Review of Fundamental Developments in Analysis
Biochemical Analysis Paul
B. Hamilton
Alfred 1. du Pont Institute o f the Nemours Foundation, Wilmingfon 99, Del.
T
HE LAST REVIEW of fundamental developments in biochemical analysis in this journal (117) covered the prriod 1954-55, The present review n-ill attempt to coaer the period January 1 through December 31, 1961. This gap in coverage of the literature is more apparent t h a n real, for last year's review of clinical chemistry (198) was extraordinarily wide and coniplcte. Major developments will be recorded, b u t niinor variants of technique or routine applications of methods n-ill be omitted. It is hardly necessary to eiiipliasize t h a t such a selective approach n-ill leave out many excellent papers which use neJv analytical tools, but nhicli do not necessarily adrance the art of the niethodology employed. The prevailing philosophy continues to eiiiphasize fractionation procedures whereby comple\ m k t u r e s of simple or coniple\ molecules are resolved conipletely, either on a preparntive or determinative basis. Other significant trends :we, however, the introduction of automation and instrumental data processing, and a n increased use of the physical properties of chemical entities for analytical purposes so t h a t chemical determinations are accomplished with increasing frequency by the measurement of physical parameters. I n many instances this leads to identification without destruction of the substance being measured, which in turn can lead to scquential analyses of divrrse types upon the same material. It is also germane to note certain communication trends within the past 5 years-namely the distribution by conimercial enterprices of brief abstracts or bibliographies of current literature n hirh are related to their interests and products. This type of commerci:ilism, season1.d with altruism c:~nbe very helpful to n orkers n ho must folloir a restricted interest very clovly. One is also hcartened to observe thc increasing numbers of reriewtype monographs and pcriodical literature devoted to signifimnt work in restricted but highly important fields. Thf. revien er has included a limited and arlitrary selection of new publications 11hich have appeared within the last few years. It seems appropriate to proffer a bouquet to those commercial companies
who for the most part have taken up and niet the challrnge of supplying the biochemist with a n e l w increasing number of new chemicals of biological interest. What were esoteric curiosities yesterday, are commonplace today, and of a degree of purity t h a t is truly remarkable. For this the biochemist is largely indebted to the universal application of the techniques of electrophoresis, ion exchange chromatography, gas chromatography, and counter-current distribution, and the willingness of the manufacturers to use them along n i t h determinations of clementary composition and optical rotation. This in turn has led to the adoption of specifications and criteria for biochemical compounds by the Sational AcadPniy of Science (266). Close cooperation between producer and consumer seems assured for the future. NEW BOOKS AND JOURNALS
Instruments, instrumentation, and niethodology continue to play a n increasingly important and expanding role in biochemistry. M a n y techniques have recently been revicn-ed in monograph form i d s ) , and a previous encyclopedic work on physical inethods in organic chemistry is slon-ly being revised (379). -211 methods concerned n ith the qeparation, characterization, and manipulation of protcins have been brought together and thoroughly documented ( 5 ) . The results to be e y e c t e d from the application of these methods arc drainatically set forth in the collected pspers of a recent symposium on biophysical studies (271 1. Chemical instrument.ition from :L general point of view arid also for continuous processes has been the subject of monographs 1329, S @ ) , and symposia (133,207'). More specific texts have been concerned n ith organic electronic spectral data (190, %7), chromatography (164, 275, 338), electrophoresis (12, t%'), inimunoelectrophoresis ( 144), and immunodiffusion (96). Oxidation-reduction phenomena in organic systems have been brought up to date (83), and free radicals in biological systems have been thoroughly discussed by many competent authors ( L O ) . This latter publication is especially valuable in bringing to the bio-
chemist the type of instrumentation involved and the results obtained in studies on electron spin resonance in bioloqical systems. It 1% ould appear that a n understanding of the importance of free radicals in biological systems is only just beginning. The appreciation of the significance of the fine structure of molecules of biological interest, together Iyith the concept t h a t polyelectrolytes impose a n orientation or structure upon the ionic clouds surrounding them (Debye ion atmosphere) seems to pave the x a y for viewing biological organisms and tissues as legitimate areas for the solid state physics approach. At any rate, the title of a recent monograph on electrolyte solutions (154) and the clemonstration of electron transport by proteins, textiles, and other polymers in the solid state (3L7),could be construed as being consistent n i t h this vier\.. Such speculations have, hon ever, little validity until demonstrated expciimentally. Flame photometry (200) and liquidliquid extraction ( 4 ) have also been treated in monographq. The yearly volume on the preparation of biochemicals of importance @25, 240, 324, 36S) and methods of analysis 1139) continue to summaiizr and collect worthn-hile preparative and deterniinative procedures in the same excellent manner a s ill the past. Uecauscl electronics i. playing a n ever expanding role in biological and biochemicd research, tu-o texts (122, 342) should prove valuable. S e w journals of interest to biochemists (46, 916, 265, d67'), clinical chemists (14, 260, 354), biophysicists 126, 51, 9 7 ) , biologists (98, 193, 194), and lipide chemists (394) have appeared in the past fen years. Commercial nonprofit serial publications of considerable interest are d e v o k d to medical electronics ( I O ) , the chemistry of collagen (235), ion exchange (310), and tissue culture (3.51).
As part of its program to improve communication n ithin the chemical Iyorld and to fend off the d a y when the individual becomes completely overwhelmed by the incessant flood of neiv literature, the American Chemicd Society cominenced publication of its neiv IBM-pon ered quick-review journal, VOL. 34,
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Chemical Titles (78). Its value should be considerable to all as this reviewer is prepared to testify. VOLTAMETRIC METHODS
Electroanalysis and coulometric analysis were reviened in 1958 ( l o g ) , and amperometric titrations (R11), potentiometric titrations (296), and organic polarography (376) in 1960, in AKALYTICAL CHEMISTRY; electroanalyticnl methods in trace analysis n ere summarized in 1956 (86-1).Oxidation-reduction \YCS masterfully covered in a comprehensive test by Clark (83). Amperometric titration of cysteine, glutathione (SH), cystine, and albumin nere studied a t the rotated dropping mercury indicator electrode (341). A continuous determination of carbon dioxide by a titrimetric method was devised (280) and applied to the study of decarboxylation of amino acids by various reactions. The time course of the evolution of carbon dioxide or the total amount of carbon dioxide were the data of interest. The possilAty of monitoring continuously the carbon dioside produced by reaction of amino acids n ith ninhydrin, after separation on a n ion eschange column n as not investigated, but such a procedure could prove valuable as a n alternate to the ninhydrin colorimetric method, provided the sensitivity of carbon dioxide determination could be made equal to tbc photonietric method. Ascorbic acid was estimated by potential coulometry (312).urea by a conductivity method (81), and the sulfhydryl groups in bovine hemoglobin by amperometric titration (31.5). Numerous substances of biochemical interest have been assaycd by po1,xrography. The polarographic behavior of kinetin, isolated from D S A 4was e\amined (221),and polarographic studies on the serum of pigs, sheep, cows, and human (female) urine (?'3), on the determination of gonadotropin on sulfur-containing proteins ( $ l a ) ,and on oxygen upt ihe in mitochondrial respiration (166) have been reported. Polarographic estimations of manganese in biological materials (291), of lead in urine ( % I ) , of cj.stine and cysteine in a001 (65), and of dinitrophenj-1 amino acids (360) were described. Oscillopolarographic determination of the components of mixtures of equilin with equilenin, and equilenin with progesterone (120), of calcium and magnesium in blood serum (182), and of deoxyribonucleic acid and apurimic acid ($77) n ere reported. Applications of oscillographic polarography to microbiological problems mere discussed (189). A nelv approach to direct current polarography which provides a n incremental approximation to the derivative current-voltage curve was developed, in which the high resolution inherent in derivative methods mas realized (13). By means of a 4R
ANALYTICAL CHEMISTRY
staircase voltqge sweep applied to stationary electrode polarography, increased sensitivity and decreased time of measurement resulted (2%). These two latter papers also provide further references to significant literature and texts on polarography. One of the most interesting applications of polsrographic analysis !vas that of continuously monitoring the alpha amino acids emerging in the effluent from a n ion eschange column (36). The column effluent ivas made alkaline and passed through a short tube of copper phosphate. Amino acid-copper chelates were formed, and after conversion to the copper-EDTA complex, the copper was determined polarographically. I n general, areas under the peaks IT. ere proportional to the amounts of amino acids, with a standard deviation of 37& Sensitivity of the method for ammonia was only about one t n o-hundreths of t h a t for amino acids, which is a distinct adrantsge over ninhydrin photometric methods, but the sensitil ity reported showed a limit of detection of the order of 5 X 10-6 mole, which is considerably loner than the detection liniit of the ninhydrin method. Potentiometric determination of chloride in hemolyzates of erythrocytes (65) and of the free amino groups of some amino acids, peptides, and proteins (369) were reported, and the quantitative analysis by a n automatic potentiometric rate method applied to the specific enzymatic determination of glucose (231) was described. A recent paper, hardly classifiable as a voltan2etric method, but still of interest in that it involved the application of a physical principle hitherto little applied in biological chemistry, described the measurement of the dielectric properties of blood (268). By measuring the capacitance and resistance of blood, early changes in coagulstion could be detected. This would seem to be a good eximple of physical measurements providing information about a chemical system, nithout interference in the chemical transformations involved. Few papers on calorimetry were noted in the literature; the most recent review encountered was on niicrocalorimetry in 1960 (201). CENTRIFUGATION
High speed centrifugation methods found their most frequent application in the separation of serum lipoproteins. For example, distribution of serum lipoproteins prestained n-ith Sudan Black B was examined by a density gradient technique a t the ultracentrifuge (SS), while separation of the lipoprotein of dog serum by ultracentrifugation was compared ivith lipoprotein precipitation with dextran sulfate (309). I n this instance, the beta lipoproteins prepared by centrifugation were con-
taminated by alpha lipoproteins; the destran sulfate precipitated the former, uncontaminated with the latter. A comparison of the beta lipoprotein prepared by ultracentrifugation and by precipitation 11ith sulfated po1yg:ilacturonic methyl ester methyl glycoside (128) indicated that the product in either case was the s2me, but starch gel electrophoretic analysis of the separated lipoprotein fraction n a s not reported. -4 single band on starch gel, obtained by standard procedures, is reasonable evidence for homogeneity. hut it should be constantly borne in mind that a single operational definition of purity is inadequate ; alteration of electrophoretic conditions may ell reveal a dissociable complex and not a sirrple entity. A modified technique for separating horse serum lipoproteins a t the ultracentrifuge described the satisfactory use of sodium sulfate in place of sodium chloride to adjust the density of solutions preparatory to centrifugation (44,. Tno papers (89, 90) on tLe charactc4zation of human serum lipoprotcins l)y ultracentrifugntion in a density gr'idient are noteworthy for their thoroughness. 111 the first 11: per, the major lipoprotein fractions nere separated quickly, but a second centrifugation at a homogeneous density \vas necessary to r e r o v e a small amount of coiitaminating high density lipoprotein from the low density fractions. The distribution of cholesterol, phospholipid, and serum lipoproteins was determined for normal hyperthyroid and hypercholesterolemic subjects. The second paper n a s concerned with serum lipoproteins in hyperlipemic subjects. It is perhaps permissible to digress momentarily to comment on the increasing use that is being made of the interaction betn een high molecular neight substances. It is probably premature to call the application of this principle a biochemical tool, yet in the separation of proteins, more particularly the serum proteins and lipoproteins, considerable success has been achieved. The coniplexing of beta lipoproteins was seriously initiated in 1958 (SO) n i t h a thorough study of the complexing properties of various high molecular n eight polysulfate esters of starch, dextran, cellulose, chondroitin sulfate, heparin, and others. Sulfonated amylopectin was the most satisfactory for the specific precipitation of the beta lipoproteins. The subseqcent developments in the isolation and characterization of plasma lipoproteins by moleculrtr coniplexing n i t h polysulfate esters w r e masterfully reviened this year, and the literature on molecular comple~esof serum protein with polyvinyl pyrrolidone was discussed (87). As a n extension of this principle, the undesirable sticking to the paper during electrophoresis of certain serum components could be abolished
by preliminary treatment of the paper with carbosymethylcellulose (176). The altered surface charge of the paper no longer bound the serum proteins. Also pre-treatment of the serum with Duponol (lauryl sulfate) enabled t h e alpha lipoproteins to be separated from other proteins in electrophoretic analysis on carboxymethylcellulose treated paper (17 7 ) . References (309) and (128) discussed above also provide examples of these interesting high molecular weight interactions. It is fair to presume that antigen-antibody reactions :ire of a similar nature and t h a t the elucidation of these intemctions will reveal interesting high molecular weight complcses. Transport n-ithin the blood stream of certain substances-e.g., calcium [cf. (SSj] may prove to be a n integral part of the complesing process. This idea finds support in the role of calcium in the union of the polysaccharide and protein moieties in Busycon (snail) mucus (383). A final example of the principle of high molecular weight interactions is selected whereby cationic agents were used to separate plasminogen act'irator from plasminogen or plasmin in guinea pig serum (269). Differential density separation of cellular suspensions, making use of organic diesters as separating layers, was reported (19), and the separation of plasnia calcium into its prot'ein and nonprotein fraetions was accomplished by high spced centrifugation (52). The sedimentation properties of native and denatured DSA u w e examined (ISl), anti a method for the rapid determination a t the ultracentrifcge of the moir n-eights of pcptidcs and proteins w:is described (3.91). An interesting discwssion of the determination of density gradients in isodensity equilibrium ultracentrifugal analysis was also reported (353). DIALYSIS, COUNTER-CURRENT SEPARATION, A N D F O A M FRACTIONATION
Fractional dialysis was reviewed in 1960 (92) and dialysis more generally in 1961 ( 7 4 ) . An interesting application involved the dialysis of phosphatides dissolved in organic solvent, in the sac, against a different organic solvent outside (153); fractionation of lipide compounds was thus effected. A factor associated with S'isking tubing capable of antagonizing insulin and a method for its removal by hot water T! ere described (222). To concentrate proteins, their solutions were dialyzed in cellophane sacs agninst polyethylene glycols or polyvinyl pyrrolidone (287). Diffusion osmotic equilibrium constants were measured in a cell where equilibria betn ccn upward diffusion and don-nward migration onto a semipermeable membrane occurred which had established across it a strong osmotic gradient (288).
As well as the testbook on liquidliquid extraction mentioned earlier a recent review of solvent extraction (243) dealt with the theoretical basis of multistage distributions in a very extensive and thorough manner. Counter-current distribution was discussed fully therein. Solvent combinations for many organic and inorganic compounds were tabulated and organized in a n exceptionally informative may, and the literature was well covered. A modification of the Craig countercurrent distribution process was described for the separation of glycosides and alkaloids (359), and the analysis of counter-current distribution d a t a by methods of curve fitting, parameter estimates, and error analysis was published (325). T h a t fractionation need not always be accomplished by two-phase organic systems or by membrane separations was fully demonstrated and discussed in a recent test on the fractionation of particles and macromolecules in aqueous two-phase systems ( 3 ) , and in a later paper on the same subject (2). Separations with foams were treated from a theoretical standpoint (372) with no application to substances of biochemical interest. Foam fractionation of organic compounds, xvhich 1%ere of themselves not foaming agents, was demonstrated (192). It would seem t h a t further development of this method should provide extremely interesting a n d valuable separations; its simplicity recommends itself to further very serious analytical investigation.
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ELECTROPHORESIS
Many sources of detailed information have become awilable in this rapidly eypanding subject in monographs (5. 12, 53. 95, 144. 164. 215, 332, 379) and in r e v i e w (209). Various supporting merlin for electrophoresis continue to be eyplored, such as porous glass (229), gelose (35?), acrylamide (276, 294). ce1lulo.e acetate (11, 55, 147, 148), agar (43, 127, 326, 3R2), starch (101. 247), and paper (29, 113, 175, 283, 320, 375). Details for preparing the acrylamide gel for use in electrophoresis were described (294), optimal staining conditions for serum protein fractions on cellulose acetate were outlined (551, and a densitometric evaluation of serum proteins on cellulose acetpte (148) was discussed. A method of ultraviolet densitometry was developed for the estimation of serum protein in agar gel electrophoresis (326),as well as a n interferometric method for visualizing the patterns (43). A very ingenious method (59) was reported of scanning paper electrophoretic strips by moving a positively charged electrode along the strip which bore, relative to the movable electrode, a negative charge. On passing over a protein
fraction, a n electromotive force was generated which was measureii. Beta lipoproteins were rendered visible under ultraviolet light by protoporphyrin staining (320). An experimental method of converting paper electrophoretic mobilities to free solution values mas developed (19). .4n improved microelectroplioretic apparatus and technique for studying free electrophoresis, or more strictly free migration of cells in a n electric field was described (138) and applied to the examination of the nature and charged state of the surface of Aergbacler aProgencs. Tmmunoelectrophnresishas been dealt with specifically in monographs ( 5 , 96, 144) and a review (1 $3). lmmunoelectrophoresis was conibined with electrophoretic analysis on cellulose acetate (1 46) and on starch gel (101). Rapid electrophoresis in density gradients combined with p I I or conductivity gradients was reviewed (209) and applied on a preparative scale (82, 354). This technique was also reviened (46 references) in its application as a new tool in T irology (93). Increasing use is being made of high voltage electrophoresis which was the subject of a review (244) and brief description (164). An apparatus for high voltage electrophoresis mas dePcribed by one of the pioneers of this technique (146). High voltage electrophoresis was also used for exnniining iodine containing compounds of the thyroid gland (249),the estimation of serotonin in serum (34$, the separation of choline esters (166), the determination of sugars (348),the separation of steroid hormones (248), and the quantitative determinations of metaboIites (349). A method of carrier-free continuous electrophoresis and its use in the scparation of dyes, amino acids, peptides, and serum proteins was described ( I 61). G A S CHROMATOGRAPHY
Gas chromatography has been the subject of texts ( 1 9 i , 2Sl). symposia (86,104,106, 819). and revien-s (162, 285). l\luch information, both on theory and application to analysis of hydrocarbons, gases, carbohydrates, steriods, alkaloids, lipides, amino acids, and other compounds of biological interest. has been brought together in a recent text (16L). Gas chromatography, as applied to separation and determinations of the fatty acids, has been revien ed separately (1841. A number of papers have hcen concerned with theoretical aspects of gas chromatography in attempts to provide greater understanding of the chromatographic process and the influence of the many variables inxolved. The van Deemter equation has been examined n i t h respect to the effect of gaseous diffusion on mass transfer in packed columns (196) and for the source of the VOL. 34, NO. 5, APRIL 1962
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velocity independent term (197). Rlodifications of the equation have been suggested (168). Consideration of the parameters involved in computing the plate height for columns was shown to lead to a theoretical basis for calculating relative efficiencies of different types of columns with different types of packings (137). The theory of solutions was applied to assist in the choice of solvent for gas chromatographic columns (236). Other papers of general interest were the determination of areas of partially resolved peaks (21), the principles of high speed chromatography (15), resolution in temperature programmed chromatography (131), low temperature chromatography (BBO), and considerations on the speed of analysis (206). A very extensive paper considered the identification of organic compounds by gas chromatography (60). Sonpolar, electron donor, and electron acceptor columns were used, and the difference in movement of any substance on these three columns provided valuable characterization data, The characteristics of 40 different compounds were detailed. I n this connection, it is interesting to note the increasing use of polar compounds for the liquid phase in gas chromatography to obtain enhanced resolving pon-er for substances whose diffusion rates probably differ very little, but whose charge affinities differ more widely. K i t h respect to automation in gas chromatography, a total analysis digital system (191) and a recording integrator (185) have been reported. The development of gas-liquid chromatography of the amino acids has advanced very considerably. To date, about 35 amino acids can be determined, including 18 of the common protein amino acids (186). These analyses were done with the K-acetylamino acid n-amylesters. Approximately 10-10 mole could be detected. X similar order of magnitude of the h*-trifluoro acetyl methyl esters of the amino acids has been detected (313), and 1-1 amino acids have been determined. A difficulty a t present is the quantitative conversion of the amino acids to derivatives suitable for gas chromatography, but one accepts the likelihood that it is only a matter of time before a good solution to this problem vrill be found. The volatilization of peptides may, however, prove very difficult or even impossible. Perhaps one of the major advances has been the determination of various steroids, sex horrnoncs, and bile acids by gas chromatography. Separation of the adrenal cortical steroid hormones (363), urinary IT-keto steroids (ISU), cholesterol and squalene (d72), bile acids (41, 366), and estrogens (390) has been described. The effect of the composition of the stationary liquid phase o n the separation of certain steroids was
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examined (624), a silicone polymer was recommended as a stationary phase (362), the trifluoroacetoxy derivatives of the steroids were examined (365),and the relation of structure to molar response in the argon ionization detector was reported (346). Group retention factors in the chromatography of the steroids were discussed (84). Gas chromatographic behavior of vitamins DB and D3 was investigated (393). The separation of the acetyl deriratives of sugars and related compounds n-as described (564))the estimation of volatile anesthetics in tissues was carried out (n),and the preparation of fatty acid esters (242, 370) for gas chromatography was reported. The influence of column support on the separation of the fatty acid methyl esters (178) mas also investigated. The determination of nitrogen with gas chromatography was reported (296). The f e x papers mentioned here merely indicate the expanding scope of gas chromatography in the biological fields; many other excellent reports had to be omitted. COLUMN CHROMATOGRAPHY: ION EXCHANGE AND GEL FILTRATION
Texts on column procedures, with emphasis on ion exchange chromatography are numerous (72, 168, 261, 3?4), including those referred to earlier (164, 275, 332). il particularly thorough treatment has been afforded the theoretical aspects of ion exchange (168). The most recent review (299) is to be highly recommended for its scope, thoroughness, and practical lucidity. It also includes considerable exposition of gradient elution theory and methodology. Mention should also be made of the summary of a recent symposium on solution Chromatography (1). It is hoped that publication of the papers presented a t this symposium nil1 be forthcoming. A sonien hat belated publication (295)also rontains much information related to ion exchange and other forms of chromatography. Several papers appearing during the past year h a r e interest from theoretical and practical niethodological points of view, The nonuniformity of crosslinking in ion exchange polymers was unequivocally demonstrated (141). Efficiency parameters in linear chromatography and their uses for analytical purposes were considered ( l b l ) ,and the basis for selectivity in chromatography, electrochromatography, and continuous electrochromatography was discussed (340). The conversion of a cationic exchanger to a ligand exchanger by placing a metal on the ion exchange resin seems a very significant observation (169), thus extending the usefulness of the exchange process to ligand exchange. By varying the metal, various types of
ligands may be absorbed or exchanged. An elample cited involved conversion to a copper ligand exchnnger. Besides the theoretical treatments of the ion exchange process previously mentioned (164, 168, d99), analysis of diffusion mechanisms led to a n equation relating the plate and rate theories, and Ivhich expressed band width of a n elution peak in terms of all column variables and diffusion phenomena ( 1 5 9 . The equation ivas tested thoroughly experimentally and shon n to be a good description of column operation. Resolution was also expressed in terms of all column variables. The elperimental verification of the theoretical equation brought out clearly the necessity for further detailed studies on the ion exchange process in order to evaluate constants whose values have hitherto been assumed. The evaluations of one constant were provided (45). This work was extended, making use of rigidly controlled particle sizes (li5), and semiautomatic procedures (158)to demonstrate the potentialities of fast flow rates in the separation of amino acids (156). Further methodological advances included a continuous polarographic determination of amino acids (36) discussed more fully in the section on voltametric methods, a semi-micro method for the determination of amino acids on cationic exchange columns ( l a l ) , and automatic analytical procedures for amino acids after separation of cationic exchange columns (204, 286, 355, 389). The first paper (204) demonstrated the feasibility of splitting the stream of the column effluent, part for analysis, part for collection for further analysis or isolation procedures. It was also demoastrated that tissue extracts could be placed directly on a n ion exchange column n ithout preliminary removal of the proteins (115), providing a nonionic detergent n as included in the developing buffers. The protein did not interfere, and the amino acid pattern was developed in the usual n a y . This observation has been confirmed in the reviewer’s laboratory (157) for whole serum placed on the column. Among hundreds of other papers using ion exchange as a laboratory tool, the folloning have been selected for their interest to the reviewer. A separation of amines from biological niaterial ivas described is@,and the water elution of amino acids from ion exchange materials was demonstrated (66). Ion exchange was also used to determine strontium in bone (371) and milk (289), and acetylated isoniazid in biological fluids (170). The anomalous behavior of arginosuccinic acid on cationic ion exchange coluinns was also elucidated (96). Silica gel was used n i t h gradient elution of 17-o~osteroids(187), and in the fractionation of lipides (225). The
standardization of silicic acid for chromatography was described (172),and the effects of silica gel pore diameter on the migration of sterol acetates were investigated (206). Blood glycerides were determined using a Florisil column ( S T ) , aroniatic fluoro derivatives 15 ere separated on a colunin of poivdered Kylon 66 (174). nucleic acids nere fractionated on a nongelatinized tertiary amino-alkyl-ether starch column (9S3), and estrone and other steroids were separated on a column packed with celite (208). Alumina served to separate lipides obtaiiicd by ethanolic extraction of brain tissue (227) and DEBE cellulose was eiiiployed to separate folic acid analogs (270),pyridine nucleotides (279), and human serum proteins by gradient elution (284). Polynucleotides and proteins were fractionated on calcium phosphate prepared in different ways and operated in column procedures (230). Gel filtration through Sephadex was used to purify urinary conjugated estrogens (63), to separate protein bound iodine from inorganic ions (33.5), and to separate unbound dye from protein bound dye (223). PAPER CHROMATOGRAPHY AND THIN LAYER CHROMATOGRAPHY
To cover the vast number of publications t h a t ha1-e made use of paper chromatography, even within the past year, nould not serve the purpose of the present review. X very few references have been selected for mention. For review discussions, the reader is referred to texts previously mentioned (164, 332) and earlier b u t excellent texts on the subject (39, 614). Improved quantitative methods and more stable ninhydrin reagents nere described in a series of papers from the same laboratory (163, 637, 350). Paper chromatography was used to test the purity of amino acids (119) and t o determine t h e dinitrophenyl derivatives of plasma amino acids (282). Separation of the enol and keto tautomers of aromatic pyruvic acids was accomplished on paper (Sly), as v a s also the separation of trace amounts of strontium and calcium in biological materials (118). High temperature paper chromatography served t o determine lysine, arginine, and histidine (368). Glass paper chromatography was used for the determination of aldosterone in urine (337), of plasma cholesterol is&) , and for the separation of glycerides of mixed fatty acid chain length (273)-the glass paper in this latter publication having been impregnated with silicic acid. Amino acids were separated on polystyrene resin loaded papers (205). A rewrsed phase chromatographic partition of steroids mas successful on a cellulose acetate foil supported on glass (7.5).
Since the original publication on thin layer chromatography (336), numerous applications of this technique have been published. Developments in equipment and application of thin layer chromatography have been recently described (388), and its use in the separation of phenglthiohydantoins of peptides (79) a n d phosphatides and glycolipids has been reported (873). INFRARED SPECTROPHOTOMETRY, FLUORIDIMETRY, TURBIDIMETRY
Recent discussions of infrared techniques (268, 379) and reviens on infrared analysis of carbohydrates ( d o ) , vitamins, hormones, and coenzymes (806), and the infrared absorption spectra of steroids (111) have now been supplemented with a companion volume to the infrared absorption spectra of the steroids (300) and a n indey to infrared absorption spectra covering the period 1945-57 (173). Discussion and interpretation of many compounds of biochemical interest, such as amino acids, proteins, polypeptides, etc., are given in Bellamy’s te\t (24). With respect to infrared methodology, a new sanipling technique has been discussed (2.57), and a method for the incorporation of biological material into agar films in preparation for infrared analysis was reported (8). X rapidly responding narrow beani infrared gaseous carbon dioxide analysis for service in physiological studies was described (17). Infrared also proved of service for the determination of deuterium in biological fluids (9L), for the determination of coproporphyrin isomers I and I11 (106), for the analysis of bile (SO),and for the microdetermination of sulfuric acid esters in sulfatide fractions (387). The dependence of the conformation of synthetic polypeptides on amino acid composition n as also reported (42). Spectrofluorimetric determination of total bile acids in bile (217) and the estimation of diethylstilbestrol in beef liver tissue (142) mere reported. Fluorescent protein tracing has recently been the subject of a monograph (76) and a review (262). This latter review covers later work, the earlier work on fluorescent antibody techniques being considered in a previous review (86). Purification of fluorescent protein conjugates by charcoal and by Sephadex were compared (124), and a new fluorescent label for antibody proteins mas described ( 4 7 ) . It is of interest to note that the initial observation of the localization of tetracyclines in bone (246) gave rise to a n in vivo technique of staining newly formed bone and mineralizing cartilage (130) which could be examined by fluorescent microscopy. Remodeling of bone haversian systems vias examined by this method (136,246).
It was recently reported t h a t oxytetracycline could be detected by fluorescent microscopy, bound to bone (260). An absolute method of turbidimetric analysis (239) and a turbidimetric determination of total serum cholesterol (200) have recently been reported. VISIBLE AND ULTRAVIOLET SPECTROPHOTOMETRY
A good discussion on spectropliotometry is to be found in a recent testbook (57). One of the most interesting reports concerned the principle and performance of a differential filter-photometer which has recently been developed (55). The absorbance difference between two cells is measured and continuously recorded. Full scale on the recorder corresponded to 0.01 absorbance unit, and the stability was such as to permit absorbance differences of the order of 0.0001 unit to be measured. The apparatus was designed to measure continuously the rate of oxidation of glucose catalyzed b y glucose oxidase, b u t both principle of operation and the 1)hotometer itself should have very wide application. A quartz flow cell for continuously monitoring column effluents containing proteins was described ( 7 ) . It was designed for installation in the Beckman DU spectrophotometer and for operation a t ultraviolet wavelengths. Among innumerable applications of spectrophotometry in the visible region, the following seemed of interest. RIethods for determining blood pH (304), bile acid conjugates (art?), cholesterol (179, 827), 17 deoxy-alpha-ketolic steroids (220), Q-alpha-fluoro-hydrocortisone (183), tocopherols ( 3 5 4 , and creatine and glycocyaniine (13.5) were reported. A s p e c t r o p h ot oinet r i c method of determining proteins (259) and a method of identifying aldolieauronic acid, free and in polymers (167), were described. Calcium was determined spectrophotometrically (64, 199, 384). An interesting spectrophotometric determination of amino acids ancl peptides using 2,4,6-trinitro-benzenesulfonic acid (picryl sulfonic acid) was worked out (314). This reagent gave orange colored products with amino acids, and molar absorptivities were of order 12,000-15,000, The use of this reagent might be developed into a n alternate to the ninhydrin reaction for monitoring amino acids in effluents from ion exchange columns providing all necessary reagents could be combined into a single solution. Otherwise a multi-feed type of reagent pump would be necessary. The far ultraviolet absorption spectra (down to 180 mp) of polypeptides and protein solutions and their dependence on conformations were reported (305). VOL. 34, NO. 5, APRIL 1962
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OPTICAL ROTATORY DISPERSION
Optical rotatory dispersion has been the subject of a book (108) and reviews (107, 109, 110), all of which demonstrate the potentialities of this procedure in application to the fields of organic chemistry and the chemistry of natural products. The method is also finding increasing application in examining substances of biochemical interest such as polypeptides and proteins (114 ) , and for studying chemical changes, as for example, following the effect of thermal denaturation of chymotrypsinogen and chymotrypsin (60). A recent publication discusses the theoretical sensitivity and linearity of photoelectric systems for polarimetry (308). Of considerable interest is the report of magneto-optical rotatory dispersion studies (322). Using a magnetic field in conjunction u-ith the optical rotatory dispersion method (a combination of the Faraday effect with the Cotton effect) extends the scope of the method to all molecules whether optically active or not. Full development of this idea ~vould seem to provide another very powerful tool for determining the fundamental properties and characteristics of molecules, but some time can be expected to elapse before such instrumentation is perfected.
tion of calcium and magnesium in urine (586). Electron microscopy as a method was reviewed in a recent text (166). Papers dealing with the routine use of the electron microscope were not sought for this review, b u t two refinements in technique were of interest. The measurement of mass, thickness, and density of the material under examination was determined (68) by the contrast obtained bctween plates made a t four accelerating voltages. The second technique involved negative staining of proteins a t p H values above their isoelectric points. This involved “embedding” virus particles in a n electron dense material under conditions which prevented the embedding material from adhering to the particlrs under examination (63). The \ irus particles were mixed with 1% potassium phosphotungstate at p H 7 . 5 , above the isoelectric point of the proteins. As the phosphotungstate did not stain the particles directly, good contrast \vas obtained. This technique was later extended to proteins a t pH values below the isoelectric point-e.g., belolv p H 5 (62); uranyl acetate was the contrast medium for the examination of hemocyanin.
ELECTRON PROBE X-RAY MICROANALYSIS FLAME PHOTOMETRY, EMISSION AND ABSORPTION SPECTROMETRY, AND ELECTRON MICROSCOPY
Besides the textbook mentioned in t h e introduction (loo), there are two earlier reviews (134, 233) of note. Some current papers of the past year discussed the value of emission spectrochemistry in nutrition research (145) (review), the factors influencing sample flow rate in flame photometry (386), the use of the oxycyanogen flame (303), and a rapid incineration method for the flame photometric estimation of tissue sodium and potassium (67). A flame spectrophotometric determination of strontium and calcium in bone (316) and a n estimation of thallium in urine (338) mere published. It is of interest to note t h a t in a n emission spectrographic determination of trace elements in human tissue (208), the presence of 20 elements was reported. -4 recent discussion of mass spectrographic analysis (160) showed t h a t this method had high sensitivity for revealing impurities in the analysis of solids; the use of this instrument both supplemented and complemented information obtained by emission spectroscopy. Recent advances in atomic absorption spectroscopy were discussed (Sod), and the method was used for the determination of magnesium in biological materials (116) and for the determina-
8R
ANALYTICAL CHEMISTRY
T o date, few papers of biochemical interest have been published in n hich this new analytical tool is used, although its use in other fields, particularly the analysis of metals, is well known and has been the subject of a recent text (34). I n principle, the instrument is a sharply focused electron microscope, whose 1 micron beam can be used to excite atoms to emit secondary x-rays. Analyses of the kind and intensity of x-rays emitted reveal the nature of the atoms activated. Two papers in a recent symposium illustrated its usefulness. The first paper (68) described the method of automatically scanning a thin section of cartilage from the epiphyseal plate of a rat tibia to determine the calcium distribution. It \vas shown that the calcium concentration decreased from about 30% in the calcification zone to 0% toward the maturation zone. I n the second paper (352), the presence and distribution of copper and iron in the cornea within Descement’s membrane were studied in eye specimens from two cases of Wilson’s disease. The distribution of silver in tissue from a case of argyria was examined, and the iron content of red blood cells and other tissues was measured. Similar experimental work with the electron probe analysis has also been reported (241) in a recent publication from another laboratory.
ELECTRON SPIN RESONANCE, NUCLEAR MAGNETIC RESONANCE
The application of electron spin resonance measurements to problems of biochemical interest could hardly receive a more impressive summary than that provided in a text embodying the proceedings of a recent symposium on free radicals in biological systems (40). I n each of 29 well organized chapters, the materials, methods, and results of ESR studies were recorded. The topics include free radicals in a number of enzyme substrate systems, resonance in polyenes, irradiated proteins, amino acids, peptides, thiols, disulfides, free radical formation in photodynamic and photosynthetic systems, etc. Concern is also given to development of new apparatus, t h e precision of free radical measurements, and the interpretation of electron resonance data. Application of nuclear magnetic resonance to biochemical problems seems a t present limited. The theory, experimental methods, and general applications have been presented in a n earlier review (149), and major developments in K l I R instrumentation have recently been discussed (266). A preliminary evaluation of nuclear spin resonance in the analysis of fatty acids (339) indicated t h a t the method could possibly be developed further for this purpose. Suclcar magnetic resonance spectroscopy of proteins also gave evidence of hydrogen bonding of water in solutions of bovine serum albumin (49). KAIR studies of aqueous solutions of DSA (162) indicated n-hich metal ions were bound to interior and nhich to exterior sites. The method employed 11 as considered generally applicable to other problems of paramagnetic ion bonding to large molecules. MASS SPECTROMETRY, X-RAY SPECTROMETRY, AND X-RAY DIFFRACTION
Instrumentation, applications, and recent developments in mass spectrometry were revielved (301). High resolution mass spectra of aliphatic esters were reported ( 3 1 ) , and quantitative analysis of amino acids as their ethyl esters by mass spectrometry n as described ( 3 2 ) . X-ray spectrometric analysis continues to serve for the determination of elements in biological materials, a s evidenced by a paper on the determination of strontium in human serum and bone (265). X-ray diffraction methods were used for further studies on the structure of collagen (229‘7) and on the structure of the protein matrix of bovine dental enamel (140). Porphyrins were identified by the x-ray diffraction powder method (203).
RADIOCHEMICAL DETERMINATIONS
The new journal (97) devoted to this field was mentioned earlier; recent texts are also of interest (9,56) ,as well as a general discussion on radioisotope techniques for chemistry students (263). Recent developments in liquid scintillation counting of biochemical samples 11ere discussed (QY), counting statistics for liquid scintillation counting were described (IYf), and a simple efficient liquid scintillation counter for aqueous solution was reported (51). A plastic spiral was used as counting chamber for monitoring the eluent from a chromatographic column (321); weak beta rays from S3j, CI4, and H3 were monitored. Tritium n as determined in urine (70, 311) by liquid scintillation counting method.. Carbon-14 and Paz I\ cre also determined in animal tissues and blood by a liquid scintillation method (16) and C14 a t high counting efficiencies in animal tissues (61). Carbon-I4 in aqueous bicarbonate solution was determined by liquid scintillation counting and applied to biological fluids (63), and a micromanometric ninhydrin-carbon dioxide method in v hich very small samples of radioactive carbon dioxide were transferred to counting tubes n a s reported (367). Other radiochemical determinations follon ed the distribution of radiosulfate in blood and cerebrospinal fluid (29S) and the amount of radium in ui iiie (37s). Application of neutron activation to biochemical work was studifd and a n ultramicromethod was dcscribed for the determination of manganese in blootl and tissues (2%’). hlnngancse n as also determined in biological materials by st~lertiveneutron activation and triple coincidmce counting (4s). INSTRUMENTAT!ON AND AUTOMATION
To the texts already referred to on insti unientation (112, 207, 329, 34R), three others should be listed (22, 103, 383), as n ell as a n interesting discussion on the automatic determination of clinic.ally important steroids (26G), on automatic refractometers ( Z j Z ) , and on new instrummts for the determination of carbon and liydrogen (254). Progrcss in instrumrnt. and equipment for microanalytitd n ork n as rex k e d (12.9). Automation tleieloped for the study of continuous reactions by refractive index measurrments (.95G), for automatically recording mclting points ( 2 7 ) , for titiation of chloride in biological fluids @ I ) , and for recording excitation spectra (330) n as reported. Automatic nieasuremtiit of enzyme actii ity was follon-ed (35, 318, S??) on a continuous basis. Automatic analysis of amino acids has already been referred to in the sections on ion exchange and gas chromatography; the analysis of condensed
phosphate mixtures separated on ion exchange chromatographic columns was also reported (228). A highly accurate continuous recording differential refractometer has also been described (361).
T h e automatic repetitive scanning
amined ( 7 7 ) ; implications for the state of the organization of the light accepting pigments were obtained. LITERATURE CITED
(1) ACS
14th -4nnual Summer Symposium, Cleveland, Wilkins, D. H., Chairman, ASAL. CHEY. 33, 1844
of absorption spectra of steroid hormone analysis (210) and a recently described steroid analyzer (6), using column chromatography, automatic analysis, and recording, is truly a remarkable piece of equipment. One of the very interesting developments was instrumentation for continuous Kjeldahl analysis for nitrogen (125). For automation applied to biochemical and clinical determinations, the numerous excellent papers on automatic chemical analysis presented a t a recent symposium (132) should be consulted. Papers on this symposium that might be singled out for further emphasis mere concerned IT ith automatic analysis of ammonia in biological materials (296), of amino acids (389), of carbon dioxide in serum (331), and of blood glucose in vivo in human subjects (381), and the general problem of automatie chemical analysis in vivo as a n approach to chemical pharmacology in vivo was reviewed (126). Elsewhere the automatic analysis of tissue culture proteins with stable Folin reagents was also described (392). MISCELLANEOUS
Under this heading, a f e n pertinent b u t unrelated topics will be mentioned. Attention is called to a series of papers reviewing the application of electronics to medicine of n hich reference is made herein to only one of these (69). It is also worthwhile to point out excellent reviews o n enzymes (219) and carbohydrates and metabolites (264) as considered from the point of v k v of the niicrochcmist. Again, in the province of microchemistry, reference should be made to microanalysis by the ring oven technique (380), a test, and a modification of this procedure for the estimation of microgram amounts of protein (123). Two papers on modified manometric vessels for special studies should be noted (238, 374), the latter paper describing a vessel designed for measuring microamounts of carbon dioxide. d new falling velocity method of density determination for small solid samples was described (SOT), and the use of a new ultrasonic generating apparatus for extracting microorganisms was reported (180) in a hich the organisms flon ed continuously through a n area of high energy density. Light scattering as a means of determining the interaction between gelatin and plasma proteins a as discussed (E%$), and the spectral dependence of scattering from a spherical algae was ex-
(1961).
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%
~
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~
~
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(127) Fine, J. hl., Uriel, J., Pathol. et bzol. Senaazne h6p. 6,1553 (1958). (128) Florsheim, JV. H., Gonzales, C., Proc. Soc. Erptl. Uiol. Jfed. 104, 618 (1960). (129) Francis, H. J., Jr., Hetherington, J. F., Mrcrocheirz. J . 5, 509 (1961). (130) Frost, H. hI., 1-illaneuva, h. R., Roth, H., Stazn Technol. 35, 135 (1960). (131) Fryer, J. J., Habgood, H. m., Harris, IT, E., CHEM.33, 1515 (1961). (132) Furness, F. I., ed., “Automatic Chemical ilnalysia,” Vol. 87, Article 2, pp,, 609-953, X e w York Academy of Science, S e w York, 1960. (133) Furness, F. S . , ed., “Automatic A
%
~
~
~
Chemical Analysis,” Ann. A’. Y . Acad. Scz. 87, Art. 2, 1960. (134) Gardiner, K. JV.) “Physical Methods in Chemical Analysis,” Bed, V. G., ed., Vol. 111, pp. 220-76, Academic Press. Kew York. 1956. (135) Gerber, G. B., Gerher, G., Altman, K. I., A X A L . CHEJZ.33, 852 (1961). (136) Ghosez, J . P., Arch. biol. (Lzdge) 70, 160 (1959). (137) Gidtiings, J. C., ASAL. CHEJI. 33, 962 (1961). (138) Gittens, G. T., James, -4. M., Anal. Uiocheni. 1, 478(j1960). (139) Glick, I)., ed., Methods of Biochemical Analysis,” Vol. VIII, Intersciencr, 1960. (140) Glimcher, >I. J., Bonar, L. C., Daniel, E. J., J . J l o l . Biol. 3, 541 (1961). (141) Glueckauf, E., Katts, R. E., .Tofure 191. 904(19(jl). (142) Goodykar, J. hI.,‘Jenkinson, S . R., A N A L . CHEJI. 33, 833 (1961). (143) Orahar, P., “Methods of Biochemical Ainalysis,” Glick, D., ed., Vol. VII, pp. 1-38, Interscience, New Tork, 1959. (144) Gr:tbnr, P., Burton, P., “hnalyse Immuno-Clertrophor&ique,” Slasson et Cie., Paris, 1060. (145) Grant, C. L., Science 134, 1207 (1961). (146) Gross. D.,J. Chromalog. 5,194(1961). (147) Grunbauni, B. \V., Fessel, IV. J., Piel, C. F., ~ A L .CHEM. 33, 860 1 1961). (138) Grunhaum, B. W.,Kirk, P. L., Atchley, JV. A , , Ibid., 32, 1361 (1960). (149) Gutowsky, H. S.,“Pkysical RIethods in Chemical Analrsis, Berl, W.G., ed., Vol. 111, pp. 304-79, Academic Press, New York, 1956. (150) Hanhti, E., Vanden Heuvel, IT-. J. A,, Horning, E. C., Anal. Biochem. 2, 182 (1961). (151) Hanrhoff, P. C., I’retorius, V., J . S. d f r i c a n Cheni. Inst. 14, 22 (1961). (152) Hagishnra, E., Biochim. et Biophys. Acta 46, 131 (1961). (153) Hnkomori, S.,Kohkichi, T., S a f z i r e 190,265 (1961). (154) Hamer, \TI. J., “The Structure of Electrolytic Solutions,” Kiley, S e w York, 1959. (155) Hamilton, P. B., A S A L . CHEAf. 30, 914 (1058). (156) lhid., 32, 1779 (1060). (157) Hamilton, P. B., unpublished esperinients, 1961, A. I. duPont Inst., Wilniington, 1)el. (158) Hamilton, P. B., Anderson, R. A, -%SAL. CIIEJI.31, 1504 (1959). (159) Hamilton, P. B., Bogue, D. C., Anderson, R. ri., Ibid., 32, 1782 (1960). (IGO) Han~iny,9.B., Science 134, 1220 (1961).
(161) Hnnnig, I-,” 2nd ed., Reinhold, S e w Tork, 1959. (196) Ilieselbach, R., ASAL. CHEW 33, 23 (1961). (197) Ibid., p. 806. (198) Kingsley, G. R., Ibicl., p. 13R. (199) Kingdey, G. R., Robnett, O., Ibid., p. 552. (200) Ibid., n. 561. (201) Kitzinger, C., Benzinger, T. H., “Xethods of Biochemical i2nalysis,” Glick, I)., ed Vol. VIII, p. 309, Interscience, Sew q o r k , 1960. (202) Klein, P. D., AXAL. CHEY. 33, 1737 11961). (203) Klesper, E., Corn-in, A. H.. Iber, P. IC., Ibid., p. 1091. (204) KnaufY. H. G., dchahert, P.. Klin. ITochschr. 38. 1206 (1960). (205) Knight, C. S., LYature 188, 739 (1960). (206) Knox, J. H., J . Chem. Soc. 1961, 433. (2ll7) Koch, von H., Ljungberg, G., eds., Instruments and Measurements,” 1-01s. ~
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VOL. 34, NO. 5, APRIL 1962
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Review of Fundamental Developments in Analysis
Chromatography Erich Heftmann, National Institutes of Healfh, Bethesda, Md.
T
review covers the period since Strain’s last review in 1960 (1143) and includes only the articles which have come to the author’s attention before December 1981, exclusive of routine applications of known methods. HIS
LITERATURE
Except for the book by Martin and James (726), all current monographs on the general topic of chromatography (209, 3213, 467, 660, 1097) have been written, characteristically, by more than two authors. This also applies to a comprehensive treatise on paper chromatography (437). I n addition to these books, a number of review articles on chromatography in general have been published in the last 2 years (222, 2613, 419, 4713, 641, 765, 769, 1070, 1143-5, 1270). R e v i e w of more limited scope have dealt with such topics as the separation of stereoisomers (522) and radioactive compounds (977), or the application of chromatography to clinical analysis ( 4 l 8 ) , or t o genetics and taxonomy (165). Other reviews will be cited under specific headings.
HISTORY
The history of chromatography has been outlined by Zechmeister (1303), and Grune (416) has written about capillary analysis, a predecessor of paper chromatography. A biographical sketch of Tswett has also been published (976). It should be recorded here t h a t the first and second Labline Awards in Chromatography and Electrophoresis have gone to Strain (204) and Zech-
meister ( I 4 ) , respectively. Both men have made outstanding contributions t o our knovledge of chloroplast pigments by the judicious application of Tswett’s method. THEORY
Giddings has been considering kinetic processes in chromatography (381, 674) with special emphasis on zone diffusion (580) (cf. also 113, 581, 605). Corrections for oyerlap in elution curves
Chromatography The rapid growth in various aspects of chromatography has been evident for the past several years. This, in turn, has presented problems in preparing reviews in this field. In 1960 the editors presented two reviews: chromatography b y Harold H. Strain and gas chromatography by Stephen Dal Nogare. In 1962, on the basis of discussions with experts in the field, it was decided to break the topic down as follows: chromatography (except electro, gas, and ion exchange chromatography), electrochromatography, gas chromatography, and ion exchange chromatography. The authors are: Erich Heftmann whose chromatography review covers solid-solid adsorption, liquid-liquid partition, Molecular Sieve processes, etc. R. P. Strickland has prepared the review on electrochromatography; Stephen Dal Nogare, gas chromatography; and Robert Kunin, ion exchange chromatography.
VOL. 34, NO. 5, APRIL 1962
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