Thin-layer gel filtration with
Sephadex SUPERFINE The advantages of both Sephadex gel filtra tion andthin-layer chromatography can now be utilized with the Sephadex Superfine. Sephadex Superfine is an important com plement to other analytic methods, par ticularly where only sample quantities of experimental material are available. It is useful also (1) for determining the optimum conditions for column experiments (2) in place of normal Sephadex in gel filtration columns when very high resolution is required (3) as a supporting medium in column electrophoresis and in partition chromatography. Thyroglobuttn 110 -Globulins — 100 Ε ,Ε
«
Serum albumin
g 90g 80-
Ovalbumin
1 S
crChymotrypsInogen Soya-bean trypsin Inhibitor rS** ^Myoglobin S ' α-Lactalbumln 60ÇOOytorhrome ç ; 10"1 101 Molecular weight 70-
Correlation between the molecular weight of 9 proteins and their migration rate in thin-layer gel filtration on Sephadex Superfine G-100 was inves tigated. Measurements from separate experiments were correlated by expression on the common basis of 6 cm. migration by cytochrome c. (Andrews, P., Biochem- J- (1964) 91,222, by permis sion of the author.]
Sephadex Superfine gels can be applied to glass plates with ordinary TLC equip ment. They adhere easily to the plates. Addition of a binder is not necessary. Six types of Sephadex from G-25 to G-200 are available in the SUPERFINE grade.The small particle size of Sephadex Superfine (between 10 and 40 microns) permits prep aration of thin layers, even with the more porous gels The various Sephadex types have the following fractionation ranges. Approximate fractionation range Polysaccharides Proteins
Type Sephadex Sephadex Sephadex Sephadex Sephadex Sephadex
G-25 G-50 G-75 G-100 G-1S0 G-200
1 0 0 - 5,000 5 0 0 - 10.000 1.000- 50,000 3,000- 70,000 1.000-100.000 4,000-150,000 1.000-150,000 5,000-400,000 1,000-200,000 5,000-800,000
For additional technical information on Sephadex Superfine, including booklet Thin-Layer Gel Filtra tion, write to:
PHARMACIA FINE CHEMICALS INC. 800 Centennial Avenue, Piscataway. Ν J. 08854 Pharmacia (Canada) Ltd.. 110 Place Crémazie Suite 412, Montreal 11, Ρ Ρ (Inquiries outside U.S.A. and Canada should b e directed to PHARMACIA FINE CHEMICALS. Uppsala, Sweden.)
Circle No. 113 on Readers' Service Card
30 A
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ANALYTICAL CHEMISTRY
Report for Analytical Chemists
acids, and various combinations in corporating hydrogen peroxide. Also included are dry ashing with sulfuric acid, with nitric acid, with nitric and perchloric acids, and with magnesium nitrate. I n the author's experience, the most generally use ful acid mixture for wet digestions is a combination of nitric, sulfuric, and perchloric acids in the ratio of about 3 : 1 : 1 . Ten ml of this mix ture will usually suffice for the de struction of 10 g of fresh tissue or blood. This is an efficient oxidizing mixture and the perchloric acid will a t t a c k all but the most resistant substances. Since oxidizing condi tions are maintained, loss of a num ber of volatile elements is prevented. Elements for which good recoveries are obtained with this mixture in clude zinc, selenium, arsenic, cop per, cobalt, silver, cadmium, anti mony, chromium, molybdenum, strontium, and iron. Lead cannot be wet digested in the presence of sulfuric acid because significant amounts are lost by coprecipitation as lead sulfate on other sulfates. The presence of perchloric acid is necessary to prevent the volatiliza tion of selenium. Simple dry ashing is satisfactory for lead, zinc, cobalt, antimony, chromium, molybdenum, strontium, and iron. Ashing in the presence of magnesium nitrate ex tends this to arsenic, copper, and silver; this salt causes chromium to be lost, however. Lead is lost in dry ashing at temperatures in excess of about 500 °C, especially if chloride is present. Mercury presents a spe cial problem since it cannot be dry or wet ashed. A somewhat incon venient method can be used to quantitatively collect t h a t p a r t of the mercury which is distilled dur ing digestion with nitric, sulfuric and perchloric acids (10). Many special oxidation procedures have been suggested for overcoming diffi culties encountered with mercury losses, and some investigators have preferred not to obtain complete de struction of the organic matter. This m a y lead to errors later in the procedure because of the ease with which mercury can combine with organic compounds. The AOAC method for destruction of organic m a t t e r involves oxidation with ni tric and sulfuric acids at a "simmer
ing" temperature, supposedly near the boiling point of sulfuric acid. These conditions will not affect the oxidation of fat, which must be re moved by filtration. B a r r e t t (IS) used a similar method involving fil tration, but he used potassium per manganate to complete the oxida tion. Policy and Miller (16) used 50 per cent hydrogen peroxide as the oxidizing agent. This has an ad vantage in t h a t much of the oxida tion is carried out without the a p plication of external heat. The amount of organic m a t t e r in urine is rather small, and Rolfe, et al. (17) have used nitric acid and potassium permanganate a t about 85 °C and under slight pressure to prepare urine samples. Nobel and Nobel (18) have described a method for quantitatively collecting mercury from urine in which all steps are carried out at room temperature. Mercury is reduced to the element with copper (I) and hydroxylamine hydrochloride, and organic m a t t e r is destroyed by potassium permanga nate at room temperature. It should be possible to develop a method whereby mercury is quanti tatively distilled from the sample, such as is often done with arsenic and selenium. A useful wet digestion procedure for selenium and arsenic incorpo rates a molybdenum (VI) catalyst (14, 19). Even under rugged heat ing conditions, these usually volatile elements are not lost. I n addition, it is much more rapid than other di gestion mixtures and organic m a t t e r is more completely destroyed. This catalyst solution should prove use ful as an efficient general oxidizing agent. Studies are currently under way in our laboratory to evaluate the effect of the molybdenum on atomic absorption measurements. There exist several dry ashing methods which can be employed for volatile as well as other elements. The Schoniger flask (20) and the P a r r bomb techniques employ oxy gen under pressure as the oxidant in place of air. The dried sample is combusted in the atmosphere in a closed system, and therefore t h e sample elements cannot be lost by volatilization. For example, the Schoniger flask (21-23) and the P a r r bomb (23, 24) have been suc-
