Effect of the gel chromatographic medium on recoveries and retention

Effect of the gel chromatographic medium on recoveries and retention volumes. Charles H. Lochmueller, and Lockhart B. Rogers. Anal. Chem. , 1969, 41 (...
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reacting with pyridoxine hydrochloride. BSA had none of these disadvantages. It was found absolutely necessary to use a silanated support for column preparation. The use of unsilanated supports and/or NaOH coated supports (8) invariably resulted in decomposition of the pyridoxine-TMS. From the above results it can be concluded that the proposed gas chromatographic method is considerably faster than, and of comparable accuracy to, those of the United States Pharmacopeia for determination of pyridoxine, ascorbic acid, and nicotinamide. (8) A. W. Decora and G. U. Dinneen, Bureau of Mines Report

5768, Laramie, Wyo., April 1961.

ACKNOWLEDGMENT The authors wish to thank F. A. Kummerow for his encouragement during this study, Gary S. Mintz for able technical assistance, and Harold Pazdara of Abbott Laboratories for gifts of commercial placeboes. RECEIVED for review July 31, 1968. Accepted October 7, 1968. Supported in part by a U. S. Public Health Service Research Grant (AM 00257), and in part by a U. S. Public Health Service Training Grant (1-T1-Gm-653-07). This work represents a portion of a thesis to be presented by Lawrence T. Sennello as partial fulfillment of the requirements for the degree of Doctor of Philosophy at the University of Illinois, Urbana, Ill.

Effect of the Gel Chromatographic Medium on Recoveries and Retention Volumes C. H. Lochmuller and L. B. Rogers Department of Chemistry, Purdue Uniuersity, Lafayette, Ind. 47907

THEuse of gel chromatography for the study of polymeric inorganic systems is well established. Ohasi, Yoza, and Ueno ( I ) noted an elution in the order of molecular dimensions for several oligophosphates on Sephadex G-25. Spiro er al. ( 2 ) have reported the separation of a high molecular weight form of hydrated iron(II1). The polymer was observed to have an average molecular weight of 1.44 x 105 and to be stable indefinitely after isolation. Ricketts and coworkers (.?) attempted a determination of the molecular weight of an ironsoluble dextran complex of pharmaceutical interest by gel chromatography on Sephadex media. It was observed that staining occurred and that total recovery of the iron sample was not achieved. The total iron recovery was shown to decrease as the total contact time with the gel increased. Alternatively, the total iron recovery was observed to increase on a more tightly cross-linked gel. Using a polyacrylamide gel in place of Sephadex was reported to have given similar results. Henry and Rogers ( 4 ) have studied the gel chromatographic behavior of aqueous ferric nitrate on polydextran gels having various exclusion limits. They confirmed that staining of the gel occurred, and found that iron recoveries were improved after successive sample injections. We wished to compare other commonly used gel media for the separation of iron polymer on the basis of resolution and recovery. We were also interested in comparing the behavior of stable inorganic and organic species on those same media. EXPERIMENTAL Reagents. A stock solution of hydrated ferric nitrate (0.3M) was prepared by dissolving a weighed amount of

reagent grade ferric nitrate (Mallinckrodt Chemical Co.) in (1) S. Ohasi, N. Yoza, and J. Ueno, J . Chrornatogr., 24, 300(1966). (2) T. G. Spiro, S. E. Allerton, J. Renner, A. Terzis, R. Bils, and P. Sattman, J. Amer. Chem. Soc., 80,2721 (1966). (3) C. R. Ricketts, J. S. G. Cox, C . Fitzmaurice, and G. F. Moss, Nature, 208, 237 (1965). (4) R. A. Henry and L. B. Rogers, Separation Science, 3 , l l (1968).

distilled water. The extent of hydrolysis was brought to OH/Fe = 2.0 by the addition of a weighed amount of potassium bicarbonate (J. T. Baker Chemical co.). Carbon dioxide was removed by bubbling dry nitrogen through the solution for 30 minutes. No precipitation was evident after 2 months. Stock solutions of Na9P7OZ2and Na2HP04(O.O5M, Monsanto Chemical Co.), Dextran 150 (mol. wt. 1.4 X 105, Pharmacia, Sweden) and dextrose (10 mg/ml; 5 mg/ml) were prepared by dissolving weighed amounts of the materials in 0.01M NaCl solution. Sephadex G-25, Sephadex LH-20 (Pharmacia, Sweden), Bio-Gel P-6 and Bio-Glas 200 (Bio-Rad Laboratories) served as the gel media. The gels G-25, LH-20, and P-6 all had exclusion limits of approximately 5000 molecular weight units. Bio-Glas 200 was reported to have an exclusion limit of 200 A. Bio-Glas 200 was also silanized with hexamethyldisilazane in a vacuum according to the manufacturer’s recommended procedure (5). The eluents were 0.01M ”OB for the iron study and 0.01M NaCl for the phosphate and organic studies. Recovery Analyses. Fractions were collected manually as they eluted from the sample cell. Iron and phosphate recoveries were determined colorimetrically using o-phenanthroline and vanadomolybdate, respectively, according to standard methods (6). Procedures. Typically, 10-pl samples were injected directly on to the column material. All the columns were 47- x 0.5-cm glass tubing fitted with Teflon stopcocks. All other parameters-e.g., fittings and pumping system-have been previously reported ( 4 ) . Void volume and total volume of the packed column were determined with Blue Dextran and either nitric acid or sodium chloride, respectively. A Beckman DU spectrophotometer fitted with a micro flow cell served as the detector for iron, and an R-4 differential refractometer (Waters Associates) was used for the phosphate and organic solutes.

( 5 ) Bio-Rad Laboratories, Richmond, Calif.

(6) G. Charlot and Denise Bezier, “Quantitative Inorganic Analysis,” Wiley, New York, 1957. VOL. 41, NO. 1 , JANUARY 1969

173

Gel Sephadex G-25 Sephadex LH-20 Bio-Gel P-6 Bio-Glas 200 Bio-Glas 2OO(s)

Table I. Effect of Pretreatment on Iron Recovery Moles Fe(NO& Moles Fe(NO& pretreatment pretreatment X los Fe recovery,

z

0 0 0 0 0

3.6 1.8 4.8 4.8 7.8

7.6 96