(352)M. Tasaka, N. Aokl, Y. Kondo, and M. Nagasawa, J. Pbys. Chem., 70, 1307 (1975). (353)A. Tateda and H. Murakaml, Bull. Cbem. SOC.Jpn, 47, 2885 (1974). (354)A. Tateda, A. Matsubara, and T. Suenaga, Mem. Fac. Scl., Kyushu Unlv., Ser. C, 0, 9 (1974). (355)A. P. Thoma, 2 . Clmermann, U. Fledler, D. Bedekovic, M. Guggl, P. Jordan, K. May, E. Pretsch, V. Prelog, and W. Simon, Chlmla, 20,
344 (1975). (356)J. D. R. Thomas, Proc. SOC. Anal. Chem., 11, 340 (1974). (357)H. Thompson and 0. A. Rechnltz, Anal. Chem., 46, 246 (1974). (358)W. Tomassl, Electrochlm. Acta, 10, 955 119741. .,. (359)L. Torma, J. Assoc. Off. Anal. Chem., 58. _ _ 477 (19751. i360) D. C. Tosteson, Mol. Mecb. Antiblot. Actlon Protein Blosynlh. Membr., Proc. Symp., 61 5 (1971)(Pub. 1972),E. Munoz, F. Garcla-Ferrandlz. and D. Vasquez, Ed., Elsevier, Amsterdam, I . - .
I
- I
The Netherlands. (361)K. Toyama and H. Hlrata, Japanese Patent 7438,478,Oct. 17, 1974; Appi. 70, 6768.
Jan. 26, 1970. (362)P. K. Tseng and W. f. Gutknecht, Anal. Chem., 47, 2316 (1975). (363)E. C. M. Van der Neut-Kok, J. De Gler, E. J. Mlddelbeek, and L. L. M. Van Deenen, Blochem. Blophys. Acta, 332, 97 (1974). (364)0. W. S. Van Osch, US. Patent 3,824,169, Jul. 16, 1974;Appl. 323,696,Jan. 15,
1973. (365)0. W. S. Van Osch and B. Grleplnk, Fresenlus’ 2.Anal. Chem., 273, 271 (1975). (366)J. Van Houwellngen, 0. W. S. Van Osch, and A. M. H. Weellnk, US. Patent 3,824,171, Jul. 16, 1974;Appl. 323,698,Jan. 15, 1973. (367)M. Vanko and J. Meola, Adv. Autom. Anal. Technicon lnt. Congr., 1, 37 (1972)(Pub 1973). (368)A. Vannl, Ann. Chim. (Rome), 63, 887 (1973). (369)P. Venkateswarlu, Anal. Chem., 46, 878 (1974). (370)J. Vesely, 0.J. Jensen, and B. NlcolaIsen, Anal. Chlm. Acta, 62, l(1972). (371)J. Vesely, Collect. Czech. Chem. Commun., 30, 710 (1974). (372)J. Vesely and K. Stulik, Anal. Chlm.
Acta, 73, 157 (1974). (373)R. Wawro and 0. A. Rechnltz, Anal.
Chem., 46,806(1974). (374)A. M. H. Weellnk, G. W. S. Van Osch, and J. Van Houwellngen, US. Patent 3,824,170, Jul. 16, 1974;Appl. 323,697,Jan. 15,1973. (375)D. Welss, Cbem. Llsty, 60, 202 (1975) (Czech).
(376)A. Wlkby and B. Karlberg, Electrochim. Acta, IO, 323 (1974). (377)A. Wlkby, Electrochim. Acta, 10, 329 ( 1974).
(378)A. Wlkby, Pbys. Chem. Glasses, 15, 37 (1974). (379)M. F. Wilson, E. Halkala, and P. Klvalo, Anal. Chlm. Acta, 74, 395 (1975). (380)M. F. Wilson, E. Halkala, and P. Kivalo, Anal. Chlm. Acta, 74, 41 1 (1975). (381)K. H. Wong, K. Yagi, and J. Smld, J. Membr. Blol., 16,379 (1974). (382)H. R. Wuhrmann, W. E. Morf, and W. Simon, Helv. Chim. Acta, 56, 1011 (1973). (383)N. Yoshlda and N. Ishibashi, Chem. Lett.. 493 (1974). (384)J. J. Zipper, B. Fleet, and S. P.-Perone, Anal. Chem., 46,21 1 1 (1974).
Electrophoresis R.
D. Strickland
Veterans Administration Hospital, Albuquerque, N.M. 87 108
This review extends the coverage of a previous one (137) by citing articles published between mid-1973 and mid1975.
METHODS AND APPARATUS Particle Electrophoresis. Particles such as cells and cell fragments carry charges when they are dispersed in a fluid. If the particles differ with respect to their ratios of charge to mass, they can be separated by electromi ration. Almost all electrophoresis of particles has been one on this rudimentary basis. This is surprising in view of the possibilities for improving separations by using more sophisticated approaches. One might, for example, use a viscous suspensions medium that would discriminate between particles with different hydrodynamic profiles. Another variation might be to impose electromigration against a density gradient; this would separate particles having the same mobility if the differed in density. One departure from simple electroprloresis, isoelectric focusing, has been used to demonstrate subpopulations of lymphocytes in the peripheral blood of both humans and rabbits (89). Even by simple electrophoresis, it has been possible to separate immuno lobulin-bearing and immunoglobulin-free lymphocytes ecause the latter migrate more rapidly (54). The observation that thrombocyte mobility in an electrical field diminishes in the preclinical phase of coronary artery disease (62, 134) is profoundly interesting; if the predictive value of this phenomenon is as high as is claimed, it will enable pro hylactic treatment of this disease. The cause of the re&ced thrombocyte mobility is not known, but it may be related to diminished phosphate or carboxylate groups on the cell surfaces (135, 136). Male and female sperm cells from humans can now be separated efficiently and without damage to the cells (128). Electrophoresis has proved to be a useful technique for preparing intact nuclei from tissue homogenates (139). Mobility measurements have shown that rat brain contains a t least two distinct classes of nuclei and that nuclei from human brain tumors migrate faster than those from normal tissue (7).
d
%
The uses of electrokinetic techniques for studying formed blood elements (86) and microorganisms (118) have been reviewed. An experiment with particle electrophoresis was done on Apollo 16. Two sizes of polystyrene particles were separated. The absence of thermal convection and sedimentation effects permitted sharper separations than were obtainable with the same apparatus on earth (129). Much more ambitious projects involving electrophoresis in space are now in progress according to a recent newsletter (Universities Space Research Association, P.O. Box 5127, Charlottesville, Va. 22903). Improved apparatuses for observin the electrokinetic behavior of cells have been described f67, 1 1 1 ) . Electroosmotic back flow can be reduced and made uniform by coating the walls of the capillary that serves as a migration chamber with agarose and plugging its ends with the same material (149). Laser doppler spectrometry has been used to measure the mobilities of blood cells; results are obtained uickly and agree with those found by direct microscopic aservation (147). Immunoelectrophoresis. The proteins of erythrocyte membranes, solubilized with Berol EMU-043 or other nonionic detergents, can be resolved into about 20 components by crossed immunoelectrophoresis in a arose gels that contain the solubilizing detergent (9,10). many as five antigens in a com lex mixture can be quantitated simultaneously if a higR titer antiserum is available; the antiserum need not be nonspecific (511. A new apparatus uses one gel for separations and another for specific reactions; it permits the analysis of proteins in dilute solutions, e.g., cerebrospinal fluid, without preliminary concentration (58). A technique called affinity immunoelectrophoresis makes use of four contiguous gels to display the characteristic zone electrophoresis pattern, the pattern following reaction with Sepharose-coupled antihuman serum albumin, the pattern in Sepharose alone, and the immunoelectrophoretic interaction of each of these patterns with free antibodies (19). A somewhat simpler version using the same principles has been used to investigate the interaction of concanaval-
1s
ANALYTICAL CHEMISTRY, VOL. 48, NO. 5, APRIL 1976
39R
lin-A with serum glycoproteins. The results obtained were equivalent to those found by the much more difficult and expensive method of affinity chromatography (13). Insulin is bound to albumin, az-macroglobulin and @-lipoprotein in both normal and diabetic serum. Diabetic but not normal serum immunoglobulins G and M also bind insulin. This shows that diabetics develop an immune response to insulin (28). A phenomenological theory of mass transport for antigen-antibody reactions has been developed. I t predicts man of the characteristic features of crossed immunoelectropioresis patterns (15, 16). Good results with immunoelectrophoresis depend upon understanding and controlling variables such as buffer composition, temperature and antiserum titer and specificity (55). Isoelectric Focusing. Ampholine (LKB-Producter) is the material most widely used to form the pH gradients needed for isoelectric focusing. It is a proprietary preparation that has been very expensive and sometimes difficult to obtain. These considerations have stimulated attempts to develop pH-gradients by other means, such as the use of concentration gradients with nonelectrolytes to alter the dielectric properties of the medium (142, 143) or thermal gradients (141) to modify the pH of a buffer and the use of casein hydrolysate as an ampholyte (130). It is doubtful that a suitable substitute for Ampholine will be found, short of synthesizing a new series of compounds with comparable properties. The method of synthesizing Ampholine itself had not been generally understood until a recent publication (120) described the process in precise detail. This preparation develops a gradient in the pH-range 3.0 to 9.5. The synthesis is easy and the product is both satisfactory and inexpensive. A companion paper (53) describes a continuous electrophoretic method for fractionating the ampholyte into preparations that develop gradients within a range of two pH units. Narrow ampholyte pH ranges are highly desirable because they permit substances with nearly equal isolectric points to be separated. Ampholine with a pH-range of 2.5 to 4 for fractionating acidic proteins has been prepared (151). Another ampholyte has been synthesized from tetraethylene pentamine and pentaethylene hexamine bases by using propanesulfone, sodium vinylsulfonate and sodium chlormethylphosphonate to introduce acidic groups. Neither the phosphonic nor the sulfonic acid groups offered advantages over the carboxylates of Ampholine, and the new ampholyte tended to form complexes both with itself and with proteins (109). Several papers cited in the previous review (137) suggested that Ampholine could form complexes with proteins, but it has been shown that this does not occur with albumin, ferritin, @glucuronidase, or serum proteins (34) or with human growth hormone or ovine prolactin (8). Recently, Ampholine has contained materials that are stained by the commonly used dyes. These artifacts are most prominent in the 6 to 9 pH interval. They do not occur with Ampholine manufactured under the original patent (106), possibly they are impurities in the Ampholine (164). The optical properties of Ampholine including fluorescence emission, ultraviolet and visible light absorption spectra, and optical rotation indicate that it contains nitrogen heterocycles. New Ampholines contain no asymmetric carbon but pre-1970 specimens do, because the pH 3-5 and 8-10 ranges were reinforced by adding lutamic and aspartic acids and lysine (119). A study to fetect possible biological effects such as toxicity and interference with bioassay systems has revealed no objectionable properties of Ampholine (156). The general methodology of isoelectric focusing (40, 153) and of isoelectric focusing in thin polyacrylamide gels (152, 154) has been described. Some, but not all, proteins show different multiband patterns and different isoelectric points depending upon whether focusing is done in a cylinder or in a slab. Artifacts and denaturation occur in slabs if samples are applied near an electrode but this does not happen with cylindrical gels (42). Improved performance results when ammonium and acetate buffers are used in electrode chambers instead of the conventional strong acids and bases (138). The isoelectric fractionation of sulfated glycoproteins can be greatly improved by coating the walls of the column with polytetrafluoroethylene (70). The isoelectric points of albumin, @-lactoglobulin,carbonic an40R
ANALYTICAL CHEMISTRY, VOL. 48,
NO. 5,
APRIL 1976
hydrase, and myoglobin have been established so that these proteins can be used as markers for measuring the isoelectric points of other proteins (14). If electrofocusing is performed in a gel that has been cast around a plastic rod that is centered in a tube, the fractions can be visualized by removing the rod and replacing with a staining solution; this reduces staining time to 90 minutes (169). The same system can be used for conventional electrophoresis (170). Proteins that have been solubilized with sodium dodecylsulfate (SDS) can be analyzed by isoelectric focusing after removing excess SDS by dialysis (100). Deuterium oxide (sp gr 1.105) can be used with water to form a density gradient. The D-isotope causes a rise in the pKa of acidic groups but pH-meter readings are lower in D20 than in water because of a shift in the asymmetry potential of the glass electrode (46, 47). An apparatus has been designed that permits the preservation and recovery of biologically active substances following determination of their isoelectric points; it uses a gradient density column that contains 1.5 ml of ampholyte (45). If Sephadex gel chromatography is performed first and then isoelectric focusing of the fractions is carried out in a polyacrylamide gel slab, the components of a mixture are seen classified in a two-dimensional display, both according to molecular size and isoelectric points (88). Antigens can be located in a slab of gel after focusing by laying strips of paper that have been soaked with antiserum on the gel (30). Preparative electrofocusing of proteins in 40-mg amounts can be accomplished in acrylamide gel slabs with a quality of resolution good enough to permit the isolation of five biologically active human gonadotropins (57). It is possible to focus proteins in gram amounts by using pH gradients stabilized in beds of a granular gel such as Sephadex or Bio-Gel (114,115). The load capacity of Sephadex is 5 to 10 mg of protein/ml of gel; H 3 to 10 ampholyte can carry 0.25 to 1 mg of proteinfml and narrow range ampholytes from 2 to 4 mg/ml (113). Equipment for continuous preparative isoelectric focusing has been designed that permits separations of 50 mg of mixed proteins/ hour (69). When preparative focusing is done in a gradient density column, there is a loss of resolution because of diffusion unless the voltage gradient is maintained while fractions are being removed (12). An elaborate apparatus permits continuous monitoring of the focusing process and also the diffusion of focused zones after the electrical field is removed (20). Data obtained with this apparatus can be used to calculate minimum focusing times (21), isoelectric points, apparent diffusion coefficients, and the slope of the pH vs. mobility curve (23). The necessary computational procedures for processing data obtained with this equipment are available (22) as are the mathematical treatments for calculating any of the parameters mentioned above (161). Some of the results obtained by isoelectric focusing are especially interesting. Human cervical mucins separate into from 3 to 5 bands in the pH range 3-6. These bands are Schiff positive but do not accept protein stains (162, 163). Heparin contains at least 21 fractions with molecular weights ranging between 3000 and 37 500. Only fractions with molecular weights of 7000 or more have anticoagulant properties. All of the heparin fractions yield identical end products when they are degraded with enzymes from Flavobacterium heparinum (99). Phenotyping of a-1-antitrypsin can be accomplished in 4 hours (2). The patterns obtained with egg white proteins are useful for taxonomic studies (48).The heterogeneity of bacterial neuraminidases can be demonstrated in a special apparatus that allows multiple samples to be analyzed at one time (59). The mechanism of antibody formation in rabbits has been investigated (18, 165). Following solubilization with a nonionic detergent (Emulphogene) rhodopsin and opsin (bleached rhodopsin) can be separated. Rhodopsin focuses at pH 6 while opsin focuses at a lower pH (74). The monoclonal immunoglobulins in the serum from patients with myeloma yield patterns that are distinguishable from the normal immunoglobulins which are polyclonal in origin (102). Bovine luteinizin hormone has two major (pZ 8.3 and 8.8) and one minor fpI 4 ) active components (167). The e~tradiol-l7/3-~H proteins from the uteri of immature rats have been studied (29). Tay-Sachs disease can
-
Rlchard D. Slrlckland is a research biochemist at the Veterans Administration Hospital in Albuquerque, N.M. He was originally employed by this institution, as clinical chemist, upon receiving his Ph.D. from the Fniversity of New Mexico in 1953. He has published extensively in the area of analytical biochemistry. A number of his papers have been concerned with electrophoresis as applied to the separation and measurement of proteins. At this time, he is investigating the modes of Interaction with polyvalent cations of proteins, peptides and amino acids.
be diagnosed before birth by demonstrating a deficiency, an altered PI and a splitting into two peaks of the hexoseaminidase in amniotic fluid (66). The methodology of isotachophoresis has been reviewed (1211. It has been used to separate soil humic acid into 5 bands and fulvic acid into 3 bands (31).The isotachophoresis of human growth hormones, both preparatively and analytically has been described in detail (27,81). Conventional Electrophoresis. The use of gels with linear (44) or exponential (79) concentration gradients enhances the resolution of complex protein mixtures. The pore gradient of dense polyacrylamide gels can be improved by varying the degree of cross-linking (95). Many gradient gels with volumes of 1 to 50 pl can be prepared simultaneously in capillary tubes (32). Gradient gels of this size can be used for measuring protein molecular weights (124). It has been reported that polarizing solutions of acrylamide monomer prior to polymerization gives gels in which proteins migrate more rapidly and in which more fractions are obtained (83). SDS-gels are superior to urea gels for separating nuclear proteins because these proteins do not enter a urea gel (50). Polyacrylamide-agarose gels made 6 M in urea promote reduced band width and superior resolution of single stranded RNA; molecules differing in size by only 5% are separable (43).The uses of molecular sieving effects to enhance separations in gel have been reviewed (71). Cross-linked hydrolyzed starch gel has resolving power comparable to conventional starch gel but greater strength and elasticity (97). The electroosmotic flow in agarose can be reduced by treating the agarose with QHE-Sephadex which is a strongly basic anion exchanger (76).Density gradients generated in small glass tubes can be used to stabilize separations of viruses and proteins during either electrophoresis or isoelectric focusing (82). Tween 20, a nonionic detergent, has been used to solubilize erythrocyte membranes selectively by varying the pH and ionic strength of the extracting buffer. Some proteins extracted by Tween 20 split when exposed to SDS (sodium dodecyl sulfate) (90). Erythrocyte membranes solubilized in SDS and subjected to two-dimensional electrophoresis, first with and then without added SDS, gave patterns characteristic for each blood group. The method is useful for detecting membrane defects such as those found in congenital dyserythropoetic anemia ( 5 ) . There has been a comparative study of the performances of the various agents that are used to solubilize erthrocyte membranes (26). Growth hormone and prolactin from rat pituitaries can be isolated and assayed following SDS solubilization and electrophoresis in SDS-polyacrylamide gel (168). The uses of SDS solubilization and electrophoresis for characterizing myelin proteins have been thoroughly reviewed ( I ) . The proteins, including enzymes, of plants can be extracted by using dimethyl sulfoxide. The extracts can be stored at -29 "C but they slowly deteriorate ( 6 ) . Comassie Brilliant Blue G250 is colorless in 3.5% perchloric acid but it reverts to its blue color when bound to proteins. This makes it possible to stain zones of protein in a gel without the need for destaining. It does not stain Ampholine (117). Proteins can be stained brilliantly prior to electrophoresis by using a derivative of Ramazol Brilliant
Blue R; the staining is quantitative but intensity varies among different kinds of proteins (33). A technique for staining proteins in agar gels with silver is reported to give excellent uantitative results (80). Fluorescamine can be used to l&el proteins without causing interfering background fluorescence; this facilitates locating the proteins when molecular weight measurements are made in SDSgels (38). Fluorescamine is also a good marker for peptide mapping (122);it detects spots that cannot be found with ninhydrin (49). Pinacryptol yellow stains SDS-protein complexes rapidly and does not require destaining (133). Ceruloplasmin staining with p-phenylenediamine is superior to that with dianisidine because of greater sensitivity and because alcohol, which causes gel shrinkage, can be eliminated from the staining procedure (911. If glucose-6-phosphate dehydrogenase is polymerized into an acrylamide gel, it remains immobilized during electrophoresis. Such a gel can then be used to detect hexokinase, glucose phosphate isomerase, and phosphoglucomutase. This method of immobilization could probably be applied to many proteins (65). Fungal carbohydrases can be detected in a gel slab (after isoelectric focusing) by allowing them to act upon a soluble polysaccharide substrate; the reducing sugars that are produced by enzymatic action are absorbed on filter paper and detected chemically (39).Cellular polypeptides that have been double-labeled with 3H and 14C can be used to detect differences in microorganisms by statistical analysis of differential isotope uptake (160). The conditions and procedures for the quantitative electrophoresis of eye lens proteins in polyacrylamide gels have been carefully worked out (166). Ribonucleic acid can be separated and measured in polyacrylamide agarose gel using samples weighing only g (85). Cyclic adenosine monophosphate, adenine, adenosine, hypoxanthine, adenosine monophosphate, inosine, inosine monophosphate, adenosine diphosphate, and adenosine triphosphate can be separated quickly by high voltage electrophoresis on paper (105) or preparatively by free flow electrophoresis (107). The electrophoretic behavior of interacting molecules (17, 104) and the electrophoretic mobility of randomly oriented cylindrical particles (36) have been described mathematically. There is a new densitometer that makes use of a soft laser beam for scanning patterns; its ability to resolve is far superior to conventional scanners (171). Another sophisticated device scans photographs of patterns and prints out relative mobilities and concentrations of the components a t the rate of one pattern per second (159). Preparative Electrophoresis. Preparative methods involving both batch (94, 148) and continuous free-flow systems (63) have been reviewed. Several excellent apparatuses for preparative electrophoresis in gels have been described (24, 72,131,150,155). One has been constructed to conform with theoretical and practical requirements that have been discussed in detail; data illustrating its performance are given (103). Most of these provide for removing fractions from the gels by electromigration and incorporate a system for concentrating the samples. A contaminant, probably oligoacrylamides with molecular weights similar to the small proteins, is present in effluents from polyacrylamide gels, it can cause difficulties in preparative procedures because it varies in staining properties, uv absorptivity, and charge, and is hard to separate from proteins (64). A simple sucrose gradient density apparatus eliminates separate buffer reservoirs, electrode chambers, and bridges; it can be used either analytically or preparatively with particulate as well as soluble samples (146). One free-flow apparatus permits continuous preparation by electrophoresis, isoelectric focusing, or isotachophoresis (112). The combined effects of convection, electromigration, and diffusion upon solute concentration profiles during electrophoresis have been discussed in a mathematically oriented paper which is intended to serve as a guide for designing and predicting the performance of free-flow preparative apparatuses (116). Two new books on electrophoresis have been published (3, 25). General electrophoretic methodology (75, 87) and the methodology of molecular mapping in polyacrylamide gels (77)have been reviewed. ANALYTICAL CHEMISTRY, VOL. 48, NO. 5, APRIL 1976
41 R
MOLECULAR PROPERTIES Structure. An interesting technique for separating circular from linear DNA even when the two configurations have the same electrophoretic properties involves forming an agarose gel in a DNA solution. The linear DNA can be eluted electrophoretically but the circular DNA is immobilized, apparently because the rings are topologically linked with the gel reticulum (35).The use of a similar technique for immobilizing glucose-6-phosphate has already been mentioned (65). Arrehenius plots of enzyme activity (log velocity (saturation) vs. temperature) are sometimes discontinuous. Such behavior is consistent with, but not demonstrative of, the existence of conformers (temperature dependent polymers). In the instance of histidine-ammonia lyase, the conformer hypothesis has been confirmed by showing by electrophoretic means that it exists either in one or two forms depending upon the temperature and that the interconversion is completely reversible (98). Immunoglobulins are made up of two heavy and two light peptide chains that are linked together by disulfide groups. Immune globulin G is monomeric, immune globulin A may be polymeric with two or more of the fundamental four-chain structures linked by disulfide bonding. The chain structure of a globulin can be resolved by a single two-dimensional electrophoresis in SDS-polyacrylamide gel. The SDS disrupts noncovalent interactions between protein molecules. Electrophoresis in the first direction serves to classify the different s ecies of globulins. Mercaptanethanol is then used to reckce the disulfide bonds in situ. Electrophoresis in the second direction separates the subunits. This method detects the absence of light-heavy disulfide bonds and the presence of light-light or heavyheavy dimers and polymeric forms of heavy-heavy dimers (93). Proteins with molecular weights greater than 300 000 cannot be separated by electrophoresis in SDS-polyacrylamide even when the gel is made as dilute as possible, but SDS-agarose has been used to classify fibrin monomer (mol wt 339 700) and its polymers up to the pentamer. Electrophoresis combined with eptide analysis was used to show that the cross-linking getween lysine and glutamine, which is the basis of the clotting mechanism, occurs between the y-chains and not the a- or ,&chains of the monomer and that it is inter- but not intra-molecular (101). The juxtaposition of layers in cell membranes can be established by causing peptide chains in adjacent positions to form cross-links and then isolating the products by electrophoresis. Identifying the linked peptides is certain evidence that they are neighbors within the membrane. Special techniques and reagents for applying this method to erythrocyte membranes have been described (157). Thermal denaturation is useful for studying the secondary structure, size, homogeneity, and base composition of nucleic acids. By using a s ecially constructed uv scanning apparatus, denaturation o both DNA and RNA can be observed directly in acrylamide gel electrophoregrams. This obviates the problems of purifying and eluting the nucleic acids as a preliminary to denaturation studies. The method is nondestructive because, under the conditions used, denaturation is reversible (123). Molecular Constants. Molecular wei h t measurements of RNA that have been based u on tteir mobilities in aqueous gels have been unreliable ecause of variations in the secondary RNA structure. By contrast all RNA molecules are homologous in formamide, and they migrate a t rates proportional to their molecular weights in nonaqueous formamide-acrylamide gels. The migration rates are not affected by nucleotide base composition (108). Molecular weight measurements of proteins can be made simply by treating the samples with SDS prior to electrophoresis; no SDS need be added to the gel or electrode compartments (132). However, omitting SDS from part of
P
g
the electrophoretic system causes electrophoretic atterns of SDS to develop in the gel (84). The behavior ofsurfactant within an electrophoretic system can be observed by uv absorption if sodium octylbenzene sulfonate is used instead of SDS (144). The cationic detergents N-hexadecylpyridinium chloride (126) and cetyl trimethylammonium bromide (96) can be used for measuring molecular weights. With cationic detergents, proteins with hydrophobic subunits are excluded from gels, a phenomenon that may prove useful for detecting or isolating such proteins. Wheat gluten polypeptides give falsely high molecular weights because their high proline content causes an exceptional elongation of their molecules and, consequently, reduced mobilities in gels (61). Histones also behave anomalously with respect to the mobilities of their SDS-complexes in gels (60). The molecular weight of histones should be estimated by plotting retardation coefficients, rather than relative mobilities, vs. molecular weight because the mobilities of SDS-histones in free solution as estimated from Ferguson plots, are abnormally low by comparison to other SDS-protein complexes (68). It has been reported that proteins with molecular weights of less than 50 000 show a nonlinear relation between their retardation coefficients and molecular weights (56). The mobility of proteins in urea-polyacrylamide gel increases with increasing content of disulfide so that accurate molecular weight measurements are possible only when all S-S bonds are broken (110). Glycoproteins from RNA-containing viruses have a low SDS-binding capacity which makes their movement in gels much more dependent upon gel concentration than is the case with other proteins (125). Mobilities are also affected by buffer concentration, when N,N,N',N'-tetramethylenediamine (TMED) buffer is us&d;increasing its concentration lowers the mobilities of different proteins in SDS-gel to an extent that varies with the protein. This complicates estimations of molecular weight (4). It has been shown by free-boundary electrophoresis that SDS-complexes of proteins ranging in molecular weight between 10-70 thousand have identical mobilities in free solution and that their different mobilities in el are attributable to sieving effects. The complexes betave more like flexible strands with SDS clustered along the peptide chains in the manner of beads on a necklace than like rigid ellipsoids (127). Su port for the string of beads structure has been obtained y! nuclear magnetic resonance studies on proteins complexed with sodium 4-(p-butylphenyl)butane-1-sulfonate instead of SDS (145). Dansylated peptides in the molecular weight ran e of 1-20 thousand show a linear relationship between mohlity and mol w t in both SDS- and 8 M urea gels (78). Chloroplast proteins (mol wt 18-90 thousand) give linear mobility plots in polyacrylamide if cross-linking is controlled; 1.2% methylene bisacrylamide yields the best results (92). The molecular weight of human erythropoetin as determined in SDS-polyacrylamide is 23 000 (37). The molecular weights of acid mucopolysaccharides can be determined in thin layers of Sephadex-cellulose (140). Polyacrylamide gels can also be used for mucopolysaccharides; the polydispensity of chondroitin sulfate is the result of varying chain len ths rather than chemical differences (73). $he molecular weights of untreated proteins can be determined by causing them to mi rate to their exclusion limits in a pore gradient gel, but gfobular and linear proteins of the same size have different exclusion limits (41). Extrapolating to infinite gel dilution does not eliminate the electroosmotic component from estimates of electrophoretic mobility; some electrically inert marker such as vitamin B12 is needed to correct for electroosmosis when mobility measurements are made (52). The advantages of SDS-gel electrophoresis over Sephadex chromatography for molecular weight measurements have been discussed (11), and the general theory of SDSgel measurements of molecular properties has been reviewed (158).
LITERATURE CITED
(1) Agrawai, H. C., Fundam. LipidChem. 1974, 511-43; Burton, R. M., Guerra. F. C.(Ed.). El-Sci. Publ. Div.. Webster Groves, Mo. (2) Allen, R . C., Harley. R . A., Talamo, R . C.,
42R
Am. J. Clin. Pathol. 1674, 62, 732-9. (3) Alien, R . C., Maurer, H. R . (Ed.), "Eiectrophoresis and Isoelectric Focusing in Polyacrylamide Gel. Advances of Methods and Theories, BiOchemical and Clinical Applications", de Gruyter,
ANALYTICAL CHEMISTRY, VOL. 48, NO. 5, APRIL 1976
New York, N.Y., 1974, 316 pp, (4) Allison, J. H., Agrawai. H. C., Moore, B. W., Anal. Biochem. 1074, 58, 592-601. (5) Anseistetter. V., Horstmann, H. J., Eur. J. Biochem. 1975, 56. 259-69.
(6) Ascher, P. D., Weinheimer. W. H., Hod. Sci. 1974, 9,219-20. (7)Badr, G. G., Waldman, A. A., lnt. J. Neurosci. 1973,6, 117-30. (8)Baumann, G., Chrambach, A,, Anal. Biochem. 1975,64,530-6. (9) Bjerrum, 0. J., Experientia 1974, 30, 831-2. (10)Bjerrum, 0.J., Lundahl, P., Biochim. Biophys. Acta 1974,342,69-80. (11) Blumberga, I., Puskarevs, I., Latv. PSR Zinat. Akad. Vestis 1974, 113-7. (Chem. Abstr., 81, 101486r) (12) Boddin, M.. Hilderson, H. J., Lagrou, A., Dierick, W., Anal. Biochem. 1975,64,293-6. (13) Bog-Hansen, T. C., Anal. Biochem. 1973, 56,480-8(14) Bours, J., Sci. Tools 1973,20,29-34. (15)Cann, J. R., Biophys. Chem. 1975, 3, 206- 14. (16) Cann, J. R., lmmunochemistry 1975, 12, 473-6. (17)Cann, J. R.. Bethune, J. L, Kegeles, G., Bid. Macromol. 1973,6,253-302. (18)Cannon, L. E., Woehler, M. E., Clark, P. D., Wilkerson, L., Lovins, R. E., Immunology
1974,26,1159-69. (19)Caron, M., Faure, A,, Cornillot. P., J. Chromatogr. 1975, 103,160-5. (20)Catsimpoolas, N., Electrophor. lsoelectric Focusing Polyacrylamide Gel, (Proc. Small Conf.) 1972 (Pub. 1974). 174-188;Allen, R. C., Maurer, H. R. (Ed.), de Gruyter, Berlin, Germany. (21) Catsimpoolas. N., Campbell, B. E., Griffith, A. L., Biochim. Biophys. Acta 1974, 351,
196-204. (22)Catsimpoolas, N.. Griffith, A. L., Anal. Biochem. 1973,56,100-20. (23)Catsimpoolas, N.. Yotis, W. W., Griffith, A. L., Rodbard, D., Arch. Biochem. Biophys.
1974, 163,113-21. (24)Cattolico. R. A., Jones, R. F., Anal. Biochem. 1975,66.35-46. (25)Cawley, L. P., et al., "Basic Electrophoresis, immunoelectrophoresis, and Immunochemistry", (Am. SOC.Clin. Pathologists, Comm. Contg. Educ., Chicago, til.) 1972,212 pp. (26)Cherry, J. P., Prescott, J. M., Proc. Soc. Exp. Biol. Med. 1974,147,418-24. (27)Chrambach, A., Skyler, J. S., Protides Biol. Fluids, froc. Coiloq. 1974 (Pub. 1975) 22,
701-13. (28)Christiansen, A. H.. Protides Biol. Fluids, Proc. Coiloq. 1971 (Pub. 1972)19,557-60. (29)Coffer, A. I., King, R. J. B., Biochem. SOC. Trans. 1974,2, 1269-72. (30)Cotton, R. G. H., Miistein, C., J. Chromatogr., 1973,86,219-21. (31)Curvetto, N. R., Balmaceda, N. A,, Orioli, G. A,, J. Chromatogr. 1974, 93,248-50. (32)Dames, W., Maurer, H. R., Electrophor. lsolectric Focusing Polyacrylamide Gel, (Proc. Small Conf.) 1972 (Pub. 1974),221-31;Ailen, R. C., Maurer, H. R. (Ed.). de Gruyter, Berlin, Germany. (33)Datyner. A,. Finnimore. E. D., Anal. Biochem. 1973,55,479-91. (34)Dean, R. T., Messer, M., J. Chromatogr.
1975, 105,353-8. (35)Dean, W. W., Dancis. B. M., Thomas, C. A,. Anal. Biochem. 1973,56,417-27. (36)De Keizer, A,, Van der Drift, W. P. J. T., Overbeek. J. Th. G.. Biophys. Chem. 1975, 3, 107-8. (37)Dorado, M., Espada, J., Langton, A. A,. Brandan, N. C., Biochem. Med. 1974, 10,1-7. (38)Eng, P. R. Parkes, Co. O., Anal. Biochem. 1974,59,323-5. (39)Eriksson, K. E., Pettersson, B., Anal. Biochem. 1973,56,618-20. (40)Fawcett. J. S.,Methodoi. Develop. Biochem. 1973,2,61-80. (41)Felgenhauer. K., Hoppe-Seyler's 2. Physio/. Chem. 1974,355,1281-90. (42)Felgenhauer, K., Graesslin, D., Huismans, B. D., Protides Bioi. Fluids, Proc. Coiloq. 1971 (Pub. 1972),19,575-8. (43)Floyd, R. W.. Stone, M. P.. Joklik. W. K.. Anal. Biochem. 1974,59,599-609. (44)Foissy, H., J. Chromatogr. 1975, 106, 51-7. (45)Fredriksson, S.,Anal. Biochem. 1974,57, 452-6. (46) Fredriksson, S., J. Chromatogr. 1975, 108. 153-67. (47) Fredriksson, S., Pettersson. S., Acta
Chem. Scand., Ser. 8 1974,28,370. (48) Frelin, C., Gasquez, J., C. R. Acad. Sci., Ser. D. 1973,277, 1817-19. (49) Furlan, M., Beck, E. A,, J. Chromatogr.
1974, 101,244-6. (50)Gallinaro-Matringe, H., Jacob, M.. FEBS Lett. 1974,41,339-41. (51)Ghanta, V. K., Hiramoto, R. N., immunochemistry 1974, 1 1 , 305-8. (52)Gosh, S., Moss, D. B.. Anal. Biochem. 1974,62,365-70. (53)Gianazza, E., Pagani, M., Luzzana, M., Righetti, P. G., J. Chromatogr. 1975, 109, 35764. (54)Gillman, C. F.. Bigazzi. P. E., Bronson, P. M., Yan Oss, C. J., Prep. Biochem. 1974, 4, 457-72. ( 5 5 ) Goetz, H., Method. Fortschr. Med. Lab.
1974, 1, 91-7. (56)Gonenne, A,. Lebowitz, J., Anal. Biochem. 1975,64,414-24. (57)Graessiin, D., Weise, H. C.. Electrophor. lsoelectric Focusing Polyacrylamide Gel, (Proc. Small Conf.) 1972 (Pub. 1974). 199-205; Ailen, R. C., Maurer, H. R. (Ed.), de Gruyter, Berlin, Germany. (58)Groc. W., Jendrey, M., Clin. Chim. Acta
1974,52,59-69. (59)Groome, N. P.. Belyavin, G., Anal. Biochem. 1975,83.249-54. (60) Hamana, K., iwai, K., J. Biochem. (Tokyo) 1974,76,503-12. (61) Hamauzu, Z., Nakatani, M., Yonezawa, D., Agric. Biol. Chem. 1975,39,1407-10. (62)Hampton, J. R., Mitchell, J. R . A,, Thromb. Diath. Haemorrh. 1974,31,204-44. (63)Hannig, K.. Methods Microbiol. 1971, 58, 513-48. (64)Harrison, A. D. R., Reid, E., Methodoi. Develop. Biochem. 1973,2,27-37. (65)Harrison, R. A. P.. Anal. Biochem. 1974, 61,500-7. (66)Harzer, K., Klin. Wochenschr. 1974, 52, 145-7. (67)Hause, L. L., Rothwell, D. J., Straumfjord, J. V., Proc. Annu. Conf. Eng. Med. Bioi. 1974, 16, 404. (68)Hayashi, K.. Matsutera, E., Ohba, Y., Biochim. Biophys. Acta 1974,342,185-94. (69)Hedenskog, G., J. Chromatogr. 1975, 107, 91-8. ( 7 0 ) Hensten-Pettersen. A,, Anal. Biochem.
1974.57.296-8. (71) Hoffmeister, H., Method. Fortschr. Med. Lab. 1974,1, 41-9. (72)Hjerten, S.,Methodol. Develop. Biochem. 1973,2,39-48. (73)Hsu. Dar-San, Hoffman, P., Mashburn, T. A,, Biochim. Biophys. Acta 1974, 338. 254-64. (74)Huang, H. V., Molday, R. S., Dreyer, W. J., FEBS Lett. 1973,37,285-90. (75)Jako. J., Hitzig, W. H., Ciin. Biochem.. Prim. Methods, 1974, 1, 105-47. (76)Johansson, B., Hjerten, S., Anal. Biochem. 1974,59,200-13. (77)Kapadia, G., Chrambach, A,, Rodbard, D., Electrophor. lsoelectric Focusing Polyacrylamide Gel. (Proc. Small Conf. 1972 (Pub. 1974), 11544; Ailen, R. C., Maurer, H. R. (Ed.), de Gruyter, Berlin, Germany. (78)Kato, T., Sasaki, M., Kimura, S., Anal. Biochem. 1975,68,515-22. (79)Kelly, P. T.. Luttges, M. W., J. Neurochem.
1975,24,1077-9. (80)Kerenyi, L., Gaiiyas, F., Ciin. Chim. Acta 1973,47,425-36. (81) Kopwiiiem, A., Chiliemi, F., Bianchi-Bosisio Righetti, A,. Righetti, P., frotides Bioi. Fluids, Proc. Colloq. 1973 (Pub. 1974)21,657-65. (82)Korant, B. D.. Lonberg-Holm, K., Anal. Biochem. 1974,59,75-82. (83)Ksenofontov, B. S.,Vilenskii, A. I., Klassen, V. I., Smysiov, P. A,, Tsimarkina, G. E., Dokl. Akad. Nauk SSSR 1974, 215, 994-5 (Chem. Abstr. 81,87354) (84)Kubo. K.. Isemura, T.. Takagi. T., J. Biochem. (Tokyo) 1975,78,349-54. (85)Kuzmin. S. V., Mickichur. N. I., Naumova, L. P., Sandakhchiev, L. S., Anal. Biochem. 1975,
65,405-11 (86)Larcan, A,, Stoltz, J. F., Biorheology 1973, 10,189-205 (Chem. Abstr., 80,35326). 187) Leaback. D. H.. Chem. Br. 1974. 10. 376-83 (Chem. Abstr., 82,9882). (88) Leaback, D. H., Robinson, H. K., FEBS Lett. 1974,40,192-5.
(89)Leise, E. M., LeSane, F., Prep. Biochem. 1974,4,395-410. (90)Liljas, L.. Lundahl, P., Hjerten, S., Biochim. Biophys. Acta 1974,352,327-37. (91)Macbeth, R. A,, Bazin, S., Allain, J. C., Clin. Biochem. 1975, 8 , 52-9. (92)Machold, O., Biochem. Physiol. Pflanz 1974, 166, 149-62 (Chem. Abstr., 82, 13268) (93)Manjula. B. N., Richards, F. F., Anal. Biochem. 1975,66,412-22. (94)Manson. W., Methods Microbiol. 1971, 58, 549-71, Norris, J. R. (Ed.), Academic, London, England. (95)Margolis, J., Wrigiey, C. W., J. Chromatogr. 1975, 106,204-9. (96)Marjanen, L. A. Ryrie. I. J.. Biochim Biophys. Acta 1974,371,442-50. (97)Markovic. O.,Kuniak, I., J. Chromatogr.
1974,91,873-5. (98)McClard, R. W.. Kolenbrander, H. M., Experientia 1974, 30,730-1. (99)McDuffie, N. M.. Dietrich. C. P., Nader, H. B., Biopolymers 1975, 14, 1473-86. (100)Miller, D. W., Elgin, S. C. R.. Anal. Biochem. 1974,60,142-8. (101)Moroi, M., Inoue, N., Yamasaki. M.. Biochim. Biophys. Acta 1975,379,217-26. (102)Mulder, J., Verhaar, M. A. T., iRCS Libr. Compend. 1973, 1, 17.22.4.(Chem. Abstr. 81, 167545). (103)Nees, S.,Electrophor. lsoelectric Focusing Polyacrylamide Gel. (Proc. Small Conf.) 1972 (Pub. 1974) 189-98: Ailen, R. C., Maurer, H. R. (Ed.). deGruyter, Berlin, Germany. (104)Nijhuis, H., Payen. T. A. J., Biochim. Biophys. Acta 1974,336,213-21. (105)Orengo, A,, Greenspan, J., Love, J. D., Anal. Biochem. 1975,63,559-65. (106)Otavsky, W. I., Drysdale, J. W.. Anal. Biochem. 1975,65,533-6. (107)Pendyala, L.. Weliman, A,, Anal. Biochem. 1974,59,634-5. (108)Pinder, J. C.. Staynov, D. Z., Gratzer. W. B., Biochemistry 1974, 13,5373-8. (109)Pogacar, P., Jarecki, R., Electrophor. lsoelectric Focusing Polyacrylamide Gel. (Proc. Small Conf.) 1972 (Pub. 1974)153-8;Allen R. C., Maurer, H. R. (Ed.), de Gruyter, Berlin, Germany. (110) Poole, T., Leach, B. S., Fish, W. W., Anal Biochem. 1974,60,596-607. (111)Popescu, A,, Dinescu, G., Stud. Cercet. Biochim. 1974, 17, 279-86 (Chem. Abstr. 83,
4116). (112)Prusik, Z., J. Chromatogr. 1974, 91, 867-72. (113)Radola, B. J., Biochim. Biophys. Acta 1975,386,181-95. (114)Radola, B. J., Chem.-Ztg. 1974, 98, 549-54. (115)Radoia, B. J., Protides Biol. Fluids, froc. Colloq. 1970 (Pub. 1971)18,487-91. (116)Reis, J. F. G., Lightfoot, E. N., Lee, H.. AlChE J. 1974,20,362-8. (117)Reisner. A. H., Nemes, P.. Bucholtz, C.. Anal. Biochem. 1975,64,509-16. (118)Richmond, D. V.. Fisher, D. J., Adv. Microbial. Physiol. 1973,9,1-29. (119) Righetti. P., Bianchi-Bosisio Righetti, A,, Galante, E., Anal. Biochem. 1975,63,423-32. (120)Righetti, P. G., Pagani, M., Gianazza, E., J. Chromatogr. 1975,109,341-56. (121)Roisman, F. R.. Castagnino. J. M., Bioquim. Ciin. 1973,7 , 183-94. (122)Rosemblatt. M. S., Margolies, M. N., Cannon, L. E., Haber, E., Anal. Biochem. 1975, 65,321-30. (123)Rosner, A., Edeiman. M., Gressel. J.. Anal. Biochem. 1974,58,602-8. (124)Ruechel. R., Mesecke, S., Wolfrum, D. I., Neuhoff, V., Hoppe-Seyler's Z. Physiol Chem.
1973,354,1351-68. (125)Russ, G.. Polakova, K., Biochem. Biophys. Res. Commun. 1973, 55,666-72. (126)Schick, M., Anal. Biochem. 1975, 63, 345-9. (127)Shirahama, K., Tsujii, K., Takagi, T., J. Biochem. (Tokyo) 1974,75,309-19. (128)Sirai, M., Matsuda. S., Mitsukawa, S.. Tohoku J. Exp. M e d . 1974, 113,273-81 (Chem. Abstr., 81,116738~). (129)Snyder, R. S.,Bier, M., Griffin, R. N., Johnson, A. J., Leidheiser, H., Micale, F. J., Vanderhoff, J. W., Ross, S.. Van Oss, C. J.. Separ. Purif. Methods 1973,2,259-82. (130)Sova, O.,Chem. Listy 1974, 68,965-9 (Chem. Abstr. 82,28052).
ANALYTICAL CHEMISTRY, VOL. 48, NO. 5, APRIL 1976
43R
(131) Stephens, R. E., Anal. Biochem. 1975, 65,369-79. (132) Stoklosa, J. T., Latz, H. W., Biochem. Biophys. Res. Commun. 1974, 58, 74-9. (133) Stokiosa, J. T., Latz, H. W., Biochem. Biophys. Res. Commun. 1974, 60, 590-6. (134) Stoitz, J. F., Larcan, A,, Thromb. Diath. Haemorrh. 1975, 33, 132-4. (135) Stoltz, J. F., Larcan, A,, Vigneron, C., Nicolas, A,, Streiff, F.. J. Angeiol. Lang. Fr. 1973, 267-71 (Chem. Abstr., 82, 13269). (136) Stoltz. J. F., Nicolas, A,, Mlrjolet, M.. Larcan. A,, C. R. Hebd. Seances Acad. Sci., Ser. D. 1974,279, 1951-3. (137) Strickland, R. D., Anal. Chem. 1974, 46, 95R-100R. (138) Swanson, M. J., Sanders, B. E.. Anal. Biochem. 1975,67,520-4. (139) Taylor, C. W., Yeoman, L. C., Busch, H., Methods CellBioi. 1975, 9, 349-76. (140) Tortolani, G., Romagnoli, E., fxperientia 1975,31,389-90. (141) Troitskii. G. .V., Kiryukhin, I. F., Zav‘yalov, V. P., Vop. Med. Khim. 1973, 19, 550-2 (Chem. Abstr., 80, 67951). (142) Troitskii, G. V.. Zav’yalov, V. P., Abramov, V. M., Dokl. Akad. Nauk SSSR 1974, 214, 955-8 (Chem. Abstr., 80, 130050. (143) Troitskii, G. V., Zav’yalov, V. P., Kiryukhin, I. F., Abramov, V. M., Agitskii, G. Yu., Biochim. Biophys. Acta 1975, 400, 24-31.
(144) Tsujii, K., Takagi, T., J. Biochem. (Tokyo) 1975,77, 117-23. (145) Tsujii, K., Takagi, T., J. Biochem. (Tokyo) 1975,77, 511-19. (146) Tsuzuki, J., Kiger. J. A., Prep. Biochem. 1974,4, 283-94. (147) Uzgiris, E. E., Kaplan, J. H., Anal. Siochem. 1974,60,455-61. (148) Van Oss, C. J., Methods lmmunodlagnosis 1973, 175-94; Rose, N. R. (Ed.), Wiley, New York, N.Y. (149) Van Oss. C. J., Fike, R. M.. Good. R. J., Reinig, J. M., AnaiBiochem, 1974, 60, 242-51. (150) Van Welzen, H., Zuidweg, M. H. J.. Anal. Biochem. 1974,59,306-15. (151) Vesterberg, 0.. Acta Chem. Scand. 1973, 27,2415-20. (152) Vesterberg, O., flectrophor. lsoelectric Focusing Polyacrylamide Gel. (Roc. Small Conf.) 1972 (Pub. 1974) 145-53: Allen, R. C., Maurer, H. R. (Ed.), de Gruyter, Berlin, Germany. (153) Vesterberg, O., Methods Microbiol. 1971,5B, 595-614. (154) Vesterberg, O., Sci. Tools 1973, 20, 22-9. (155) Vogel, O., Reinmuth, H., Anal. Biochem. 1974,62,461-71. (156) Waldstrom, T., Mollby, R., Jeansson, S., Wretlind, B., Sci. Tools 1974, 21, 2-4. (157) Wang, K., Richards, F. M., J. Biol. Chem. 1974,249,8005-18.
(158)Weber, K., Osborn, M., “Proteins,” Third Ed. 1975, 1, 179-223; Neurath, H., Hill, R. L., Boeder, C. (Ed.), Academic Press, New York. N.Y. (159) Weidekamm, E., Wallach, D. F. H., Neurath, P., Flueckiger, R., Hendricks, J.. Anal. Biochem. 1974,58, 217-24. (160) Weisberg, S.,Anal. Biochem. 1974, 61, 328-35. (161) Weiss, G. H., Catsimpoolas, N., Rodbard, D., Arch. Biochem. Biophys. 1974, 163, 106-12. (162) Wetzel, V., Sturm, G., Daume. E., lRCS Libr. Compend. 1973, 1, 3.2.9. (Chem. Abstr.. 81, 132415). (163) Wetzel, V., Strum, G., Daume, E., Fert. Steril. 1974, 25, 45-50. (164) Yapa, P. A. J., J. Chroflatogr. 1973, 87, 311-13. (165) Yeger, H., Freedman, M., Anal. Biochem. 1975,64,450-4. (166) York, A. T.. Manski, W., Ophthalmic Res. 1974,6, 245-58. (167) Yoshida, T., Ishii, S.,fndocrinol. Jpn. 1973,20,629-33. (168)Zanini, A,, Giannattasio, G., Meldolesi, J., Endocrinology 1974, 94, 594-8. (169) Zeineh, R. A., Fiorella, B. J., Abdulkarln, K., Am. J. Med, Techno/. 1975,41, 10-12. (170) Zeineh, R. A., Mbawa, E. H., Fiorella, B. J., Dunea, G., Anal.cm. 1973, 45, 2159-60. (171) Zeineh, R. A., Nijm, W. P., AI-Azzawi, F. H.. Am. Lab. 1975, 7, 51-8.
Gel and Affinity Chromatography V. F. Gaylor” and H. L. James The Standard Oil Company, 4 4 4 0 Warrensville Center Road, Cleveland, Ohio 4 4 128
H. H. Weetall Corning Glass Works, Research and Development Laboratories, Corning, N. Y. 14830
GEL PERMEATION CHROMATOGRAPHY Liquid Exclusion Chromatography V. F. Gaylor and H. L. James
(“rigid gels”). Some of each are included. Similarly, we’ve included some material on small molecules along with work on synthetic and natural polymers.
GENERAL REVIEWS This review covers the period of approximately December 1973 through November 1975. The literature surveyed includes major analytical, chromatographic, and polymer journals, Chemical Abstracts, and various liquid chromatography literature abstracts. Publication of the ASTM D-20.70.04 “Bibliography on Liquid Exclusion Chromatography” was a major event of the past two years (13). This document includes literature on gel permeation chromatography from the early 1960’s through 1972, and will be updated regularly. Annual seminars on gel permeation chromatography, sponsored by Waters Associates, Inc., were a second major event. These meetings in late 1973, 1974, and 1975 were an excellent communications forum for specialists in the field. The American Chemical Society short course on gel permeation chromatography was made available in tape cassette form ( 4 6 ) . Two additional American Chemical Society short courses on liquid chromatography also contain training material useful in the practice of liquid exclusion chromatography. The authors of this review concur with ASTM D20.70.04 in preferring “liquid exclusion chromatography” to the less definitive “gel permeation chromatography” nomenclature. But, nevertheless, we’ve used the common abbreviation, GPC, for ease of reading the review. However, we’ve not necessarily drawn any rigid distinction between size separations from soft gels, (“gel filtration”), semi-rigid gels (“GPC”) and porous, inorganic column packings 44R
ANALYTICAL CHEMISTRY, VOL. 48, NO. 5, APRIL 1976
A number of general discussions on theory and practice of GPC and gel filtration chromatography were published (27, 117, 124, 129, 183, 247). Steric exclusion technology is also covered in other documents dealing with practice and applications of modern liquid chromatography (58, 93, 306). GPC source material is also included in general reviews of characterization methods for polymers, such as Keiner’s review of automated methods ( 1 7 0 ) .One third of the book, “Polymer Molecular Weight Methods,” edited by Ezrin (107)is devoted to selected topics in GPC, and several of these are referenced elsewhere in this review. Chapter 6 from a book of the same name, and edited by Slade, is also recommended reading for the novice (253).
APPARATUS Considerable improvement in liquid chromatography equipment was made during the past two years, and the number of suppliers of such equipment increased. Many of these developments are useful in GPC. The 1974/75 International Chromatography Guide (147)is a particularly useful equipment guide for both novice and experienced chromatographer. A number of general reviews (50, 176, 221, 327) covered all hardware requirements and available equipment, and a fifth review focused on solvent delivery systems (150).Additionally, Rossler, Schneider, and Halasz successfully tested a syphon-type flow meter for high pressure liquid chromatography (285), and Palyza described a
