Separator column pharmaceuticals

Figure 5 Separation of pharmaceuticals, including amines, on strong cation exchange. Column 0.46 x 15 cm Merckosorb SI-60-SCX, 5 p. Eluent 50 mM aqueous ammonium formate-10% ethanol, pH 4.8. Flow 1 ml/min. Temperature 50°C. The peaks are (1) aspirin, (2) paracetamol, (3) phenacetin, (4) caffeine, (5) phenylephrine, (6) salbutamol. (Reproduced with permission of Elsevier Science from Cox, G. B., Loscombe, C. R., Slucutt, M. J., Sugden, K., and Upheld, J. A., /. Chromatogr., 117, 269, 1976).

HPLC has transformed quality control in the pharmaceutical and chemical industries, as it provides a rapid means of checking the purity of samples and even allows for the purification of small amounts of samples by preparative HPLC. The majority of such systems use L V to detect and quantify substances as they elute from the separative column. UV detectors are usually variable wavelength and can be used to detect molecules with absorption maxima above 210 nm by measuring the absorbance of the eluent. When a UV-absorbing substance is eluted from the HPLC column, it absorbs UV radiation at the appropriate wavelength for its chromophore. The amount of UV absorbed is proportional to the quantity of substance being eluted, and is converted into a peak on a chart recorder. Integration of each peak allows the relative quantities of the components of the solute to be determined. 

N. Wu, J Dempsey, P. Yehl, A. Dovletoglou, D. Ellison, and J Wyvratt, Practical aspects of fast HPLC separations for pharmaceutical process development using monoUthic columns. Anal. Chim. Acta 523 (2004), 49-156. 

Dear, G.J. Mallett, D.N. Higton, D.M. Roberts, A.D. Bird, S.A. Young, H. Plumb, R.S. Ismail, I.M. The potential of serially coupled alkyl-bonded silica monolithic columns for high resolution separations of pharmaceutical compounds in biological fluids. Chromatographia 2002, 55, 177-184. 

In pharmaceutical development, the determination of APIs and counterions are two important assays. Due to the charge and/or hydrophobicity differences, APIs and counterions are usually analyzed by different chromatographic techniques that require different separator columns and/or detection methods. For example, reversed-phase liquid chromatography is most commonly used for analyzing APIs with intermediate to higher hydrophobicity, but it fails to provide adequate retention for hydrophilic counterions. In contrast, ion chromatography provides a selective and highly sensitive solution for the analysis of counterions. 

Figure 6.90 Simultaneous isocratic separation of pharmaceutical counterions on Acclaim Trinity PI, 3 pm. Column dimensions 50mm X3mm i.d. column temperature 30°C eluent MeCN/20 mmol/L ammonium acetate, pH 5 (60 40 v/v) flow rate 0.5 mL/min detection ...

Figure 10.274 Anaiysis of methyi suifate in a pharmaceutical sample. Separator column lonPac AS4A-SC eluent 1 mmol/L TBAOH flow rate 2 mU/min detection suppressed conductivity injection volume 50 pU peaks (1) chloride and (2) methyl sulfate [450].

Another approach to resolve this issue is to preconcentrate the sample dissolved in 100% organic solvent on an anion-exchange concentrator column and then eliminate the matrix with deionized water prior to analysis. This procedure was successfully used to determine anionic impurities in a proprietary pharmaceutical compound with limited water solubility [454]. An lonPac AS15 column was chosen as the separator column, because it is a high-capacity column specifically developed for the rapid and efficient separation of trace inorganic anions in matrices with varying ionic strength. 

Figure 10.279 Isocratic separation of pharmaceutically relevant inorganic and organic anions by mixed-mode liquid chromatography. Separator column Acclaim Mixed-Mode WAX-1, S m column dimensions 150 mm X4.6 mm i.d. column temperature 30°C eluent 50 50 (v/v) MeCN/buffer (2.68 g potassium monophosphate-I-0.2 g sodium...

Figure 10.282 Analysis of amylamine in a pharmaceutical drug. Separator column lon-Pac CS14 eluent 50 mmol/L methanesulfonic acid/MeCN (95 5 v/v) flow rate 1 mlVmin ...

Fig. 9-184. Analysis of tert.-butylamine as a counter ion of a pharmaceutical drug. - Separator column lonPac CS14 eluant 10 mmol/L methanesulfonic acid - acetonitrile (99 1 v/v) flow rate 1 mL/min detection suppressed conductivity injection 20 pL drug solution (1 mg/mL BMS-188 494-04) solute concentration 10.6% (w/w) tert.-butylamine (1) (taken from [280]).

The table also shows that the optimal flow rates for these rapid gradients Ue outside the pressure limit of current H PLC technology, if one wants to use columns of length 5 cm or more. It should be pointed out that the estimates assume that we want to separate typical pharmaceutical compounds with molecular weights between 250 and 600 with a water/acetonitrile gradient The exact values are somewhat dependent on the solvent composition at which the compounds are... 

Wu, N., JilUan, D., Dempsey, J., Yehl, P, Dovletogolous, A., Ellison, D. and Wyvratt, J. Practical aspect of fast HPLC separation for pharmaceutical process development using monolithic columns. Anal. Chim. Acta. 523 149-156, 2004. 

Chiral separations have become of significant importance because the optical isomer of an active component can be considered an impurity. Optical isomers can have potentially different therapeutic or toxicological activities. The pharmaceutical Hterature is trying to address the issues pertaining to these compounds (155). Frequendy separations can be accompHshed by glc, hplc, or ce. For example, separation of R(+) and 5 (—) pindolol was accompHshed on a reversed-phase ceUulose-based chiral column with duorescence emission (156). The limits of detection were 1.2 ng/mL of R(+) and 4.3 ng/mL of 3 (—) pindolol in semm, and 21 and 76 ng/mL in urine, respectively. 

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