Water acetonitrile mobile phase

Several other methods have been published using RP-HPLC for the determination amphetamines and related derivatives. Studies have shown the determination of amphetamine and related derivatives in plasma, urine, and hair by RP-HPLC with precolumn derivatization and either UV/VlS or fluorescence detection. Various methods are employed by SPE technologies using Cl8 cartridges for sample cleanup prior to derivatization. The derivatized compounds were separated on analytical columns of various Cl 8 bonded phase materials. The methods generally used water/acetonitrile mobile phases operated in gradient mode. All studies reported extraction recoveries of 85-102% for all the analytes, with LLOQs ranging from 5 to 60 ng/ml (Tedeschi et al., 1993 Ealco et al., 1996 Hernandez et al., 1997 Al-Dirbashi et al., 1997 Al-Dirbashi et al., 2000 Soares et al., 2001). 

Petrick and Wilson [137] recently developed an HPLC method using both UV-Vis and ESI-MS detection able to separate and detect 15 basic dyes and 13 disperse dyes. Separation was carried out in RP-mode using an acidified (formic acid) water-acetonitrile mobile phase in gradient mode. The method enabled the discrimination of fibers with the same apparent colour based on their different chromatographic and mass spectrometric profile. 

Figure 2 Comparison of HPLC (top panels) and chrom-NMR (bottom panels) for a series of homologous aromatic molecules, recorded under RPLC (left panels ODS solid phase and water/acetonitrile mobile phase) and HILIC conditions (right panels bare silica solid phase and water/acetonitrile mobile phase). From Ref. 49. Copyright 2006 Wiley-VCH Verlag GmbH Co. KGaA, Weinheim.

Figure 10.3. Examples of chromatograms. (a) The separation of 20 essential amino acids (in derivatized form) by RPLC using 1 mm inside diameter by 150 mm long microbore column packed with 4 ft m silica support (with C18 CBP) particles and a water/ acetonitrile mobile phase gradient. (6) The fractionation of gasoline using SFC with C02 mobile phase (temperature and pressure programmed) in 0.25 mm x 50 cm packed column with 5 fim polymeric support particles. (Courtesy of Frank J. Yang.)...

In [84] a variable size simplex is u.sed to optimize the mobile phase composition in RP chromatography. The fractions of methanol and water in a methanol/water/acetonitrile mobile phase were optimized. The feasible experimental area was determined by carrying out a gradient elution separation and then calculating the boundaries of the variable space in which the simplexes should move to find an optimized isocratic separation. 

Normal phase/reverse phase If the stationary phase is more polar than the mobile phase, this is normal phase chromatography. An example of this is a silica column with its polar silanol groups and a mobile phase of an organic solvent. When the stationary phase is less polar than the mobile phase, this is reverse phase chromatography, exemplified by the hydrocarbons bound to the silica support and a water/acetonitrile mobile phase. Reverse phase chromatography is very widely used as a form of HPLC (see Chapter 6) and most natural products have a region of hydrophobicity that leads to their retention to some extent on a reverse phase column. 

Figure 4.12. A typical plot of the excess adsorption (irmol/m ) of acetonitrile by an octadecylsilox-ane-bonded sorbent from a water-acetonitrile mobile phase.

To illustrate the effects of mobile phase composition, results for four different water/acetonitrile mobile phase compositions are discussed here (1) pure water, (2) 33% molfraction acetonitrile, (3) 67% molfraction acetonitrile, and (4) pure acetonitrile, (hereafter referred to as systems WAT, 33A, 67A, and ACN, respectively the results for system WAT are taken from [32] and those for the other three compositions from [34]). Each system contained 1,200 solvent molecules and 16 analytes (2 each of Cl to C4 normal alkanes and alcohols) and utilized a stationary phase with a surface coverage of 2.9 pmol/m (9 ODS chains on each surface) which resulted in a residual sUanol density of 4.8 pmol/m (15 silanols on each surface). The temperature and pressure used in this study were 323 K and 1 atm, respectively. For each solvent system, four independent simulations were carried out. Each simulation was equilibrated for 2 x 10 Monte Carlo (MC) cycles (one MC cycle corresponds to N MC moves, where N is the total number of molecules in... 

A series of nine alkyl and alkylphenol ether carboxylates were separated on a Cig column (A = 276 nm or RI detector) using a 75/15/10 methanol/water/acetonitrile mobile phase containing 4 mM tetrabutylammonium hydrogensulfate and 1 mM tetrabutylammonium hydroxide [137]. A linear range of 0.01-10 mg/mL for UV detection and 0.1-10 mg/mL for RI detection was reported. Although baseline resolution was not achieved for all analytes, various mixtures used to create surfactant formulations could be readily identified and characterized using this method. Elution for the longest-retained analyte, oleth-6-carboxylic acid, occurred in <25 min. 

Diazepam, three metabolites (A -desmethyldiazepam, temazepam, oxazepam) and clonazepam [IS] were isolated from plasma or urine and separated on a base deactivated C g column (2 = 232nm). A 50/10/40 methanol/acetonitrile/water (50 mM KH2PO4 pH 3.5) mobile phase generated baseline resolution in 12 min [552]. Calibration standards ran from 10 to 2000ng/mL with detection limits of lOng/mL reported. Similarly, diazepam and three metabolites (nordiazepam, oxazepam, temazepam) were baseline resolved in 12min on a Cjg column (A = 257 nm) using a 60/35/5 methanol/water/acetonitrile mobile phase [553]. 

A -Acyl- and 7/-glycolylneuraminic acids (sialic acids) were isolated fiom serum and tissue samples, per-O-benzoylated, and analyzed on a C,g column (2 = 231 nm) using a 67/33 water/acetonitrile mobile phase [907], Good resolution was achieved and elution was complete in <20 min. Calibration standards of 0.05-50 pg injected were used and detection limits of 10 ng injected were reported. 

Gill disease in fish is treated with chloramine-T. The residue marker compound, p-toluenesulfonamide, is extracted from fish meat and analyzed on a 50°C Cjg column (A = 226 nm) using a 77/23 water/acetonitrile mobile phase [979]. An impressive list of retention times for 20 potentially interfering compounds was tabulated. Elution was complete in 11 min and excellent peak shape was achieved. A concentration range of20-1000 ng/g and detection and quantitation limits of 4 ng/g and 13 ng/g, respectively, were reported. 

Anatoxin-a, homoanatoxin-u and four degradation products (dihydroanatoxin-a, dihydrohomoanatoxin-a, epoxyhomoanatoxin-a, epoxyanatoxin-a) were isolated fiom blue-green algae, derivatized with NBD-F (4-fluoro 7-nitro-2,l,3-benzoxadia-zole), and separated on a 35°C C g column k = 470 nm, ex 530 nm, em). The use of a 55/45 water/acetonitrile mobile phase generated baseline resolution, excellent... 

Thirteen urinary anabolics and corticoids (e.g., cortisone, 11-ketotestosterone, hydroxyprogesterone, epitestosterone, androstenolone) and synthetic anabolics (bolderone, bolasterone) were extracted from urine and analyzed on a C g column X = 200 nm) and 245 nm) using a 60/40 water/acetonitrile mobile phase [1507]. Baseline resolution was not achieved for all compounds and elution was complete in 24 min. Calibration data for each compound were generated from 2 to 10 (ig/mL. Detection limits were reported as 0.05 (ig/mL. It is interesting to note that the authors used an optimization triangle approach to maximize resolution and minimize analysis time. For 10 of the compounds, a <20 min analysis was achieved using a 56.7/20/13.3/10 water/methanol/acetonitrile/THF mobile phase. 

Thalidomide levels in raw material and finished products was detomined using a C]g column (2 =237nm) and an 85/15 water/acetonitrile mobile phase [1527]. Elution was complete in 8 min. The authors note that past mobile phases used a 0.1 % H3PO4 additive that caused column deterioration much fiister than the mobile phase described here. The effect of changing percent acetonitrile on retention time was also reported. A linear curve was established between 10 and 200pg/mL. 

Triiodophenol (Bobel-24, anti-inflammatory) was extracted from plasma, synovial fluid, and tissue and analyzed on a C g colunm (2 = 277 nm) using a 62/38 water/acetonitrile mobile phase [1531]. Elution was complete in 5 min. Interestingly, the highly substituted phenol should be quite acidic and yet excellent peak shape and acceptable retention time did not require a buffer. Standards of 0.5-20 pg/mL were used and a quantitation limit of 0.1 pg/mL was reported. 

The exo and endo forms of iodixanol (X-ray contrast agent) were isolated from plasma and analyzed in 16 min on a C g colunm (2 = 244nm) using a 91/9 water/acetonitrile mobile phase [1532]. Good resolution between the isomers and co-extracted peaks was obtained. Standards ranged from 0.5 to 500pg/mL. Detection and quantitation limits of O.lpg/mL and 0.3pg/mL, respective, were reported. 

Patulin and 5-hydroxymethylfurfural (5HMF) were extracted from apple juice and analyzed on a Cig column (photodiode array detector, 2 = 250-300 nm). Water alone as a mobile phase did not resolve the peaks, but a 99/1 water/acetonitrile mobile phase resolved and eluted the analytes in <9 min. Concentration ranges of 0.05-10 mg/L for 5HMF and 5-300 pg/L for patulin were reported. Detection limits of 5.4 pg/mL were reported for 5HMF [1578]. Patulin was also isolated from apples and quantitated using a 40°C Cjg column (2= 276nm) and a 95/5 water/ acetonitrile mobile phase [1579]. Patulin is well resolved fiom other extractants and eluted in 12 min. A linear range of 1-100 xg/mL and detection limits of 2 xg/kg were reported. 

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