Parabens column

Eigure 13.57 discusses in some detail the use of mobile-phase modifiers to prevent adsorption on PDVB resins. These concepts are very valuable in developing methods. Eor example, note how the observed column efficiencies improve for paraben analysis in the order of methanol < acetonitrile < 50/50 methanol/acetonitrile < THE. Eurthermore, when THE is used the chromato-... 

Superheated water at 100°-240 °C, with its obvious benefits of low cost and low toxicity, was proposed as a solvent for reversed-phase chromatography.59 Hydrophobic compounds such as parabens, sulfonamides, and barbiturates were separated rapidly on poly(styrene-divinyl benzene) and graphitic phases. Elution of simple aromatic compounds with acetonitrile-water heated at 30°-130 °C was studied on coupled colums of zirconia coated with polybutadiene and carbon.60 The retention order on the polybutadiene phase is essentially uncorrelated to that on the carbon phase, so adjusting the temperature of one of the columns allows the resolution of critical pairs of... 

Before releasing a process column for chromatography, it is advisable to perform some test to measure efficiency, such as calculating height equivalent theoretical plates (HETP), both to forestall any problems in the column bed and to provide a benchmark by which to measure column reproducibility and predict degradation of the bed or material. Examples of compounds that are relatively innocuous for use in pharmaceutical applications are 1% NaCl (for gel filtration), concentrated buffer solutions (for ion exchange), and benzyl alcohol and parabens for reverse phase LC.10... 

FIGURE 6.17 Chromatogram overlay for 24 consecutive runs performed on a single column. (A) results of overlay for the chromatograms obtained with UV absorbance detection. Peaks are identified as (with increasing retention time) uracil (dead volume marker), methyl paraben, and propyl paraben. (B) results of overlay for chromatograms obtained from fluorescence detection (peak identified as rhodamine 110 chloride). 

To evaluate linearity, limits of detection (LOD), limits of quantitation (LOQ), and sensitivity, an experiment assessed the responses for different concentrations of two analytes of interest. The analytes employed were methyl paraben and rhodamine 110 chloride. Consecutive 5.0 /jL injections of a series of serial dilutions (four replicates) of this standard mixture containing the analytes described were carried out via a cartridge packed with C18 stationary phase and per-column dimensions of 0.5 mm circular cross section and 80 mm length. 

Signals for methyl paraben were monitored with UV detection at 254 nm. The signal for rhodamine 110 chloride was monitored via fluorescence detection with an excitation filter of 482 nm (35 nm bandwidth) and emission filter of 535 nm (40 nm bandwidth). A gradient method (same as the one in Figure 6.16) was used. The compositions of mobile phases A and B were 5 95 H20 CH3CN with 0.1 HCOOH and CH3CN with 0.085% HCOOH, respectively, with a total flow rate of 300 fiL/ min (corresponding to 12.5 /rL/min for each column). 

Ye X, Tao LJ, Needham LL, Calafat AM (2008) Automated on-line column-switching HPLC-MS/MS method for measuring environmental phenols and parabens in serum. Talanta... 

T. T. Nguyen, R. Kriugstad, and K. E. Rasmussen, Use of extraction columns for the isolation of desonide and parabens from creams and ointments for HPLC analysis, J. Chromatogr., 366 445 (1986). 

Fig. 10.5. Separation of parabens with the poly(TBAAm-co-AMPS)-coated column. Conditions column, 750 mm x 25 pm i.d. (600 mm effective length) mobile phase, 20% acetonitrile (v/v) in 50 mMTris-HCl buffer field strength, 400 V/cm injection, 12 kV for 3 s at the side of the anode detection wavelength, 254 nm. Peak identification 1, methylparaben 2, ethylparaben 3, propylparaben 4, butylparaben 5, amylparaben. Reproduced with permission from Sawada and Jinno [11].

One example that illustrates this is the GPC separation of aspirin and propyl paraben. These two compounds have identical molecular weights (MW = 180) but can be separated using GPC (e.g., on a 100-A jiStyragel columns). From the structures (Fig. 11-3) it can be hypothesized that propyl paraben acts as a larger molecule in solution than does aspirin. Since the side chain on propyl paraben would give the molecules a larger end-to-end size than the more compact aspirin molecule, propyl paraben should elute before aspirin. 

Eigure 17-17. Theoretical plates versus retention factor for series of parabens separated by isocratic chromatography. Extra-column effects result in decreased efficiency for early-eluting components (low values of k). 

The principal advantages in the use of superheated water are that it is relatively easy to attain and the back-pressures required on the column are small. Thus even a modest length of narrow bore tubing can be employed to provide sufficient resistance to prevent boiling in the column and at these pressures many conventional spectroscopic flow cells can be used. Because of the high temperatures, there have been concerns about the thermal stability of the analytes, but of the numerous examples, there have been few reports of instability or a tendency for accelerated hydrolysis or oxidation, of the reported examples, only aspirin has hydrolyzed. Compounds which might be expected to be labile to oxidation or hydrolysis, such as the paraben antioxidants, have chromatographed without problems even up to 200°C [59]. 

Figure 18-12. Separation of test mixture and fractions after extraction and trapping and sequential elution at increasing temperatures. Separation on PS-DVB column at 75-185°C at 15°C/min. Analytes 1, paracetamol 2, salicylamide 3, caffeine 4, methyl paraben 5, phenacetin 6, ethyl paraben. Separations a, direct injection of original mixture of 1-6 without trapping b, fraction untrapped at ambient temperature c, fraction released from trap at 70°C d, released at 90°C e, released at 110°C. (Reproduced from reference 76, with permission from Royal Society of Chemistry.)...

Sulfacetamide and prednisolone acetate in a compound sulfacetamide ophthalmic ointment have been determined by HPLC. The peak of sulfacetamide sodium is well separated from those of the other constituents when using methyl cyanide-1 % acetic acid solution (15 85) as the mobile phase on /t-Corasil Cis column. The two compounds are determined by calculation of the peak area ratios using thiamine hydrochloride and methyl paraben as the internal standards. The maximum errors are 0.39% and 1.2% respectively (96). 

Figure lb. Separations of parabens and tartrazine (0.5-1 mg each) on an analytical column (Whatman Partisil Prep 40 0DS-3, 37-60 ptm, 2.5 x 50 cm). Mobile phase 607o methanol. Flow 20 ml/min. Detection 254 nm (1 AUFS). 

Figure 6.16 Methyl, ethyl, and propyl paraben—50/50 methanol water, column—GraceSmart RP-Cis 150 mm x 4.6 mm, 5 pm.

With only two chromatograms at appropriate isocratic conditions, the retention times for all other isocratic conditions can be predicted provisionally. Assume that we have a 15 cm SunFire column in the drawer as well as a test mixture of methyl-, ethyl-, propyl-, and butyl-paraben. Then we can start with, for example, 55% methanol and 45% water and obtain the chromatogram in Figure 3.12. 

The main problem immediately recognizable, the separation of the nine substances on a SunFire column, is due to the very similar manner of interaction of toluene and chlorobenzene, which coincide by 55% methanol. Only at %B = 35% or at = 75% are the lines separated by ln(/r) = 0.05 (one box vertically). At 75% the front peaks have almost no retention. Therefore, in the isocratic mode this separation problem can only be solved with a methanol content of 35% or lower. Then, however, the last peak butyl paraben has a ln(Ar)-value of 4.75, that is. A = 115.6, and a retention time of 175 min. So here there is no acceptable isocratic chromatogram. 

The earlier gradient start from %B = 45% down to 10% in steps of five causes a significant change in the separation. The two peaks for ethyl paraben and benzene even move past each other. Interesting is that the last peak is shifted toward the end only by almost a minute (differences between 0.97 and 1 are shaded gray), this means that butyl paraben in contrast to all earlier peaks remains stuck at the front of the column. The peak width of butyl paraben remains constant, although the retention times increase. The elimination concentration does not change either. 

Ye, X. Bishop, AM. Needham, L.L. Calafat, A.M. Automated on-bne column-switching HPLC-MS/MS method with peak focusing for measuring parabens, triclo-san, and other envirorunental phenols in human milk. Anal. Chim. Acta 2008, 622,150-156. 

J.F.G. Jimenez, M.C. Valencia, LJ. Capitan-Vallvey, Parabens determination with a hybrid FIA/HPLC system with ultra-short monolithic column, J. Anal. Chem. 65 (2010) 188—194. 

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