Biochemical analysis - Analytical Chemistry (ACS Publications)

Biochemical Analysis Anthony 1. D’Eustachio, Development Department, E. 1. du Pont d e Nemours and Co., Inc., Wilmington, Del.

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HIS REVIICW will discuss most of the significant developments in biochemical analysis which appeared in the literature during the period from January 1966 through December 1967. Undoubtedly, many worthwhile contributions have been overlooked, but scanning the literature quickly brings one to the conclusion that the field of biochemical analysis is undergoing a “publication explosion.” The deluge of pertinent reports encompasses journals in chemistry, biology, physics, electronics, and engineering; thus, the sheer physical volume demands that this review be selective, rather than comprehensive, and precludes incorporation of routine applications or minor modifications of accepted techniques and methods. This approach is in keeping with the philosophy of the previous reviews of the same title, as established by Dr. P. 13. Hamilton, and appears to be well suited for the continuity of this series. Accordingly, the emphasis will be on new developments and trends having potential applications.

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BOOKS AND JOURNALS

Recent texts pertaining entirely t o the subjects discussed in the following sections will be mentioned as they arise. Of general interest to biochemists will be a n ‘iEncyclopedia of Biochemistry” (663), which stresses methodology in relation to the rapid developments in this science, and an annual series “Essays in Biochemistry” (108, 109). The first volume of a n “Atlas of Clinical Laboratory Procedures” (405) was published and may well be the primary reference work for clinical biochemists. Specialized fields of biochemical analysis have been treated in the continuing editions of annual monographs by Dr. D. Glick (234, 235). Analytical methods for nutritional biochemistry (5), medicinal chemistry (182), general biochemistry (83, 84), vitamins (210), hormones (273, 485), microbiology (630), virology (399), cell physiology (495), carbohydrates (133, 134, 670), proteins (8, 17, 18, 32, 444), immunology (661),enzymes (134,452,453), lipids (403, 471), flavonoids (269) and nucleic acids (134, 1481149) have been published during the period under review. Many of them have been new editions to annual monographs. A text

on hallucinogens which has a significant bearing on today’s biochemical, pharmacological, and sociological problems was published (295). The parasympathetic biochemistry of known hallucinogenic compounds was described in a manner indicating the importance of establishing methods for their analysis. Inorganic biochemistry was surveyed in a fundamental treatment of trace elements (79). Quantitative cytochemistry was reviewed with respect to advances in combining instrumentation and cytochemical techniques (658). Methods used in cancer research were the subject of a monumental fourvolume publication which presented a comprehensive review of all analytical methods, their application, and interpretation (103). A significant book on “Separation Techniques in Chemistry and Biochemistry” ($42) was published which included all chromatographic techniques used to date. Other monographs devoted to the general field of chromatography appeared as serial editions of highly respected reviews (228, 281, 374 , 375). A general survey of biochemical analysis, as well as continuous systems, has been elegantly presented in the publications of the symposia sponsored by the Technicon Instrument Corp. (338, 586). The papers presented at these symposia cover all aspects of accepted chemical methods and afford a onesource reference book. The impact of new technology in physics and mathematics, as applied to the organization of living systems, is evidenced by a book on “Molecular Insights into the Living Process” (244) and a new annual series “Progress in Biocybernetics” (660). The application of chemical engineering to medicine and biology was the subject of another book (286). These volumes do not directly bear on biochemical arialysis, but are included because of their general interest and as a n indication of the trend of biochemistry. New journals to appear during this review period include a Russian publication 011 molecular biology (185) and a chemical journal from mainland China (127). Journals have also appeared that are devoted t o nucleic acids (553), lipids (34), virology (646), molecular pharmacology (237), separation techniques (229), biomedical electronics (458), protein sequence and structure

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(153),and natural compounds (67’4). A journal concerned solely with applied biochemistry and microbiology has been published (,98), The end of 1967 saw the publication of two journals aimed at providing a medium of rapid publication of short commuriications in the entire area of analytical chemistry (254) and spectroscopy (526), The importance of instrumentation as the basis for modern chemical analysis was recognized by the publication of a new journal devoted to presenting an integrated interdisciplinary medium for chemical and biochemical techniques (28). The tradition and excellence of Biochemische Zeitschrift, which terminated in January 1967 has been continued in the new European Journal of Biochemistry (383). DIALYSIS, ULTRAFILTRATION

RIany biochemical methods involving separation of large volumes of material require later removal of salt from the fractions and concentration of the desired components. Dialysis systems designed to meet this demand are a system for dialyzing up to 98 fractions using a single membrane (542),dialyzers for continuous countercurrent flow (151 398), and a method for halting inadvertent loss of excess volume during concentration (625). A new automated analysis method was described (601) that enabled study of the quantitative binding of small species to macromolecules with results comparable to classical equilibrium dialysis procedures. A competitive situation was shown to exist between sodium and oligolysines for polynucleotide phosphates in equilibrium dialysis studies at pH 7.0 (368). A dialysis method was proposed that did not use a membrane (675); hemoglobin was separated from salts in a system that allowed diffusion across the interface between the flowing solution and a solvent. The use of ultrafilters has been proposed for resolving complex mixtures of biological material into size classes without the use of precipitating agents, cumbersome electrophoretic methods, column chromatography, or gel filtration (65, 66, 152). A novel approach to protein determination in the presence of interfering low molecular weight substances proposed the removal of protein by ultrafiltration and subsequent assay of the filter (48). One report appeared VOL. 40, NO. 5, APRIL 1968

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that cautioiied against the use of Salgeiie filter units because of toxic iiiilnwities (583). -1paper appeared on the use of foaming as a iiieaiis for separating aiid coiicentrating catalase and amylase (119). CENTRIFUGATION

The theory of centrifugation as applied to biological situatioiis was reviewed (52), in addition to a comprehensive review of ultracentrifuge techniques (634). Optical systems, determinations of sediineiitatioii coefficients, and molecular weights of macromolecules were also reviewed (636). The majority of individual reports have dealt with the application of absorption optics to analytical ultracentrifuge studies. Dye-protein mixtures were found to provide data for sedimentation velocity aiid sedimentation equilibrium that compared with that obtained by equilibrium dialysis (602). The introduction of split-beam photoelectric scanning optical systems with monochromators (76, 121, 552, 637) has allowed sedimentation coefficients to be obtained with as little as 3 pg of material/pl (652). Protein aggregates in dilute solutions have been resolved at levels as low as 0.2 mg/pl (54). An automatic system for control of sequencing and photographing of four different samples was described (221). h niulticell rotor has been devised (121) for use with six, four, or two cells with absorption optics and single or double sector cells. An automatic masking assembly for such rotors has been developed (317 ) . -4 continuous-source nionochroniatic ultraviolet optical system was described (141) aiid a new system using a senoii-mercury lamp that yielded monochromatic light from 235-700 nip (530). Gouy interference optics were shown to allow determination of diffusion coefficients in 3 hr (250). Molecular weights were determined by a combination of schlieren and interference optics (125, 364, 365). The effects of concentration dependence on sedimentation velocity was esamined by generating solutions to the Lamm equations (164). .1 report cautioned that sedimeiitatioii behavior of chemically reacting systems precludes t'he determinat'ion of inolecular weights by use of sedimentation coefficients (341). Unless the sedimeiitation is independent of speed and pressure, erroneous changes and effects on the dissociation of bimolecular complexes will be recorded. Some iniiior modifications include an electronic micrometer for recording coordinates on plates (19), a four-step synthetic boundary cell ( 2 @ ) , analysis of two specimens (73), and a simplified means for analyzing patterns (157). The application of digital computers to

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ANALYTICAL CHEMISTRY

ultracentrifuge data was reported (129, 595). The preparative ultracentrifuge was shown t o yield data that could be used in sedimentation studies of serum proteins (82). A unique sector-shaped cell was described for use in swinging-bucket rotors to aid in determining sedimentation coefficients (610). Zonal centrifugat,ion has evolved to the state of a practical technique for isolating nuclei, mitochondria, niicrosomes, polysomes, ribosomes, ribosomal subuiiits, viruses, and macroglobulins (14, 16, 17, 59, 110, 137). The technique has been fully reviewed (12,13,37) and the design of rotors and flow systems described (38, 39, 251). Specialized rotors containiiig transparent end-plates allowed observation of organelle sedimentation (496). Gradient formation for use with this type of centrifugation was reported (16, 115). -4 procedure for digit'al computation of sedimentation coefficient's in the zonal centrifuge was described (60). Density gradient, separations in the ultracentrifuge were reviewed (118). Kew devices (468) and methods were reported (451). 4 simple device for preparing linear and comples linear sucrose density gradients was described (85). Substitutes for sucrose appeared, such as colloidal silica (480), sulfolane, trimethyl phosphate, and urea (472, 473). An automatic optical scanner for density gradients was described (287); tables for estimating sedimentation coefficients in linear sucrose gradients (415), and a mathematical model for density gradient centrifugation showed how gradient perturbations effect the zonal shapes of separations (596). A method was described for determining sedimentation coefficients in polyacrylamide gel which was photocatalyzed after the component's were separated (327). X novel "coil planet" centrifuge was reported (319) that was capable of separating and analyzing cellular particles, A helical sample tube was centrifuged, containing two miscible solvents which established a countercurrent distribution of sample components. ELECTROPHORESIS

Several excellent reviews on electrophoresis have appeared (414, 576), as well as a new edition of a comprehensive reference work describing both theory and practice (58). Others have dealt with the application of immunoelectrophoresis to diagnosis of disease (347,392, 413), electrophoretic behavior of cells, (11), and gel electrophoresis in ureacontaining buffers (491). Electrophoresis in stabilized media was the subject of the first volume to be published in a new series on this analytical technique (683).

Because of its high resolving power, polyacrylamide gel electrophoresis has received the most attention during the past 2 years (324, 561, 522, 579). Several worthwhile applications and modifications have been reported; the characterization of different strains of a virus by electrophoresis in two sucrose gradients (35), the estimation of molecular weights using two different gel concentrations (682), determining molecular weights of polypeptide chains in sodium dodecylsulfate-polyacrylamide gel ( ~ 5 7 2 production )~ of speciesspecific patterns in 5ill urea and acetic acid (514, determination of molecular weights of viral aiid bacterial nucleic acids (61),electrophoresis across a molecular sieve gradient (401), electrophoresis in an acrylamide concentration gradient (400),and the use of schlieren optics to study migration rates and zone diffusion in vertical, flat-bed discontinuous systems (9). Polyacrylamide electrophoresis was reported to be 100 times more sensitive than starch gel and has allowed separation of human sera into more than 20 fractions (145). Discontinuous acrylamide gel plate electrophoresis has been shown to be superior to disk systems for separating serum proteins (297). A multiple-sample operation using vertical gel slabs has allowed up to 49 samples to be separat,ed with high reproducibility (626). Use of a single, slotted gel slab allowed up to 16 samples to be run in parallel with no deformation of patterns (240). A single gel system with continuous elution was shown to facilitate a preparative method (331)and to afford a means for purifying beef serum albumin with a minimum of extraneous polymers (196). The technique has recently been ext'ended to the micro scale where 1-3 pg of protein could be debected (199, 205). Starch gel electrophoresis appears to be falling out of favor as a medium of choice; it was unfavorably compared to polyacrylamide (223). Innovations were limited to improving separations by manipulating the thickness of the slabs to less than 2 mm (668),employing slrict molding requirements (300), and detecting protein migration photometrically (307). The most noteworthy innovation was the microelectrophoresis of samples as small as 0.1 pl under petroleum ether in 40 min (YO). Ribonucleic acid (RNA) nucleosides and nucleotides were separated with agar gel electrophoresis in 15 to 30 minutes (654). I t s use for mobility studies of proteins (294) and dyes (304) was reported. Agarose gel has been used for evaluation of multiple runs for lactic dehydrogenase isoenzymes, protein, and hemoglobin because of minimum endosmosis, low fluorescence, and clarity (180). It has been used to separate immunoglobulins showing slight charge

differences (369) and for in vivo proteinbinding studies of I31I (416). Electrophoresis in fibrinogen-containing agarose has been used to demonstrate and identify factors of the blood-clotting system (288). Disk electrophoresis has been described as a preparative means (612) and as an analytical method for histones (575). Multiple analyses on a single disk were reported (611) using differential staining methods. A method of destaining disk electrophoresis gels appeared (192) in which current was applied perpendicular to the gel axis. I t was also found that ammonium persulfate, which is often used in polymerization of gels, produced an artifact that could inactivate yeast enolase and increase heterogeneity with 8 M urea (88). Either the use of riboflavin and light, or the use of thioglycolate, was recommended for polymerization, as a replacement for persulfate. The inevitable has occurred and a report appeared describing microdisk electrophoresis using only lo-’ to 10-9 grain protein in 200-fi diamet’er glass tubes (313). This latter approach is particularly useful when separating and analyzing radioactive-labeled protein disks. A variety of supporting media can be used for t,he method known as thinlayer electrophoresis (519). I t encompasses the deposition on glass plates of alumina (589), starch gel (%I), cellulose acetate gel (373), polyvinyl chloride (627), and polyacrylamide gel. An apparatus was described for a stable high-voltage supply (337). Several reports proposed combining thin-layer electrophoresis with chromatographic separation (44, 57). A particularly impressive report on this approach showed that tryptic digests of glyceraldehyde-3phosphate dehydrogenase, viral coat’ proteins, hemoglobin, and ribonuclease yielded more spots than anticipated (102). The use of cellulose acetate as a support for zone electrophoresis continues (122, 332), but with few innovations. Separation and analysis of lipoproteins on this medium can be improved by removing the lipophilic character with alkali causing a conversion to cellulose (641). Another material, nitrocellulose, has been proposed as superior for microelectrophoresis of proteins in sit’uations where cellulose acetate is now used (498, 499, 502). -4 review appeared (484) that showed paper to be the best medium for microelectrophoresis. High-voltage electrophoresis continues as an area of interest. Mobilities of protonated amino acids can be determined (174, 4 6 f ) , and mononucleotides from RN.4 hydrolyzates were separated and characterized (131). A new apparatus for two-dimensional separation in potential gradients up to

100 V/cm was described (467). A new technique for objective examination of peptide fingerprints employed a scanning densitometer and automatic taping of recorded absorbance (232). The combination of high-voltage electrophoresis and circular paper chromatography allowed a defined separation of acid, basic, and neutral peptides (212). The large-scale purification of proteins using membrane electropartition (372), reversed electrophoretic fields in flow columns (512), countercurrent electrophoresis (91), elution convection ( 5 l S ) , a new power supply for maintaining constant voltage across a separation field (163), and a theoretical appraisal of continuous electrophoresis and electrochromatography (511), all showed an awareness of the need for minimizing denaturation and binding effects for obtaining more valid separation of components. Xovel approaches to electrophoresis included the separation and characterization of particles by the use of electromagnetic fields (552) and continuous electrophoresis in free media (609). A “flowing junction’’ method which allows external control of boundaries in a modified Tiselius cell was described (592). COLUMN CHROMATOGRAPHY: ION EXCHANGE AND GEL FILTRATION

While tests on column chromatography, or liquid chromatography as it is now being called, have appeared during this period, most of them are repetitive presentations of the techniques reviewed earlier (262). The most useful reference texts for keeping abreast of the developments in this field are those in the series “ildvances in Chromatography” (227, 228). The separation and analysis of amino acids by ion exchange chromatography continues to draw the major effort in this area of analysis. Reviews have appeared that describe new methods (20, I n ) , application of anion and cation exchange resins to specific assays (320), and an international comparison of methods (409, 428). Recent advances in analytical determinations using ion exchange chromatography of amino acids were reviewed in depth in an excellent presentation that discussed innovations in technique and hardware (261). The significance of cellulose structure was discussed in a paper stressing direct application to biochemical situations (349). In another report (@a), emphasis was placed on accelerating an automatic single-column procedure for performing three complete analyses of hydrolysates of collagen and elastin in 1 day. The AutoAnalyzer system has been the basis for many reports (353, 524) which will probably add impetus to wide acceptance of this

technique. The importance of amino acid profiles in the diagnosis of metabolic disease prompted the development of rapid short-column chromatography (578). Using a single column and a spherical cation exchanger, analysis of amino acids associated with inborn metabolic errors could be assayed in 1 hr (49). Determination of hydroxyproline was shown to be useful for detecting bone disease and collagen turnover (204). A method for separating peptides from amino acids in urine by ligand exchange chromatography was reported (97) Noteworthy innovations in amino acid chromatography to appear during this period pertained to improvement of resolution by the use of sodium cyanide (62.9) and 2,4,6-trinitrobenzenesulfonic acid (550); and the use of 3to 5-nim bore multiple columns under 500-600 psi, lvhich allowed three samples to be run in 24 hr (270). This latter method showed sensitivity to the nanomole level. A method was described for recovering tryptophan in 90 min (523). A new continuous system of automatic amino acid analysis was described (170) which could operate unattended for 96 hr by virtue of automatic sequential applications of 24 samples to a single column. Polyethylene glycol 400 (15% v/v) was suggested as a means for improving sample application of amino acids to columns (543). h nomagrani was described for calculating results of an analysis in 5 min (563). Recent reports discuss the use of computers to calculate amino acid analysis data (188, 625). Two papers describe computer programs that are compatible with several qystems (396, 599) and can edit for instrument noise, compensate, split partially resolved peaks, calculate peak area and convert peaks to mole per cent, weight per cent and/or residues. Chroniatography of ninhydrin-negative compounds was described (646), using radioactive tracers. The automatic high resolution analysis of nucleotides and other ultravioletabsorbing compounds by ion exchange chromatography was the subject of several reports (213, 431, 679). Analysis of urine samples showed differences occurring between normal and diseased states (565). An apparatus was described for high pressure column chromatography of nucleotides, bases, and nucleosides a t 4000 psi (246). The use of “pellicular” column material consisting of a basic ion exchange resin coated onto small glass beads and high inlet pressures was reported as a means to faster analysis of nucleotides (306). I n this apparatus, separations of nanomole amounts of nucleotides could be assayed in less than 2 hr. Ligand exchange chromatography of bases, nucleotides, and nucleosides on ChelexVOL. 40, NO. 5, APRIL 1968

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100 was described (238). Many nucleotide analyzers were readily converted to anion exchange systems for separating and analyzing organic acids (305, 346, 64.8, 677). Soluble RNA was separated on ion exchange cellulose (591) and denatured deoxyribonucleic acid (DNA) could be separated from native DNA on columns of silk fibroin (310). Proteins could also be separated by liquid chromatography (492, 577, 620). Carbohydrates and mucoid substances were separated by ion exchange chromatography (162,454,653). Automatic chromatographic systems were described for analyzing sugars on anion exchange resins in a few hours (245, 3S0, 345). Lipids, phospholipids, and lipoproteins were the subject of several reports on column chromatography (440, 447, 497, 536, 551, 606), as were steroids (105, 642). Detectors for liquid chromatography were reported during this period and included a sensitive, linear, flowingstream photometer (263) which showed only 2% drift for the range of 0-0.01 absorbance. d n inexpensive electronic multiple-wavelength selector was described (268) for continuous spectrophotometric monitoring of column effluents. The use of volatile eluting solvents allowed column effluents to be deposited on a continuously moving metal chain which carried the nonvolatile samples to a pyrolysis chamber where they were volatilized in nitrogen. The resulting vapors were then aspirated into a flame ionization detector. This concept was discussed in the previous review (262) but now has been shown to be practical. It is capable of separating microgram quantities on silicic acid columns and eluting with buffers of increasing polarity (335). A potentiometer detector for monitoring effluents for catecholamines was described (419). Some worthwhile instrument modifications were noted. A simple density gradient generator composed of two syringes was described (544) as was a hydrostatic gradient generator capable of forming either simultaneous or sequential linear gradients (55). Another paper proposed an electronic photoelectric cell-controlled system for dispensing solutions to amino acid analyzers (26). It was reported that the sensitivity and capacity of automatic amino acid analyzers could be increased by introducing a rotary valve system that allowed automatic sample application and column regeneration (169). Thus, six complete analyses could be run unattended every 24 hr. The volumn of reports in the area of column separation using gel filtration has increased a t such a rate that it demands separate consideration. Several excellent critical reviews on molecular sieve theory ( S l 4 ) , new methods (355),

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and comparative evaluation of gel materials (397,404,477)have been written. The dextran gels have received the most attention by workers in this field. Systems have been derived for analyzing protein mixtures and showing that a correlation exists between molecular weight and elution behavior (159, 161, 165, 607). A layer technique was described for estimating differences in the degree of dissociation of closely related proteins (230). A simple method was proposed for concentrating proteins by “stacking” on a dextran gel column by electrophoresis prior to filtration (417). Dextran gels have been used to remove pyrogenic material from parenteral solutions (482),to identify iodohistidine and phosphoroiodohistidine (478), and in molecular weight distribution analysis of pharmaceutical dextrans (241) and lignin (93). Proteins and enzymes associated with disease were separated and identified by gel filtration (209). Histones and DKA could be gently isolated (42) and RNA and DSA separated (40). Complex mixtures of polyhydroxyphenols and mixtures of flavonoids were separated on dextran gels (671). The technique was shown to be capable of separating inorganic ions from blood proteins and allowed the determination of protein-bound magnesium and calcium (388). The problem of bacterial growth contaminating long-standing dextran gel columns was considered and it was suggested that 2.5% iodine in 0.5 saturated potassium iodide was an effective sterilizing agent (167). Lipophilic dextran gels are now available which allow synthetic peptides to be rapidly separated from reaction byproducts by chromatography on methylated gel columns (184, 433, 456). A method was proposed for evaluating gel filtration data on systems subject to chemical and physical interaction (445). Zonal gel filtration was discussed in terms of solute heterogeneity from analysis of ultracentrifugal data (665, 666). A new material appeared in the literature as a possible competitor to commercial dextran gels (Sephadex). This reaction product of dextran and epichlorohydrin (Epidex) was shown to perform as well as dextran (464). Polyacrylamide gels continued to gain wider acceptance by offering the same exclusion limits as the dextran gels, but often possessing more uniformity in bead size and flow rate (664). Agarose gel filtration also gained in use for separation of lipoproteins (402, 656). The use of hexafluoroacetone as the solvent for chromatographing large peptides from chymotrypsin on dextran gel caused fragility of the beads and slower flow rates (102). Granulated polyacrylamide gels were found to be extremely effective in separating complex protein mixtures (194). The prep-

aration of gels of varying porosity was described for use in columns having a plate count within the range of 7001300 plates/ft (476). Elastin fibers mere proposed as a medium for gel filtration of solutes with molecular weights to 1500 (475). The calibration of gel filtration columns must be considered if standards are to be used in analytical column development. Two papers dealt with this problem: a new procedure that was not based on the hypothetical structure of the gel matrix ( l ) ,and the homogeneous fractionation of dextran and inert, nonionized polymers used as calibration standards (370). A sample applicator was described which reduced both the chance of disruption of the gel bed and the time required to add the sample (528). Sucrose was used as the suspending medium. Column effluents were monitored for five hydrolytic enzymes in a single automated flow system (46). A new development in column chromatography, with potential for wide application, is the technique of reversed-phase column chromatography (RPC). This technique has resulted from the intense research on transfer RNA (tRXA) and the need for preparative and analytical separation of individual tRNA species. h complete and excellent review brought this subject up to date (455). Another innovation in column chromatography was the use of dry columns (5’87), claimed to have the resolution of thin-layer methods. PAPER CHROMATOGRAPHY, THIN-LAYER CHROMATOGRAPHY

Paper chromatography has seen few innovations during the period of this review. Apparently it no longer attracts the attention and effort that would advance it to the stage of yielding the type of separations offered by other media. A high-resolution procedure was described for paper chromatography of purines and pyrimidines using acetonitrile solvents (50). Other reports dealt with the separation of plant sugars (11 4 ) , adenine metabolites using six solvent systems (533), or a continuous system (4467, and a rapid detection of amino acid abnormalities in whole blood or urine (619). A sensitive method for detecting 0.1 to 40 pg of amino acids with 10% accuracy was described (280). Thin-layer chromatography (TLC) has been the subject of several complete reviews (192, 436, 659). Specific methods have been described, such as chromatography of tricarboxylic acid cycle compounds (67, 291, 435), plant alkaloids ( S I I ) , amino acids on starch (74, 481), hemoglobins on Sephadex

(608), phosphate esters from erythrocytes (147), methylated tRNA (62) tetracyclines (23), imino acids (71), macroglobulins (51), and antibiotics (421). Silica gel is often noted as the medium of choice for chromatography of porphyrins (128, 166); mixed peptides (438); chlorophylls and related plant pigments (394); nucleic acids (639); nucleotides, nucleosides, and bases (222, 367, 427, 520, 554, 629); and aflatoxins (488). For N-terminal and sequence analysis, fluorescent amino acid derivatives of l-dimethylaminonaphthalene-5-sulfonyl chloride were prepared and readily separated on silica gel plates (430). Lipids were analyzed frequently by TLC. A good review evaluated this field and showed that glass fiber was superior to other media (27). Other reports described quantitative methods for human tissue lipids (21, 149, 343 357, 560). Improved methods for detecting lipids on chromatograms by charring were noted (328, 329, 407). These were further improved by viewing the spots under ultraviolet light (441) or by photographic densitometry (580). Innovations in TLC adsorbents have included cellulose powder for nucleic acid bases (271), plaster of Paris for amino acids (4), talc (647), and nitrocellulose membranes (600, 501 505) Specific color reactions for amino acids. with metal salt-ninhydrin mixtures allowed easy identification of components in a complex mixture (356). Cellulose could be treated with polyethylenamine which allowed nanogram levels of purines to be detected by ion exchange TLC (509). A report proposed the use of glass rods or test tubes to support TLC adsorbents, thus removing the need for special applicators (316). A simple method was described for removing salt from samples (208). An automatic device for adding solvents to chromatography tanks (443) and an apparatus for simultaneously applying three different samples to TLC plates (633) were described. A technique for conserving TLC plates was described (107). Supporting instrumentation for TLC included reflectance spectroscopy of resolved nucleotides (384) and a spectrophotometric scanner for large plates (264). .4n automated system for sample collection and computer analysis of thin-layer radiochromatograms was described (593). Electrogel filtration was the basis of a report (640) which combined cellulose acetate electrophoresis and gel filtration. A new method for determining base ratios in RXA using electrophoresis and chromatography in thin-layer cellulose was described (289). ]

GAS CHROMATOGRAPHY

I t is difficult to select a few noteworthy reports from the many that have appeared in this rapidly expanding area of analysis. Two new books dealing with biomedical techniques were published (672, 680). Two reviews were noted, one updated the developments of gas chromatography (GC) for determining bile acids and steroids (359) and the other described ultrahigh pressure GC at 2000 atm (434). Direct GC was extended to analysis of acetyl groups in proteins and peptides (649), aliphatic oxygen-containing substances in water (558), deoxycorticosterone and aldosterone (510), phenolic acids (63), homogentisic acid (75), indole auxins (155), 2,4-dinitrophenylhydrazones of amino acids (316), lipids (NO), tricarboxylic acid cycle compounds (361), and simultaneous analysis of blood carbon dioxide and oxygen (412 ) . Gas chromatographic techniques are undergoing expansion and innovations, particularly the modification now known as gas-liquid chromatography (GLC). Two excellent reviews discussed the present state of this powerful, new analytical tool (146, 333). Reports describing the preparation of trimethylsilyl (TMS) derivatives of a variety of compounds have appeared during this review period. The scope of application of this technique is illustrated by the list of TMS derivatives of catecholamines ($OS), carboxylic acids (302, 537), blood glucose and galactose (136), hexosamines (507), sugar phosphates (277), nucleosides and bases (549), adenosine derivatives (265), and ribonucleotides (276). Further reports describe GLC analysis of cyclopropenoid acids (517), neutral glycoses (429), dehydroepisterone (585), 17-ketosteroids and progesterone (2 42) , estrone (539), cholesterol (390), fatty acids (144), bile acids (463), and mevalonic acid (252, 292). A new method for determining base ratios of nucleic acids was proposed] using GLC to detect nanomolar levels (224, 395). Vitamin BIZ deficiency could be determined by detecting methylmalonic acid in urine samples (296). The analysis of amino acids and peptides has been the subject of increased effort in GLC. An excellent, up-to-date review emphasized this effort on derivatives of amino acids (140, 597, 655). The use of N-trifluoroacetyl (TFA) butyl esters yielded 98% of the amino acids and allowed detection of 0.1 ng (225). Further use of TFh butyl esters allowed diagnosis of homocystinuria (248) and the determination of the stereospecific hydrolytic action of acylase (260). Gas chromatography has been used

in the differentiation of microorganisms by pyrolysis of the organisms (518), analysis of ether extracts of the cells (283), or analysis of the extracts of the growth medium (424). I n these applications, the electron capture detectors provided the sensitivity for analyzing multicomponent mixtures (117). A new detector was described as a combination membrane-gas detector (334). A thin rubber membrane is placed so that one side is in contact with the solution to be monitored. A stream of gas passing over the other surface sweeps any volatile material that had been dissolved, and subsequently permeated the membrane, to a hydrogen flame ionization chamber. A continuous GC apparatus (613) and a means for introducing blood samples for volatile gas determillations (559) were proposed. Improved precolumn techniques were proposed for high temperature GC (190). h simultaneous determination of mass arid radioactivity of labeled steroids by GC was described (615). Computer programs have been presented to allow rapid processing of GC data (323, 657) by allowing for base line anomalies, linearity, Gaussian resolution, and column variation.

SPECTROPHOTOMETRY: ULTRAVIOLET, INFRARED, ATOMIC ABSORPTION, FLUORESCENCE, AND LUMINESCENCE

Ultraviolet and visible spectrometry are admirably presented in a new text which presents practical techiiiques for all aspects of absorption analysis (172). Reviews on microphotometry in biological research (617), cytophotometry (156, 548), and adsorptioli characteristics of carotenoids (150) and nucleic acids (432) were published. Photometric methods mere reported for determining purine and pyrimidine bases from RNA (36), XAD :?JADH in tissue honiogenates ( 9 9 ) , hemoglobin (183), uroporphyrins (200), proteins (100), amino acids (257, 376), free fatty acids (%), lactone bodies (89),polypeptides (326), and lactic dehydrogenase (598). A method for determining DX.1 coiicentration and composition by spectral changes was described (293). Maxima a t four wavelengths were recorded and a 5-mill calculation produced the required information. Another method suggested that D S h possessing the four usual bases could be used for simultaneous spectral determination of base composition (687). Spectrophotometric titration equipment was described using a single-beam instrument for application to nucleic acids (678). A unique detection and digital conversion system allowed precision to be attained with less than 1% coefficient of variation. Ultraviolet VOL 40,

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spectra were utilized in a computer analysis of oligonucleotides (494). A prograni was preseiited which used a curve-fitting approach in a series of equations. A differential adsorptionspectrophotometric assay method for nucleotides was described (3) using a charcoal column and determining the molar absorptivity of eluted nucleotides. The problems in low temperature spectrophotometry of turbid specimens were discussed (181). Multiple reflection of forward scattered light decreased the amount traversing the sample; a means for minimizing this loss in a Dewar device was described. Turbidity corrections were also noted when spectra of heme proteins were determined (256). Total lipids and lipoproteins were assayed turbidometrically (193). Difference spectra were employed to distinguish gelatin clots from fibrin clots and fibrinogen (531). A spectrophotometric assay of mitochondrial oxygen uptake was described (41). Infrared (IR) spectrophotometry has been shown to be applicable to determining the secondary structure of globular proteins (470), studying nonheme iron proteins a t liquid nitrogen temperatures (664),and determining the base composition of D S A (217). Attenuated total reflectance (.ATR) has been shown to be useful for infrared studies on human tissues to 3000 cm-’ (474). iinother report described a “Skin Analyzer” for in vivo analysis of tissue by ,ITR (64). A semiautomatic analysis of serum triglycerides and cholesteryl esters using I R \vas described (211). A double-beam I R spectrometer was described for monitoring column effluents in an automated liquid chromatograph (2%). Infrared spectra of microorganisms were shown to be masked by the cellulose material of bacterial filters, but were readily apparent when the bacteria were deposited and scanned on microporous silver filters (365). Atomic absorption spectrometry has been applied to the rapid measurement of lead in urine (354), cadmium and mercury in urine and blood (53, 504), and sulfur (529). I n cobalt analysis, amine interference was reported to be significant and could be minimized by use of a nitrous oxide-acetylene flame (274). Fluorescent spectrophotometry was the subject of a review describing the techniques used in clinical pathology and their interpretation (483). Methods were described for determining nucleic acids (378, 6361, inulin (644), catecholamines (168), and histidine and spermadine (358). A semiautomatic flow system was described for fluorometric estimation of plasma pyruvate, lactate, acetoacetate, and P-hydroxybutyrate in enzymatic reactions (282). Another enzymatic, fluorometric

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method was suggested for determining

0.2 mg of glucose in 100 ml of urine (555). Fluorometric assays were proposed for 6-mercaptopurine in serum (202),xanthine oxidase (258), and urea in nanoliter samples (527). Fluorescein isocyanate was used to label antiserum for determining and identifying microorganisms (614). A versatile device was proposed for microscopic spectrofluorometry to study excitation, emission, and absorption spectra of nerve cells (621). X-Ray fluorescence was the subject of a report describing a method for measuring nanogram quantities of zinc in biological specimens (676). Many reports have appeared during the period of this review describing the application of rarely used light-emitting reactions to analytical chemistry. The state of present knowledge and practice was presented in a monograph ($85) and a review (284) and definitely pointed toward the increased use of these techniques. The simplicity and sensitivity (nanogram levels) of luminescent methods of assaying organic compounds was described in an ozoneinduced method (80). A method for studying lipid and lipoidal substances in yeasts and animal cells was described ( 7 ) . The application of sensitive luminescence to biological processes was reviewed with attention being given to oxidative and radical-forming reactions (29). I n many instances, free-radicals could be detected at lower levels than is possible with ESR techniques. Automation for luminescent measurements of blood iron, indole, and adrenalin in biological samples was described (652). An excellent monograph was published which bridged the gap between chemiluminescence and bioluminescence by surveying the present state of theory and applications (568). Perhaps it is because of this book that a surge of papers has recently appeared describing the use of the unique light reaction of the firefly for measuring adenosine triphosphate (ATP). The reaction has been applied to the assay of ATP in enzymatic processes ( 2 ) , erythrocytes (6, 56, 87), plasma (299), and bacteria (132, 160, 298, 379). I n most of the reports, attention has been given to instrumentation and to extending the limits of sensitivity for ATP to the picomole level (393). I t seems safe to predict that such activity portends increased use of these methods in biochemical analysis. RADIOMETRIC ANALYSIS: ELECTRON MICROPROBE, NEUTRON ACTIVATION ANALYSIS

Two worthwhile texts covering this area of analysis were published: One was an updated third edition of a wellknown standard (120); the other, a continuing series ( 5 3 4 , emphasized detailed

applications rather than principles and will be of most interest to the specialist, The use of liquid scintillation systems in laboratory medicine (175, 651) was reviewed. A bibliography was published which listed all new isotope techniques and instruments used in the biological sciences (650). Liquid scintillation techniques of note to appear during the period of this review included a new 4~ detector that permitted high sensitivity and repeated counting (590) and a new method for counting labeled nucleic acids (628). New solubilization procedures for tissues were described which allowed low energy beta emitters to be counted with true solution efficiency (266). The determination of picogram quantities of bound estradiol (43) and a method for I4CO2 combined with heme or copper proteins (638) were proposed. A new method for measuring 14C uptake in photosynthesis was described (557). d new type of incubation system for measuring I4CO2 (SO) in which an air stream transported the gas directly to the scintillation vial and allowed rapid counting was proposed. Two relatively simple methods for measuring the radioactivity of labeled proteins after paper chromatography (113) and disk electrophoresis (81) were described. A radioactive-trapping assembly was proposed for monitoring aerobic microbial cultures and allowed 1 0 0 ~ orecovery of labeled I 4 C (662). A radiometric determination of pyridine nucleotides to 10+ mole in enzymatic processes was described (570, 571). A method for determining radioisotopes based on simultaneous distillation, separation, and gamma spectrometry was the subject of another paper (547). The techniques of radiochemical analysis lend themselves readily to computer treatment and reports have appeared describing programs for calculating spectrometry data from dual-labeled isotope studies (86,410). A review was published describing the current methods for combining autoradiography and electron microscopy (111).

The usefulness of an electron microprobe X-ray analyzer was discussed (259, 318). .All elements down to magnesium in the periodic table could be detected in concentrations of 0.01% or 10-l~gram in a k 2 . Its application to electrolyte analysis of biological fluids is obvious. The technique was also reported to have been used for estimating the phosphorus and calcium content of dentine (336). The increased number of references to activation analysis attests to its reliability and sensitivity in biology and medicine. Several general reviews have appeared (77, 95), in addition to others that have described applications and limitations of the technique (78, 94, 130). I t has been used to assay extra-

thyroidal iodine down to 10-9 gram (344, 425). Combined with electrophoresis or chromatography, it has been extended to the determination of base ratios in nucleic acids (206, 5@), phosphoryl peptides ( 5 7 4 , and proteins (203, 546). Activation analysis has been a great aid in determining essential and nonessential elements in humans (198, 288, 385). Specific techniques are described for fluoride (197), copper and zinc in lung tissue (340),manganese in blood (138, 624), selenium (603),copper in plant tissue (92), and zinc in human enamel (449). A freezing technique to minimize radiolysis of water in biological specimens was described (96). The use of a semipermeable membrane as a preconcentration matrix for ions was reported as a distinct aid in the analysis of protein samples (179). As is the case for most analytical techniques, activation analysis data can be processed directly by computers (278) and a general program for most biological systems has been written in FORTRAN (618). MASS SPECTROMETRY

Few reports appeared during the period of this review that possessed sufficient novelty to be cited. The major interest was focused toward the combination of gas chromatography and mass spectrometry. The combined technique has been suggested as the best approach to studying human metabolic pathways where numerous significant compounds are present in complex mixtures (501). Reviews have been written that present the application of this combined technique to the analysis of proteins, peptides, and amino acids (420), bile acids (584), deuterium-labeled organic acids (177), lipids (377), and plant sterols (350). Instrumentation was described (189) and commercial systems are now available. The mass spectrometer was used in the automatic determination of the amino acid sequence in peptides (569). Peptides were used in which the Nterminal amino acid was labeled with a readily identifiable group. The data were read directly from photographic plates into a computer and the sequence calculated. ELECTRON MICROSCOPE, LASERS

Quantitative electron microscopy was adequately reviewed with reference to dry mass determinations (31). A mass range of lo-" to lo-'* gram allowed investigation at the level of individual particles. The newest advance in this area has been high resolution electron microscopy with superconducting lenses at liquid helium temperatures (201). This technique imparts exceptional stability to high quality images. Elec-

tron micrographs of catalase were recorded 5t 4.2' K at the resolution of 10-20 A. Cholinesterase distribution in neurons was demonstrated (382) and copper deposits in tissue (556) were shown to be detectable under the electron microscope. A laser microprobe for elemental analysis in histochemistry was described (235) and application to in vivo measurements discussed. MAGNETIC RESONANCE

Reports have appeared with increasing frequency of the routine application of nuclear magnetic resonance (NNR) and electron spin resonance (ESR), as biological systems are being probed at submolecular levels. Three excellent texts cover this field of analysis: one in a general manner (176), and the others aimed a t ESR (25, 489). The latter area was also reviewed (561), where the determination of paramagnetic ions, Fe(II1) high spin and low spin complexes, copper complexes, free radicals, charge transfer complexes, and triplet states were discussed in relation to biochemical compounds and processes. A novel low temperature control device for ESR studies was described for temperatures of 80-273' K (267). The report showed that these temperatures could be maintained for optimum study of signals a t g = 1.94. Xuclear magnetic resonance spectrometry of naturally occurring polyphenols was reported (195). Halide ions were suggested as chemical probes for NMR studies of proteins (604) and shown to yield information analogous to that obtained by ESR studies on spinlabeled molecules. Proton spin-lattice relaxation times were reported in a paper on pulsed N M R (45). I t was shown that prior removal of solvent protons by exchange with deuterium was necessary for precise measurement of relaxation times. A review on micro-h'MR in high permanent magnetic fields discussed the application of this technique to medical diagnoses (459). Another significant paper discussed the determination of aqueous proton magnetic resonance shifts in biomedical samples, using an external calibration system (460). A coaxial tube system was described and different calibration systems discussed with regard to studies in fields up to 37 kG. The same paper discussed studies in fields up to 200 kG for NMR investigation of water molecules in biological systems. ELECTROCHEMICAL METHODS

Polarography and its biological applications were reviewed with emphasis on the oxygen cathode (582) and cysteine-containing proteins (505, 542). A theoretical study of the relation between the polarographic current and

the oxygen tension in tissue and suitable instrumentation for the study were presented (509). A sealed polarograph suited to slow respiration studies of 5 mpmoles of oxygen/hr in 0.2 ml (goy), as well as a diffusion exchange respirometer using a carbon dioxide electrode, was described (522). Highly sensitive pulse polarography was shown to be applicable to the study of small conformational changes in proteins (124) and DNA (469). A method for detecting and measuring dextran sulfate, heparin, polyphosphate, and hyaluronate by ac oscillopolarographic techniques was described (72). The oxygen electrode was used to rapidly mea, sure blood oxygen content ( Q l l )monitor microbial cultures (5867,and determine activity of catalase and peroxidase (253).

The biochemical application of cation-sensitive glass electrodes has been the subject of many papers. A comprehensive survey was published which updated the theory and practice of all aspects of the glass electrode (178). A recent review describes these devices as the interface between man and machine (243). The measurement of sodium on the skin surface has provided a rapid diagnostic test for cystic fibrosis (567). -4glass electrode method for determining blood sodium and potassium was shown to compare favorably with flame photometry (490). Several applications have been described for use with cerebrospinal fluid (545) and urine (521, 437). An inert, polyvinyl chloride membrane impregnated with tributylphosphate and thenoyltrifluoroacetone was used to make a calciumspecific electrode that was not affected by sodium, magnesium, or barium (68). Indeed, these new approaches to electrode technology hold a great deal of promise for rapid measurement of many ions. A precipitate-impregnated silicone rubber membrane was used in the construction of an electrode sensitive to sulfate and phosphate (516). Liquidliquid membrane-type electrodes have been described for measuring calcium (552) and copper (515). Probably the greatest impact will be felt from the introduction of the enzyme electrode (631). This device is a miniature glucose transducer composed of a polymerized acrylamide gel membrane containing glucose oxidase on an oxygen electrode and is used to measure the concentration of glucose in vivo. The measurement of intracellular p H of muscle using microelectrodes was described (112). One-, two-, and threebarreled electrodes allowed the measurement of pH, transmembrane potential, and extracellular potential, and showed the p H of a resting cell to be 5.99. A transistorized battery-operated pH meter was described (159) which incorporated a fast response pH electrode VOL. 40, NO. 5, APRIL 1968

25 R

(2 msec) with a noise level of 0.001 p H units. Amperometric titration of thiol groups in imidazole buffer (588) and improved apparatus for the titration (535) were reported. A novel differential titration method was described which allowed determination of pK values of phenolic groups in tyrosine and related compounds (215). A rapid, sensitive amperometic titration of native proteins was described (125). A coulometric determination of nonprotein nitrogen was proposed (126). d simple rapid method for measuring methionine with a platinum electrode was described (863). Direct measurement of oxidation-reduction potentials in bacterial cultures (589) and biological solutions (406) was reported. A paper was noted where high frequency reflectometry was used for measurement of small variations in electrolytic conductance and could be applied to dynamic measurements (256). GASOMETRIC ANALYSIS

The older methods for biochemical analysis continue to be used in spite of the sophisticated instrumentation now available in most laboratories. A new device was described for accurately and rapidly calibrating the Warburg respirometer (673). The quantitative determination of ammonia, urea, and ketone bodies with a modified microdiffusion vessel was described (643). Microdiffusion vessels were also used to determine protein nitrogen (450) and blood oxygen (135). A technique was proposed for measuring respiration in electrically stimulated cerebral cortex tissue (465). Monitoring the carbon dioxide, oxygen, and pH of cell cultures was the subject of a paper describing the construction of suitable control units (272). A gasometric-electrometric method was described for determining oxygen in photosynthesis reactions (47). A calibrated oxygen electrode was employed that enabled initial reaction rates to be studied. INSTRUMENTATION AND AUTOMATION

Many instruments having reference to a particular application have been described in the preceding sections where pertinent. General texts have been published describing instruments for biochemistry (239) and clinical medicine (605). Reviews have examined new instruments (348), automated analysis in nutritional research (187), automation of blood lipid analysis (BO), kinetic parameters of continuous flow analysis (622), and the definitions and limitations of clinical chemistry “standards” (508). A survey of analytical chemistry instrumentation

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as applied to aerospace research was published (669) which reviewed advances in spectroscopy and chromatography. An inclusive reference guide to all scientific instruments was again available (442). Its usefulness in searching for a specific instrument, or a general survey of a class of instruments, is based on a coding and classification system which categorizes instruments and their components. A review of advances linking the medical and measurement sciences was published (493). Improved multichannel flame spectrometers (218, 314) were described, including an integrating model for simultaneous microanalysis of sodium and potassium, without the use of photomultiplier tubes (457). Another instrument was designed for the rapid analysis of biological fluids and tissues (255). Only one detector and amplifier were required for the simultaneous determination of sodium, potassium, and calcium a t the rate of one sample/min. A microspectrophotometer was reported for measuring absorbing substances in individual biological cells (10). il recording microfluorospectrophotometer capable of detecting low light levels for tissue fluorescence was described (538). A nanosecond fluorometer to measure emission kinetics of chromophores in excited states was described, in which the analog output of the sampling oscilloscope was digitized, averaged on a LINC computer, and transferred to magnetic tape for processing by an IBM 7090 computer (312). iinother paper described a recording fluorescence polarization photometer which incorporated an analog computer to generate a variety of functions (258). An electromagnetic shutter, with a total open-time of 35 psec, was suggested as being applicable to short-duration absorption spectroscopy (486). Complete p H titration curves on protein complexes could be determined within 1 hr on a new recording potentiometric-spectrophotometric titrator (462). The study of photoinduced reactions was facilitated by the report of a differential flashphotolysis kinetic spectrophotometer (2.47)with a response time of 1.4 psec. A high precision magnetic float densitometer (423) and a stable linear nitrogen analyzer (70) were reported. A device was suggested for measuring voltage-current relationships in biological membranes based on ion flux through them (366). The calorimetry of subcellular particulate enzyme systems was made possible by a differential calorimeter, which measured the enthalpy of oxidation of NADH (487). A new mass spectrometer, composed of two electrostatic and two magnetic 90’ lenses, was described (667). I t could be programmed for mms and energy resolution of primary and secondary ion species.

Rapid, automatic protein analysis has been described (154) and a “protein sequenator” instrument reported (173). The latter instrument uses the Edman degradation of proteins to automatically split off and identify the sequence of constituent amino acids. The novel use of existing instrumental techniques was reported infrequently. Differential thermal analysis has not been applied to many biological materials; a single report on denatured DNA and nucleosides showed that the bases did not adsorb on the double-stranded helical structure (308). The heat capacity of cream, milk, and skim milk was shown to be directly indicative of their fat content (219). A photoelectric technique for measuring coagulation of human plasma was described (426). Optical rotary dispersion was shown to be capable of distinguishing base-paired double-stranded RNA from the singlestranded structure (104). MISCELLANEOUS

This section presents those methods that, by virtue of their novelty, simplicity, or complexity, are not easily categorized in the preceding sections. The determination of sulfhydryl groups was described, using either 4,4’-or 2,2’-dithiodipyridine to detect less than 0.05 l g (242). Radioactive mercurials (371), tetraethylthiurain disulfide (439), and 2,2‘-dihydroxy-6,6’-dinaphthyldisulfide (681) were also suggested as useful, sensitive reagents. Cytochemical methods for determining RNA were reviewed (616). Methods for studying chemical mechanisms of nerve transmission a t the cellular level were reviewed, with specific reference to catecholamines and cholinesterase (186). Another review (391) presented methods for obtaining and weighing single neurons and for analysis of enzymes and substrates in the lo-‘* mole range. A simple method was described for determining the water content and protein concentration in subcellular elements (232). Several reports described new sensitive methods for determining tryptophan in proteins ( 5 9 4 , acetyl groups in proteins and peptides (GOO), protein in dried herbages (506), humic acid in humus and soil (573), total serum protein by refractometry (408), protein with membrane filters (362, 562), and iron in biological samples ( 2 4 ) . Titrametric assays were described for nonpolar acids in plasma and depot fat (352) and sulfur in biological material (106). The determination of lysine in foods was proposed using chromatographic and polarographic methods (69). Microbiological techniques are often ignored in today’s analytical laboratories because of the time required for growth. Nevertheless, the following are of interest; a method for detecting as few as 23

bacterial cells in 1 h r using 32P-labeled nutrients (418); growth methods for detecting trace metals at the picogram level (446); a review of the large-scale growth of tissue cultures (479); and an automatic system for preparing culture media for long-term continuous-flow cultures (275). An interesting paper described a computer-based analysis of biochemical data (220). Particular reference was made to the treatment of systems undergoing transient changes between steady states. This method enabled the investigator to witness the manifestations of metabolic control, The report stated that control during a transient process was subject to two constraints; the direction of change of a supposed control substance and the time of duration of the change. Another noteworthy report dealt with the studies of rapid reactions by flow and relaxation techniques (279). Methods were described using temperature-jump, pressure-jump, electric field pulse, continuous perturbation, and rapid quenching. It was suggested t h a t such an approach could be applied to reaction kinetics and mechanistic problems in any branch of chemistry. It is perhaps fitting to end this survey with a discussion of the new development of preparing insoluble derivatives of biologically active proteins. The author considers this process to be the most exciting and promising technique developed for significant application and research in biochemistry. Binding enzymes to insoluble matrices has proceeded from an interesting theoretical effort to a practical technique. A comprehensive review of past research, present applications, and the potential of covalently binding enzymes to polymeric lattices has been written by the pioneering scientists in this area (581). Insoluble enzymes have been used as heterogeneous, specific catalysts in batch or columnar form because of the ease with which they can be removed from the reaction mixture. The stability of the enzymes may allow repeated use of the material for treating large volumes of substrates. Immobilized enzymes may be prepared in several ways. Recent reports describe coupling the enzyme to cellulose (339) and entrapping the enzyme within the lattice of polyacrylamide gels (290) or dextran gels (116). The latter report describes a method for imparting heat stability to the enzymes. Bound enzymes have been used in the production of peptide products from proteins (466), the isolation of enzyme inhibitors (216),separating antibodies from sera (566), and in reagentless analysis of enzymatic processes (632). The concept of immobilized enzyme activity offers many advantages to biochemistry, aside from the aspect of economy when using costly enzymes. A most exciting aspect is the

possibility of embedding the insolubilized, active material in membranes or tissues for in vivo study or therapeutic purposes. LITERATURE CITED

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(671) \Voof, J. B., Pierce, J. S.,J . Chrom a t o g , 28,9.2 (1967). (622) n’otiL. If. H.. Claik. S. J.. Eds.. “Gaq Chrornatogrnphy ii; the knalysis of Steroid IIormoiies,” Pleiiiim PLtblishitig Corp., l-ew York, 1966. ( 6 7 3 ) 1-amagiichi, )I., rIoward, F. D., Pratt, 11. K., dnal. Biochem., 16, 338 (1966). (6i4)Yiinosov, S.Yii., Ed., “Chemistry of Satiiritl Compoiuids,” Yol. 1, Y o . 1, The Faraday Press, Sew York, 10fi.i. - _

(67.5) Zasepa, IV., I)obry-lliiclaus, A., J . Chinz. Phys., 63, 675 (1966). (676) Zeit/, L.. Lee. lt., .-trial. Biochem.. 14. 101 (1966). ( 6 i 7 j Zerfing, It. C., S’eeriing, H., ANAL.

CHEW,38, 1312 (1066).

(678) Zimmer, C., Ileiiiert,, II., :lnal. Bz‘ochein., 14, 1 (1966). (679) Ziporiii, Z. Z., Ilaiiion, 11. W., Ibid., p 78. iGS0) Zlat,ki*. h..Ed.. “,ldvaiices iii Gas

Chromatography l!j67,” Pre;itoti Techiiical iibstract.;, Evaiistoii, Ill., 1967. (681) Zwaau, J., dnal. Biochem., 15, : (682) Ibid., 21, 1.53(1967). (683) Zweig, G.j Whitaker, J. li., Eds., “Paper Chromatography atid Electrophoresis,” Vol. 1, Academic Press, New York, 1967. j - . , - - / .

Gas Chromatography Richard S . Juvet, Jr., Department o f Chemistry and Chemical Engineering, University of Illinois, Urbanu, 111. 6 7 80J Stephen Dal Nogare, Department o f Chemistry, Virginia Polyfechnic Institute, Blacksburg, V u . 24060

T

lication of the last review in this series (120) and covers the years 1966-67. Gas chromatogra1)liy continues to be one of the most active areas iii alialytical rheniistry. .i recent survey by the Satioiial Ycimce Fouiitlatioii (93) iiitlicates that 15.9y0of all analytical chemists hliecialize i i i the area of chromatogralihic aiialy.~is,second o n l ~ . iii numbers to those specializing iii absorptioii spectroscopy. Oiie marketing groul) (413) has estimated that 70,000 gas cICS a i r and I3oiielli i,5 ai-ailable through Variaii -4erograph Corp. (41.4), atid a nioiiogral~h by Votiz aiid Clark (653) gives detailed procedures used iii the gas chromatographic anal) hormones. Referelice texts iiiclude a coinliilation by 11cReyiiolds (415) of retention data for over 300 solutes, mainly aliphatic compouiids, on 80 commoiily used liquid phases expressed a t two temperatures as specific retentioil volume and retention index; a sequel to ail earlier AISTNcoinpilation by Schup~iaiid Lewis (630) providing data on reteiitioii indices, relative retention, and liquid phases for some 3800 compouiids; and a comprehensive handbook on gas chromatography published in East Germany (866). Four volumes of “.ldvaiices in

C h r o i i i a t o ~ r a ~ ~ ledited i y ’ ~ by Giddings and I d l e r have appeared during this bieiiiiium (193). Each of these volumes contailis several sections 011 gas chromatography. The pal)ers are generally of a revielv nature writteii by authorities iit the various areas of cliromatogral)hy. .I secoiirl ctlitioii of Heftniaii’s “C1irornatogral)h~”has appeared (242) and 14 of the 20 chapters of the book coiitaiii iiiformation of direct interest to the gas chromatographer. 1he ~iroceediiigsof two major symposia 011 gas chroniatography were pubed including the proceedings of the th Iiiterliatioiial Symposium on Gas Chroiuatogm~~liy :iiitl Aqsociated Techniques held iit Iioiiic, Italy (36‘3),aiid sponsored 11y t’he I{ritish Institute of Petroleum aittl the 1)roceedi~gsof the meetiiig, JouniCes Ilell&nesd’Etude des Met hodes de S i.para t io1i Imm6tlia t e e t de Cliromatograj)hie, sponsored by G.A.1I.S. (Groupeiiieiit pour l’.ivanceinerit des Methodes Spcctrographiques) aiid the union of Greek Chemists (466), Volume I of which cotitailis 30 papers on gas chromatography. Published rev i e w of‘ various sy1iq)osia devoted t o gas chromatography iiiclude the Sixth r ,

VOL. 40, NO. 5, APRIL 1968

33 R

Biochemical analysis - Analytical Chemistry (ACS Publications)

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