be able to query the user about the specific requisites for each sample (scale, solubility, etc.) and generate rules which will indicate a ranked list of CSPs as well their most suitable experimental conditions (mobile phase, temperature, pH, etc.). 

Acetochlor and its metabolites are extracted from plant and animal materials with aqueous acetonitrile. After filtration and evaporation of the solvent, the extracted residue is hydrolyzed with base, and the hydrolysis products, EMA and HEMA (Figure 1), are steam distilled into dilute acid. The distillate is adjusted to a basic pH, and EMA and HEMA are extracted with dichloromethane. EMA and HEMA are partitioned into aqueous-methanolic HCl solution. Following separation from dichloromethane, additional methanol is added, and HEMA is converted to methylated HEMA (MEMA) over 12 h. The pH of the sample solution is adjusted to the range of the HPLC mobile phase, and EMA and MEMA are separated by reversed phase HPLC and quantitated using electrochemical detection. 

The HPLC elution pattern is affected to some extent by the pH of the mobile phase. Moderate pH adjustment to optimize the resolution between EMA and MEMA may be performed. Retention time can be affected greatly by the history of the HPLC column and also the buffer/methanol ratio. The mobile phase ratio should be adjusted to provide adequate separation and retention. Control and fortified samples should be run in the same analytical set with treated samples. 

Concentration and MWD of F-PHEA After Absorption. F-PHEA was determined in perfusate samples by quantitative GPC relative to a freshly prepared F-PHEA standard run on the same day. Either a mixed-bed column (12 x 300 mm Sephacryl S-200 Sephadex G-25 SF 3 1, Pharmacia LKB) or a Separon HEMA-Bio 40 column (8 x 250 mm 10 pm particle size, Tessek A/S, Aarhus, Denmark) was used with a 20 pL injection volume. A mobile phase of pH 7.4 phosphate buffered saline (0.05 M phosphate, 0.15 M NaCl) was supplied (Model LC-7A Bio Liquid Chromatograph, Shimadzu Corporation, Kyoto, Japan) at 0.5 or 1 mL/min. Fluorescent detection was employed (Model RF-535 Fluorescence HPLC Monitor,... 

Retention of solutes in ion-exchange chromatography is determined by the nature of the sample, the type and concentration of other ions present in the mobile phase, the pH, temperature, and the presence of solvents. Because there are so many variables, it is often not easy to predict what will happen in an ion-exchange separation if we change the experimental conditions. There are some useful guidelines, and to see how they work we will look at the ion-exchange separation of two weak acids (see Fig. 3.3c). 

Smith et al (76,773 analyzed hydralazine in a drug mixture containing hydralazine hydrochloride, hydrochlorothiazide, and an impurity derived from the latter. The column was 1 m x 2.1 mm (ID) stainless steel, packed with a strong anion exchanger on 30 Jm Zipax . The mobile phase was pH 9 2 borate buffer containing 0.005M sodium sulfate (5 methanol), at 1.7 ml per minute. Detection was by ultraviolet absorption at 25 nm. 

Aliquots (0.25 mL) of plasma samples were mixed with 50 pL of the IS (Pfizer Global UK-115794, 20 /ig/mL in water) followed by 0.5 mL 0.2M ammonium acetate buffer (pH 9.0), extracted with 7 mL ethylacetate diethylether (1 1 v/v), vortexed for 90 sec, centrifuged at 1500 g for 3 min, and frozen. The organic layer was collected, evaporated to dryness at 40°C under a stream of nitrogen, reconstituted with 0.2 mL of mobile phase, and centrifuged at 10,000 g for 6 min. The supernatant was collected and assayed. The injection volume was 30 pL. Figure 11.2 shows chromatograms of voriconazole and the IS in plasma. 

The simultaneous determination of LAS and its potential biodegradation intermediates of low molecular weight (C < 8), as sulfonated and non-sulfonated, has been performed by RP-HPLC-UV using a gradient regime with the mobile phase at pH 2.2 [29]. Compound elution with the same number of carbon atoms in the carboxylic chain was as follows SPCs, hydrophenyl carboxylic acids and finally phenylcarboxylic acids. 

Global LSER calculations have also been applied to the study of the retention of ioniz-able analyses in RP-HPLC. While the retention of neutral analyses does not depend on the pH of the mobile phase the retention of analyses with one or more ionizable substructures considerably depends on the pH even at the same concentration of organic modifier in the eluent. The relationship between the retention and pH of the mobile phase and pK value of the analyte can be described by... 

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