ANALYTICAL CHEMISTRY Kebbia, L., Guerrieri, F., Chimica e industria ( M i l a n ) 35, 896-9 (1953). Neu, R., 2. anal. Chem. 143, 254-7 (1954). ANAL.CHEY.26, 600 (1954). Oita, I. J., Conway. H. S., Oliver, F. H., Analyst 80, 593 (1956). 27, 1177-8 Owens, 11. L., Jr., hIaute, R. L., ANAL. CHE~I. (1965). Pasini, C., Vercellone, b.,Z . anal. Chem. 143, 172-7 (1954). Perlin, A. S., ANAL.CHEX 26, 1053-4 (1954). Pesez, &I., Bull. soc. chim. France 1954, 1237-8. Peters, E. D., Jungnickel, J. L., ANALCHEW27, 450-3 (1955). Zhur. A n a l . K h i m 8, 302-5 (1953). Polyakov, K.K., , 64, 1913 (1931). Ponndorf, W ~ Ber. Potterat, 31., Eschmann, H., M z t t . Lebensm. H y g . 45, 312 (1954). Prillinger, F., M i t t . Hoheren Bundeslehr- 7 1 . T’ersuchsanstaZt Wezn- u. Obstbau Klosterneuburo, Hoheren Bundeslehr- u. T’ersuchanstalt Bienenkunde Wien Grinzino 2. -, 20 (1962). Radford, A. J., Analyst 79,501-4 (1954) Reifer, I , Tarnowska, K , Przemysl Chem 31 ( 8 ) , 58 (1952). Reznikov, I. G., Farber, E. L., Maslobotn- Zhiroraya Prom. 18, SO. 5, 13-16 (1953). Ricciuti, C., Coleman, J. E., Willits, C. O., ANAL. CHEM.27, 406-7 (1955). Roudier, ~ A . Eberhard, , L., Mens. serbices chini. &at. ( P a r i s ) 36, 383 (1951). Ibid., 37, 227 (1952). Sakakibara, S.,Komori, S . , J . Chem. SOC.J a p a n , I n d . Chem. Sect. 56, 429-30 (1953). Salomaa, P., S o r d . Kemistmotet Helsingfors 7, 201-2 (1950). Salomaa, P., Suomes Kemiastilehti 27B, No. 2, 12-14 (1951). Sant. B. R.. 2. anal. Chem. 145. 267-60 (1955). Satterfield, C. N., iT7ilson, R. E., Le Clair, R.’lI., Reid, R. C., ASAL. CHEM.26, 1792 (1954). Schivizhoffen, E. Y., Dana, H., 2. anal. Chem. 1 4 0 , 8 1 4 (1953). Schonfeldt, N.. h’ature 172,820 (1953). Schoniger, W., dfikrochim. Acta 1955, 123-9. Schomberg, XI., Compt. rend. acad. agr. France 40, 271-3 (19543. Schormuller, J , Walter, J., 2 . anal Chem. 134, 337-53 (1952) Schulz, 6. F., ANAL.CHEX 25, 1762-3 (1953) Segal, W., Starkey. R. L., Ibid., 25, 1645 (1953). Shostakovskil, AI. F., Bogdanova, A. Y., Z h u r . A n a l . Khim. 8, 231-4 (1953). Shostakovskii, 11. F., Sidel’kovskaya, F. P., Ibid., 9, 105-S (1954). Siegel, H., U‘eiss, F. T.. ~ X A L CHEM. . 26, 917-19 (19641. Siggia, S.,Ibid., 19, 972-3 (1947). Siggia, S.,Edsberg, R . L., Ibid., 20, 938-9 (1948). Siggia. S., Stahl. C. R., Ibid., 27, 550-2 (1955). Sjostrom, E., Acta Chem. Scand. 7, 1392-4 (1953). Skell, P. S.,Crist. J. G., .Vatwe 173, 401 (19*54).
-
REVIEW OF FUNDAMENTAL DEVELOPMENTS IN ANALYSIS
~I
I 1
I Biochemical Analysis
’’
I
B
(138) Smirnov, 0 . K., Beahentseva, V. M., Zavodskaya Lab. 21, 414 (1955). (139) Spencer, D., Henshall, T., A n a l . Chim. Acta 11, 428-30 (1954:. (140) Spencer, W. R., Duke, F. R., ANAL.CHEM.26, 919-20 (1954). (141) Spliethoff, W. L., Hart, H., Ibid., 27, 1492 (1955). (142) Stakheeva-Kaverzneva, E. D., Salova, A. S., Zhur. A n a i . K h i m . 8, 365-9 (1953). (143) Stalcup, H., Williams, R. W,, A N ~ LCHEX . 27, 543 (1955). (144) Staudinger, H., Siessen. G., Chem. Ber. 86, 1223-6 (1953). (146) Stepanov, B. I., Sergienko, V. A., T r u d y Komissii A n a l . K h i m . , A k a d . Nauk S.S.S.R. Otdel. K h i m . Nauk 5 (8), 274 (1954). (146) Sternglanz, P. D., Thompson, R. C., Savell, W. L., AN\TAL. CHEAI.25, 1111 (1953). (147) Suda. K., Yamamoto, S., Kusagawa, T., Research Repts. Fac. Textiles and Sericult., Shinshu Univ. 1, 63-6 (1951). (148) Svirbely, W. J., Roth, J. F., ANAL.CHEX 26, 1377-8 (1964). (149) Swan. J. D.. Ibid.. 26. 878-80 (1954). i15Oj Takahashi. T.. Kirno;o. K.. Kimoto. 11..J. Chem. SOC. Ja~an. I n d . Chem. Sect. 55, 283-5 (1952) (151) Takahashi, T., Kimoto, K., hlinami, S., Ibid., 55, 805-6 (1952). (152) Ibid., 56, 491-3 (1953). (153) Takahashi, T., Kimoto, K., Takano, Y., Ibid., 56, 571-3 (1953). (154) Takeda: K.,Senda, J., .Yogaki K e n k y u (Rept. Ohara Inst A g , . B i d ) 41, 97-118 (1964). (155) Takiura, K., Takino, Y , J . Pharm. SOC.Jupale 74, 971-4 (1954). (156) Tanaka, Y., Ibid., 75, 653-5 (1955). (157) Terent’ev, A. P., Zabrodina, K. S., Doblady A k a d . S a u k S . S. S. R. 95, 85-7 (1954). (158) Tomicek, O., Krepelka, S., Chem. Listy 47, 526-30 (1953). (159) Tomicek, O., Vidner, P Ibid., 47, 521-5 (1953). (160) YanEtten, C. H., Wiele, 11. B., ANAL.CHEY 25, 1109-11 (1953). (161) \-iallard, R., Corval, 11.. Dreyfus-Alain, B., Grenon, 11.. Hermann, J., Chim. anal. 36, 102-4 (1954). (162) Wagner, P T., Lew, AI., .\SAL. CHEM.26, 575 (1954). (1631 Katanabe. H.. J . Chem. SOC.J a v a n . Pure Chem. Sect. 76. 1-3 (1955). (164) White, T. T., Penther, C. J., Tait, P. C., Brooks, F. R., . I N ~ L . CHEM.25, 1664 (1953). (165) Wickbold, R., Angew. Chem. 66, 1 7 3 4 (1954). (166) Wilson, H N.,Peterson. R. h l . , Fitagerald, D. lI.,J . A p p l . Chem. (London) 4, 488-96 (1954). (167) Wojahn, H., Arch. Pharm. 286, 278 (1953). (168) Kojahn, H., Wempe. E., Ibid.. 285, 375-82 (1952). (169) Tamagishi, AI., Tokoo, 11.. Inoue, S., J . Pharm. SOC.J a p a n 75, 351-3 (1955). (170) Tudasina. A. G., Yysochina, L. D., IYauch. Zapiski Dnepropetrocsk Gosudarst I-niv. 43, 53-6 (1953) ; Referat. Zhur. K h i m . 1954, S o . 15.061.
EDWARD L. DUGGAN D e p a r t m e n t o f Physiological Chemistry, University o f California M e d i c a l School, University of California, Berkeley, Calif.
EFORE proceeding to the review of major developments in
biochemical analysis for the past 2 years, i t is well to state the general requirements and restrictions of biochemical analysis, in contrast t o those of analytical chemistry “Biochemical analysis is inherently a more complex and broad field than classical chemical analysis. Because the major activitj- of the biochemist is always to find out what and how much, biochemical analysis actually miist include virtually every biochemical technique” (7’3). Because the biochemist directs his analysis toward the understanding of structure and content of biological units, his analJ ses
must be accomplished on ssniples JThich vary enormously in interfering substances, such as proteins, nucleic acids, and lipide. Rarely can an analysis be transposed without modification from analytical chemistry into biochemistry; this is apparent even in the case of the relatively simple technique of flame photometry. The biochemist manifests proper concern with fractionation and sample preparation before analyses are attempted, as the analyses have meaning only in their biological contest. Thus, developments in fractionation procedures are eagerly accepted, whether the fractionation is that of simple molecules, m a c omolecules, or cellular particulates.
V O L U M E 28, NO. 4, A P R I L 1 9 5 6 The recent development of methods for disassembly and reassembly of the molecular units of collagen ( 5 2 ) , and a similar reassembly of isolated nucleic acid and protein subunits t o form infectious tobacco mosaic virus ( 4 6 ) , are of extreme importance. The breakdown of other macromolecules t o molecular subunits would ease the restrictions on present attempts t o relate analytical units (amino acid, nucleotide, etc.) t o polymer structure. One consequence would be to expect the amino acid analysis of the virus protein to fit a subunit array of 104 weight, rather than the unmanageable total protein weight of more than a million. Another consequence is that the hydrophobic residues and charged groups of such proteins cannot be arranged in random fashion; the concentration of charges. especially, must be stereospecific t o allow polymerization t o occur. Thus, the directed crystallization of protein molecules, such as collagen, must result from highly “ordered” shapes and surfaces of the fundamental units. A development represented by the findings described above is that the electron microscope has matured as an analytical instrument. I t now appears the inst,rument of choice for examination of biological macromolecules, prior t o the use of the analytical ultracentrifuge, or light-scattering, The review period presents a picture of maturation of ion exchange, paper chromatography, and zone electrophoresis. S e a methods of separation or analysis have been less apparent, greater emphasis being placed upon the correct use of consecutive fractionation methods folloived by simple analysis. A great tendency exists a t present for workers t o consider all chromatography as incidental to the biochemical study concerned. .4ny scheme of fractionation is the servant of the process of discovery of biochemical concepts. However, the way of a reviewer is eased if brief attention is paid to novel fractionations or techniques, as incidental findings. Instrumental analysis continues t o emphasize absorptiometry. Titrimetry is neglected, with little usage of potentiometr>-, amperornetry, or high frequency end points. Coulometry or polarography is rarely used. Respirometry, with the techniques of Warburg or Holter and Linderstrom-Lang, is used only when less sophisticated procedures have failed to yield the desired d a h .
PREPARATIVE METHODS CHROMATOGRAPHY AND ION EXCHANGE
Chromatography continues to bring ease and convenience to fractionations in every field of biochemical research. Adsorp tion chromatography, partition, and ion exchange have achieved various degrees of success. Universal use of paper partition chromatography occurs throughout the biological sciences. Ion exchange is perhaps second in importance, as it allom a rational separation of contanlinants, or the fractionation of similarly charged isomers or related compounds. The burden of review of these fields is eliminated by the recent appearance of a variety of monographs (8, 18, 26, 23, 85, 97, 145) which correlate past work and project the future advances of chromatographic methods. The prior developments in the field of ion exchange are revien-ed by Lederer and Lederer (85), in the previous reviem- (?’,$), and in the practical manual by Brimley and Barrett (18). In the experience of this reviewer, the number of papers devoted to Separations or new uses of resins has fallen to a small fraction of the papers appearing during 1952-54. Absorbents and Resins. The utility of chromatographic separations is completely dependent upon the existence of materinl which allows the desired separation, whether the separation uses reversible adsorption, partition between stationary and mobile phases, or ion exchange. Paper chromatography and ion exchange suffice for the major separations of amino acids, carbohydrates, or nucleotides. The use of natural adsorbents is minor a t the present time. Silica gel or alumina is used as column adsorbent in a few specialized separations of steroids (20, 55, 82) or carboxylic acids ( 1 9 .
715 121). The volatile organic acids d l be separated in the future by gas phase chromatography, rather than by partition on silica. A new adsorbent for mixtures of polystyrene or poly(viny1 acetate) is urea ( 6 9 ) , with benzene as the developing solvent. Though the process is of no immediate use t o biochemists, a certain interest in the work remains. The question arises: Is it possible to form a hydrocarbon-specific urea complex, capable of repeated use in column separations, along the lines described by Marschner (91)? The resins in major use in ion exchange eontinue to be the commercially available styrene-base resins, either sulfonatede.g., Dowex 50 X 8-or containing a particular quaternary amine group (Dowex 2 X 8). The designation X 8 identifies the percentage of divinylbenzene in the original polystyrene latex. This degree of cross linking allows approach of small ions, without, not,iceable quantitative adsorption of proteins or other macroions. A popular resin for protein separations continues to be the self-buffering carboxylic resin, IRC 50, of unspecified resin matrix. Attempts to produce resins active in oxidation-reduction continue, the latest product being a polythiol resin, prepared from polystyrene (50). Resins of great interest to biochemists are formed by treating cellulose so as t o introduce a number of phosphoryl groups for cat,ion exchange, or esterified taurine (free monoalkylamine groups) for anion exchange (70). Phosphorylated cellulose in the form of an endless cotton belt has been proposed as a continuous cation exchanger ( 103). Finely powdered cellulose has been modified by Sober and Peterson (126) to provide either anionic carboxyl or cationic amine groups. These workers are proceeding toward “resins” specifically t.ailored t o allow the ion exchange of proteins or other macro ions. The preliminary results obtained by several grorips of workers are described in a following section. Amino acids may now be fractionated by gradient elution from the n e v Dowex 50 X 4 resin (101), or on Zeokarb 225 ( 2 1 ) . Both resins have sulfonate groups and a polystyrene base, though the latter has unspecified cross linkage. Hirs, Moore, and Stein (56) describe a preparative fractionation of amino acids which uses both Dowex 1 X 8 and Dowex 50 X 4, with acetic and hydrochloric acids as eluents. The volatile acids simplify the later steps toward preparat’ion of pure amino acids. A disturbing finding, not yet confirmed, has been the discovery of partial s e p aration of carbon-14 amino acids from their natural counterparts, during fractionation on a Dowex 50 column (107). Solute Recovery Techniques. The common method of solute recovery, in methods based on adsorption or partition, is the use of solvent mixtures of an indifferent solvent and a specific solvent. d useful procedure is that of continuous variation of solvent composition, gradient elution. Gradient elution using solvent pairs is a common procedure ( 2 , 15, 47, 74, 86, fdf). The technique may also be used to supply graded increases in acid ( 19 ) or buffer electrolyte concentration ( 101 ) in ion exchange. Drake (36. 38) indicated, from theory and experiment, t h a t gradient elution need not increase resolution between peaks of material, although the peaks are sharpened and more symmetrical. Column Monitoring. Anyone attempting column chromatography of amino acid or nucleotide mixtures is soon awire of the necessity for rapid proximate analysis of the multitude of fraction volumes obtained, before serious analysis is possible. If the peaks may be identified by any method, the number of serious analyses may be cut by 2- to 100-fold. Ideally, one method should suffice for the location of all volumes representing a “peak,” folloR-ing which these volumes could be combined, adjusted to k n o m volume, and analyzed for the actual average solute concentration. In preparative work, it is sufficient t o judge the identity and limits of a particular peak, thus collecting the pure solute for further purification or crystallization. Light absorption, in the visible or ultraviolet, remains one of the most sensitive and convenient methods of monitoring col-
716 umn output. One of the most ambitious attempts to separate nucleotides routinely, using adsorbance ratios for 260/280 mp, is that of Deutsch, Zuckerman, and Dunn (35). These workers relied on electronic decision for delivery of the nucleotide fraction to the correct receiver. For the analysis of ribose nucleic acid, for example, the curve of absorbance os. fraction number obtained from the monitor would represent the quantities of nucleotide present, after integration under the peaks (26). Some correct’ion for background absorbance may be necessary before integrat,ion. These analyses are unique in their simplicit)once precise fractionation is obtained. As nucleoside monoand polyphosphates are important compounds in carbohydrate metabolism and nucleic acid synthesis, such fractionations continue t’o provide information of dynamic importance (59, 118). Khere absorbance measurements are impossible or inconvenient, monitoring is sometimes possible on the basis of increments of refractive index (68, 138), conductance (34, 37), p H (64,66,147), or dielectric constant (85). Unpublished work in this laboratory, as well as recent. reports (25, 154), indicates that the fraction collection and analysis may be simplified, on a small “pilot” column basis, a t least. The innovations consist of collection of one or several drops of eluted fract,ion in a single area of filter paper t,ape. The tape may be continuously moving (25), or a number of drops may be collected and evaporated in one region, the paper moving discont,inuously (40). Following collection, the amino acid spots ma)- 1~ detected with ninhydrin treatment and color development in a hot paraffin bath (25). If nucleotides are fractionated, the regions of interest may be detected by ultraviolet reflex photography (40). Det,ection of radioactive peaks on such paper tapes is possible, using techniques already developed for paper chromatography (42, 53, 88, 144). CHROMATOGRAPHIC SEPAR.4TION OF PROTEINS 4 S D h-UCLEIC ACIDS
I n keeping with the present trend t o focus once more upon the solutes being fractionated, rather t,han on detail of fractionation, d a t a bearing on the purification of several natural macromolecules are grouped together. I t xyas reported in 1954 ( 7 4 ) that only ribonuclease, lysozyme, and one cytochrome had yielded t o fractionation. Ribonuclease was capable of isolation and fractionation by either partition or ion exchange. The other proteins were adsorbed and eluted from the carboxylic resin, I R C 50. Some purificat.ion of thrombin occurs during similar treatment (111). Separation of h - 0 bovine hemoglobin derivat,ives, the carbonyl derivative and the oxidized methemoglobin, is also possible (10). The rational separation of histones on the barium ion form of the same resin has been accomplished by Crampton, Moore, and Stein (SO). These aut’hors find three types of histone from calf thj-mus which can be identified on t’he basis of arginine or lysine content,. Several similar histones exist in calf liver, or in guinea pig testes. In one of the first report,s on the use of the resin, treatment with Dowex 2 has resulted in purification of several phosphat,ases (11). Partition on Celite has separated various immune globulins from bulk yglobulin (108). Partit.ion on silane-treated Celite has yielded purified insulin A and B polypeptides ( 3 ) . Fractionation of a variety of dissimilar prot,eins has been attempted by Tiselius (136, 137). He used a simple calcium phosphate gel, and prot,eins ranging from egg albumin to tobacco mosaic virus. The results are preliminary, for simple known mixhres, but they promise a separation method for proteins. Tiselius regards the method as a “gentle” one, as no organic phase is present in the adsorbent. It may be possible to form the gel about molecules of a desired protein, so as to mold the gel for later specific use. The synthesis of several cellulose derivatives as adsorbents for prot,ein purification has been reported by Sober and Pet’erson (1d6). These workers find that their low-capacit,y adsorbents are equipped to provide separation of numerous enzymes from
ANALYTICAL CHEMISTRY heart’, spleen, or kidney extracts. They also report (106) that the various serum proteins are separated. Pet.erson, Wyckoff, and Sober (106) recognize an element of ion exchange in t’hese fractionat’ions, although adsorption by hydrogen bonding must also operate. These new “resins” have been used also in nucleic acid separations, as described below. Many xorkers in nucleic acid chemistry consider that t,he ribose ( R S A ) and deoxyribose ( D N A ) nucleic acids are classes rather than individuals. Until the present time, nothing approaching a separation of individual ribose or deoxyribose nucleic acids has been reported. The preliminary reports (6, 1 4 ) may represent a beginning t o such fractionations. The first workers ( 1 4 ) used a cellulose amine resin formed from cellulose, epichlorohydrin, and t’riethanolamine, synthesized by Sober and Peterson (126). The amino groups are contributing a certain amount of ion exchange; capacity may be as low as 0.2 meq. per gram. Various ribose nucleic acid specimens provided a different spectrum of eluted fractions, with recoveries of 90 to 100% of the ribose nucleic acid taken ( 1 4 ) . Comparative fractionat’ion attempts for deoxyribose nucleic acid on Dowex 1, Amberlite 4B, and two modified celluloses have sholvn the superiority of the same cellulose amine resin (6). Thus far, the only test applied is the reversibility of adsorption of the long fibrous deoxyribose nucleic acid molecules to the resin. The first true fractionat,ion of one deoxyribose nucleic acid molecule from anot,her may be realized during the purification of a “transforming principle,” a deoxyribose nucleic acids of pneumococcal origin which exerts a genelike action on ot,her pneumococci. Here, success in fractionat,ion of deoxyribose nucleic acid will be demonstrated, if increased biological activity is achieved through fractionation of analyt.ically similar deosyribose nucleic acids. Some purification of one “transforming principle” has been achieved using a column of Celite-albumin mixture as t,he adsorbent (87). PAPER CHROMATOGRAPHY
The review of this field 2 years ago required consideration of hundreds of articles, just to outline the invasion of all fields of biochemistry by the technique. One may report a t this time t,hat the diverse growt,h of the method is complete; few chemical compounds refuse to separate by the technique, n-ith the exception of the proteins and nucleic acids. The apparatus used may vary from a covered glass jar to a shiny Chromatocab. The allglass or polyethylene atomizers for the inevitable spraying of corrosive test reagents are now cheap and commonplace. Conipounds may be chromatographed on one of several filt,er papers, with one of many solvent mixtures, as the native compound, or as a derivative. The experience in this field is now amply described by monographs or manuals ( 8 , 29, 85, 89, 97, 145). A fen- recent articles on paper densitometry or detection of zoncs are reviewed. The natural desire to extend paper chromatography from isolstion-identification to an absolute det,ermination has not been realized. The complexity of absorbance measurement directly on filter paper is outlined by Price (109). Densitometers of several types are available, finding use also in the techniquerelated field, paper electrophoresis. Densitometry does suffic$e t,o establish appr0ximat.e quantity and relative concentration of plasma proteins, after electrophoresis and staining (8, 28, 4,9, 67). The technique may also serve for the location and identification of ultraviolet-absorbing compounds, such as the barbiturates ( Y l ) , or purines and adrenal steroids (110). RIcReady and l l r Comb (93) suggest that reflectance, rather than absorbance, may he used for the est,imation of carbohydrates after chromatography. They use a commercial densit,ometer and attain a precision of 574 in the determination of 50 y of the common hexoses. I t is seldom possible t o detect zones on a chromat,ogramwithout the destruction of the compounds involved. Intrinsic light absorption provides an exception to this rule. Two new methods
V O L U M E 28, NO. 4, A P R I L 1 9 5 6 lmsed on capacitance or conductance are also capable of locating zones without, destroj-ing the compounds or ions responsible. Blake ( 7 ) suggests the application of radiowaves a t 1-mc. frequency to the paper by two electrodes to outline the zone. The current passed by the electrodes is rectified and read on a suit’able meter. The method has general application where electrolytes or polar compounds are concerned. An alternative method has Ixen proposed for detection of electrolytes in inorganic separations. This method ( 3 5 ) suggests passage of direct current between rollers Khich come in contact n.ith the chromatogram, GAS PARTITION CHROMATOGRAPHI-
This technique extends the advantages of chromatography t.o niicro samples of compounds commonly separated by complex distillation procedures. The subject was reviewed briefly in 1954 ( 7 4 ) , but the importance of t,he method is considered such that a fekv references are repeated t o permit more complete coverage of the subject,. The method, developed by James and RIart,in (62) for analysis of volatile acids, requires a passage of the solute mixture a t “distillation” temperahre through a 4-foot column packed with M i t e moistened with a high-boiling silicone or ot,her solvent. The necessary mobility is provided by a nitrogen stream. The Ir:ict,ions are carried into a collection vessel, where they are periodically titrated with standard alkali to a photometric end point,. A plot of microequivalents of alkali required against time of titration would provide a st,epped or smooth sigmoid curve, \vhose midpoint location in time corresponds t o the appearance of pnrtirular acids in the observation vessel. The method has also l i ~ e napplied to amines and pyridines (60, 65))and presumably could also be ext.ended to substituted phenols. The aromatic amines may be separat,ed on Celite-paraffin (61, 6 3 ) . Kit8hmasterful understanding of t,he principles involved, James anti Martin ( 6 3 ) have ext,ended t,he procedure to include sqxiration oi the aliphat,ic hydrocarbons and the aromatic hj-droc:irhons. The separat,ion of the hydrocarbons required the development of a n e x detection device, as the common thermocouple device did not, prove sensitive enough for t.heir purposes. The general detector is an aut,omatic gas density halance (631. LIartin considers the advantages of gas phase chromatograph>-to be: negligible solute viscosity, rapid equilibration, and simple solute detwtion. Other workers have provided an additional detection device, the infrared analyzer, following oxidation of the Ernctions to carbon dioxide ( 9 6 ) . I-ndoubtedly, the general procedure described is capable of gre:tter refinement and extension of application. Even in it,s Ilresent stage of development it is capable of separation of the isomeric hydrocarbons from pentane through oct,ane, in an analysis requiring about 30 minutes. One does not wonder that commerunits embodying these separat.ion principles are already announced by Burrell, Perkin-Elmer, and Podbialniak in this rotintry. Chromat,ography has invaded one of the last untouched iical(ls of chemistry, v i t h a tool capable of providing rapid micro:in:ilyses of complex mixtures on a “simple“ apparatus. ZOYE ELECTROPHORESI S
This field, like that of paper chromatography, has “come of :lee” n i t h the appearance of reviews (81, 105, 125) and monographs (8, 86, 92). The necessary growth of knowledge concerning apparatus and technique has been more restricted, since the siwcifications for routine applicat,ion of the method are more i,igorous than t,liose required for paper chromat,ography. The teiahniques used by Durrum (a), McDonald ( 9 2 ) , and Iiunkel (81 remain distinctive, just as the commercial assemblies are produced in all three forms. The three variants use the filt.er paper in a t’aut, inverted V form, a t a u t horizontal form, or a horizontal form between pressure plates of glass. An apparatus using the paper in the last arrangement with pressure between glass cover and metal lower support, is described by Werkheiser
711
and K i n d e r (142). The recently described apparatus of Crestfield and Allen (31) represents a very useful form of the horizontal, glass-supported apparatus. I t routinely uses water cooling, which allows higher volt,age gradients and shorter times of separation. St the end of a run, hot water is directed into t.he cooling manifold to dry the paper in situ. All forms of zone electrophoresis differ from the “ancestral” free-boundary technique, in that the fract’ions are obtained free of the other compounds of the mixt’ure, given sufficient time for separat,ion. The advant,ages of the new approach include: inexpensive apparat,us, isolated fractions, micro quantity or micro concentration scale, litt’le time and effort per run. The disadvant,ages include: mobility values are not absolute, though standards can be run simultaneously; adsorption on the anticonvective material is possible; electro-osmosis must’ be measured under the actual p H conditions used. The importance of elect,ro-osmotic effects is emphasized by Strain (129). This author uses hydrogen peroxide or hydroquinone as indicat,ors of movement,. Caffeine has been proposed as a standard for measurement, of elect’ro-osmosis, with picrate ion providing a mobility standard over the pH range, 4 to 10 (31). These substances are located on ultraviolet, “print,s“ of the dried paper. The amino acids and peptides may be separated by zone electrophoresis into acidic, neutral, and basic fractions (51, 143); t h e three fractions are commonly subjected t o paper chromatography thereafter. The unusual ninhydrin spray may be used for their detection. The serum proteins and hemoglobins are separable, according t o their differing net charges ( 8 , 28, 67, 72, 81, 92, 116, 1 1 7 ) . The proteins are usually stained with a mixture of mercuric chloride and bromophenol blue. The adsorbed dye may be eluted from cut fractions, or the dye-protein spots may be scanned with visible light (28,115). Sucleotides, nucleosides, and free purine or p-yrimidine possess charge in sign and degree conferred by nucleotide phosphate, or bl- the amino and enol groups of the bases. These materials may be separated by one of the types of apparatus described. T h e particular zones are located by ultraviolet “prints” (32, 125, 141, 142). The nucleotides of the ribose nucleic acid in single nerve cells have been determined by Edst.rom and H y d h ( 4 3 ) , using elect,rophoresis on a rayon fiber, followed by microspectrophotomet,ric estimation of the isolated nucleotides. Presumably, the separation of carbohydrates (as borates) and aldehydes or ketones (as bisulfites) is also possible by electrophoresis as by ion exchange. A stimulating article on the separation of amylase-digested amylose has appeared (102). These authors were able t o obt8ainseparation in agar gel of the various fract,ions of the degraded amylose, using the familiar iodine complexes of these polymers as the migrat,ing species. The combination of electrophoresis and descending chromatography has not received the wide use accorded the single techniques. Most devices available for bhis continuous separation are similar in principle t o the original ones described by Durrum (41 ) and by Strain (130), with two commercial models in production. Here a hanging paper curtain is used. Modified apparatus of the same type has been described (58,116). The t.echnically difficult apparatus described by Svensson and Brattsten (132) continues in use for the fractionation of serum proteins in quant,ity ( 1 3 , 1 6 , 1 7 ) . This apparatus employs avertical tank filled with fine glasspowder, with careful delivery rates of buffer into and out of the tank. Gradient Electrophoresis. A new approach toward preparative electrophoresis has been taken in the past 2 years by Kolin (76-79) and Svensson (131, 133). Kolin has approached the problem of separation of proteins from a physical point, of view. He already has achieved rapid separations in a type of freeboundary apparatus, through the use of density gradients t o ensure the maintenance of any protein zone obtained. A p H gradient may be superimposed on the density gradient as well. The proteins taken (hemoglobin, catalase, cytochrome c ) are
718
ANALYTICAL CHEMISTRY
separable on the basis of migration in a p H gradient until the isoelectric p H is reached ( I spectra), or on the basis of differing mobility ( M spectra). Since Kolin is using a narrow zone of application and migrations of about 1 em., the separations are extremely rapid. The difficulties which exist during present use of the apparatus are several: location of zones, careful removal of a zone unmixed n-ith other zones, surety t h a t the particular p H gradient is acceptable to the desired protein without denaturation. Undoubtedly, ways mill be found to circumvent these difficulties. One could separate proteins from a complex mixture into acidic, neutral, and basic fractions. Thereafter, in a shallow p H gradient, the exciting method of migration of proteins to their individual isoelectric zones could be applied. For electrophoresis in columns of starch or in a density gradient, several assemblies have been devised (81, 131). The apparatus developed by Svensson and Valniet (133) has been designed to deliver the isolated fractions by gravity flow to an automatic fraction collector, after the electrophoresis. Sorof, Ott, and Young ( 1 2 7 ) propose the use of the conventional Tiselius apparatus for density gradient electfophoresis. Such use is possible where the apparatus is not in continuous analytical use. The schlieren system should aid greatly in the location and removal of particular fractions.
DETERMINATIVE METHODS bdvances in methodology for the determination of individual components of biochemical systems have not kept pace n-ith the advance in fractionation techniques. This trend is logical, as the precise fractionations, now commonplace, demand less of the determinative methods. The widespread use of a few “family reactions” is apparent now, as it was in 1954 (74); thus a single ninhydrin photometric procedure suffices to determine all a-amino acids, once fractionation has occurred. ABSORPTIOMETRY
The determination of materials by quantitative absorbance measurement in the ultraviolet (90, 120) or visible ( 5 7 ) spectral regions continues t o be of paramount importance. The trend is toward automatic determination of spectra by various recording spectrophotometers. More routine control of quantitative methods will result, as small spectral regions are identified as a means of determination of one component in complex mixtures. As in 1954 ( 7 4 ) , the use of infrared absorption continues to be an isolated special field. The use of infrared spectrophotometry has been discussed in a recent monograph on optical techniques (84). The findings regarding the detailed structure of biological macromolecules, such as deoxyribose nucleic acid (9) are of great interest. Infrared spectra will continue t o provide information regarding “fine structure” of such polymers. New Apparatus and Techniques. The announcement in 1954 and 1955 of three new recording spectrophotometers illustrates the modern trend toward automatic spectrophotometers of reasonable price and high routine accuracy. The nearinfrared region, to about 3 microns, may be used for analysis in the Beckman instruments. The availability of the Keston ( 7 1 ) polarimeter attachment for the familiar D U spectrophotometer should increase the use of polarimetric analysis on a micro concentration scale, with small sample volume. More data regarding the optical rotation and rotatory dispersion of biochemical compounds should appear (15). Cuvette modifications which serve t o increase the utility of absorptiometry include the use of Teflon inserts for microvolume absorbance measurements (123), an anaerobic cuvette with provisions for additions or transfer of solutions (84), and half-height cuvettes for alternate p H adjustment and absorbance reading (99).
The extension of absorbance measurements to include turbid systems, such as cell suspensions, is possible in several ways.
Shibata, Benson, and Calvin (184) use the expedient of interposing a diffusion plate, such as filter paper, following the emergent beam from the cuvette. Presumably the plate serves as a source of uniformly diffused light for the photomultiplier or other phototube. Batenian and Monk ( 4 ) approach the turbid systems from the standpoint of uniform reflectance of light not absorbed by the system. These authors use a round quartz flask, surrounded with magnesium oxide, as their sample holder. Light enters in one dimension and reaches the phototube after multiple reflections The quantitative measurement of fluorescence in biological systems has been used as a means of estimation of thiamine ( 6 4 ) since 1939. However, fluorescence has not been used as a general method of investigation until the present time. Two types of instruments have been developed which use the extreme sensitivity possible with the method (18, 155). The first instrument uses filtered light (360 mp), detects the light emitted by the solute with a photomultiplier tube, amplifies the signal through a number of stages, and uses a variable sensitivity recorder. These authors estimate that the method is a thousandfold more sensitive than corresponding absorption methods. They report studies of the combination of flavin mononucleotide with the protein portion of Warburg’s “old yellow enzyme.” The free flavin is fluorescent; that combined with protein is not. The second instrument is a true spectrophotofluorometer (12), since the activation light is supplied by one monochromator and the emitted light is subjected t o refraction in a spectrograph before intensity measurement. Udenfriend, Weissbach, and Clark (139) indicate that a large number of organic compounds, varying in complexity from aniline to morphine, are determinable in the instrument. Such determinations would be subject to interference by other fluorescent solutes in the anaIysis mixture. A second report indicates the determination of serotonin (5-hydroxytryptamine) in blood platelets by quantitative fluorescence. The estimation of less than 0.1 y of the compound was possible. The application of absorption, fluorescence, and other optical methods t o the location and estimation of important compounds in tissue sections has been described by ten authors, under the editorship of Bfellors (99). A second section of the book is devoted t o methods using electrons and other radiation. The power of the fluorescence microscope is shown by the report of Mellors and others (100) on the location of antibody-antigen complexes at the site of pathological action. Localization in the glomeruli of rabbit kidney precedes the development of glomerulonephritis in these animals. OTHER METHODS OF ANALYSIS
It is beyond the scope of this revien- to discuss the methods required for those determinations necessary t o only a few analysts. Rather, the time and effort have been directed toward recognition of approaches or methods which promise general use in biochemistry. Titrimetry. As indicated in the introduction, the use of methods based on titration (44) has been negligible in the usual organic analyses done by the average research biochemist. The exceptions to this would include any method which determines the appearance or disappearance of hydrogen ion as an indirect estimation of ammonia, or of the compound concerned. Similarly, the determinations of two biological cations (calcium and magnesium) frequently use titrations, n-ith Versene as titrant (118). Alternative procedures, using the same reagents, may be absorptiometric (1, 146). The Versene titrations or their absorptiometric equivalent will remain important in biochemistry. Their utility will be for the determination of micro quantity of bound metal ions which have been found as necessary components of important enzyme systems. Many of these ions can now be determined by the application of indicator titrations, using an azo-dye end point, and a chelating agent as titrant (23, 96, 112, 1W).
V O L U M E 28, NO. 4, A P R I L 1 9 5 6 Because many biochemists are no longer satisfied with subjective indicator end points, the trend is either t o determine such ions as calcium by spectrophotometry of the calcium-dye chelate ( 1 4 6 ) ,01 t o determine the ion by Versene titration to a particular absorbance end point (95, 126). Such developments have led to the design of photometric titration units (162), or t o the use of assemblies which permit titration in the confines of the cell compartnient of the DU spectrophotometer ( 7 5 ) or of the hlodel B instrument (45).Photometric titrations m e capable of geneial use, without excessive investment in capit a1 equipment ( 1 4 0 ) . Thus such titrations may be used for determination of a weak acid or for estimation of ionization constants (49,113). The coulometric generation of oudative titrant in such titrations is also possible (45). Coulometric generation of titrant ( 5 , 1f9) or the newer ampeiometric or voltammetric methods of end-point detection ( 8 0 , have found few important uses in biochemical analysis. Surh neglect of chemical advance is temporary, as acceptance a\wit* merely the need for the use of such methods, in preference to existing methods. Thus, polaiography, M hich can discriminate among the related coenzymes ( 6 2 ) (diphosphopyridine nucleotide, triphosphopyridine nucleotide, and niacin mononucleotide 1, must still compete with existing methods, surh as t h a t liasrd on chromatography followed by ahsorptiometry (85). SOURCE iIIATERIALS FOR BIOCHEMICAL ANALYSIS
While time does not permit individual identification of chapters or authors, recent years have seen the appearance of a number of book serials devoted to the niethodology of organic analysis (98), and biochemical analysis (48)for medicd rcsearch (138) and for clinical chemist’ry (114). The three volumes on “Physical Techniques in Biological Research,” devoted to optical techniques (104), physical chemical techniques, and cells and tissues, should prove of lasting value t o the harassed research worker. The four-volume set of ”Methods in Enzymologj-” ( 2 7 ) should prove of universal value, as the ideal is t,o present the method in enough detail t,o make repetition possible without’ return t o t,he original literat,ure. The published volume of this set, provides a large section on preparative procedures for enzymes. This sect,ion includes authoritative summaries hy various aut,hors of advances in tissue homogenate preparation and diff erentiul centrifugation of cell particulatm. For this reason, the subject of difl‘erential centrifugation is not rrvien-ed here, alt,hough the literature was rovered in previous I evieivs of this sei ieq LITERATURE CITED
Aconsky, L., JIori, AI., . ~ . L I . . C H E L f . 27, 1001 (1955). Ibid., 27, 476 (1955). Allen, R.R . , Eggenberger, D. S., Carlsberg 29, 49 (1954). hnderaen, IT’., Compt. r e n d . Lrais. /a/,, Bateman, J. G., Monk, G. IT.. S c i e n c e 121, 441 (1954). Bell, K., Sock, IT., Morris, G . , .4nalUst 79, 607 (1954). Bendich, d.,Fresco, J. l t . , ltosenki,anz, H. S . , Beiqer, S. 11.. J . Ani. C h e m . Soc. 77, 36i1 (1955). Blake, G . G., Che7nistr.u and Iridiistry 1955, 701. Block, R. J., Durruni, E. L., Zweig, G., “SIanual of Paper Chromatography and P a p e r Electrophoresi-,” Academic Press. h-ew York, 1955. Blout, E. K., Lenorinant, H.. Iliochin,. et B i o g i i ~ s Acta . 17, 325 (1955). Boardman, X . II., A?IgeW. Chem. 63,280 (1951).
There have also been some good papers on precision laboratory fractionation, Mair’s use of fluorochemicals as azeotrope formers with hydrocarbons is also worthy of special mention (60). VAPOR-LIQUID CHROMATOGRAPHY
Vapor-liquid chromatography involves establishment of composition differences between a liquid phase on the surface of a stationary solid adsorbent, and a moving vapor or carrier gae phase. The liquid phase of the mixture undergoing separation
