Anal. Chem. 1984, 56,300 R-349 R
...
(48) Mulay, L. N.; Mulay, I.L.; Worden, R. “Microtechniques Instrumentation for Biol. Cells” P499 in “Automation and Instrumentatlon”; Sagglatore and Mondadori, Eds. (FAST) Federation delle Associazloni Scientifiche Techniche”, Milan, Italy, 1988. (48a) Nature (London), 190, 1019 (1961); see also citatlons In ref 49. (49) Mulayk L. N.; Mulay, I.L. Anal. Chem. 38, 404R (1984). (50) Mulay, L. N.; Mulay, I.L. Anal. Chem. 38, 501R (1968). (51) Mulay, L. N.; Mulay, I.L. Anal. Chem. 40, 440R (1968). (52) Mulay, L. N.; Mulay, I.L. Anal. Chem. 42, 325R (1970). (53) Mulay, L. N.; Mulay, I.L. Anal. Chem. 44, 324R (1972). (54) Mulay, L. N.; Mulay, I.L. Anal. Chem. 48, 490R (1974). (55) Mulay, L. N.; Mulay, I.L. Anal. Chem. 48, 314R (1976). (56) Muiay, L. N.; Mulay, I.L. Anal. Chem. 50, 274R (1978). (57) Mulay, L. N.; Muiay, I.L. Anal. Chem. 52, 199R (1980). (58) Mulay, L. N.; Prasad Rao, A. V. Digests (Abstracts) of Intermag. Conf. I€€€. Mag. Trans. 19, 1998 (1983). Two papers related to thls work will be published In the near future by Muiay, L. N.; Prasad Rao, A. V.; Walker, P. L., Jr.; Rlvlera, R.; and Vannlce, M. A. (59) Mulay, L. N.; Oskooie-Tabrlzi, M.; Lo, C. Dlgests (Abstracts) of Intermag. Conf. I€€€. Mag. Trans. 19, 2001 (1983). See also: Ph.D. Thesis, Oskooie-Tabrizi, The Pennsylvania State University, University park, PA (1983). (60) Mydosh, J. A. Lect. Notes Phys. 149, 87 (1981). See Also: J. Appl. Phys. 53, 2211 (1982). (61) Novak, P. I n “Proc. of 4th Inti. Seminar on Magnetlsm-Hochschule Verkehrsues, “Friedrich Llst””; Dresden, German Democratic Republlc. (a) Onufrionokv, V. Geomag. Aeron. (Rws.) 21, 895 (1981). (62) O’Connor, C. J. Prog. Inorg. Chem. 29, 203 (1982). (62a) Orrell, K. G.; Sik, V. Anal. Chem. 52, 567 (1980). (83) Pandey, A. R.; Kumar. Y.; Verma, V. P.; Slngh, D. R. Acta. Cienc. Indica, Ser. Phys. 7 (1-4), 39 (1981). (64) Pesch, J. A. Rev. Sci. Instr. 54, 480 (1983). (65) Pollock, D. D. “Physical Properties of Materials for Engineers”; CRC Press: Boca Raton, FL; Voi. 11.
(86) Prasad Rao, A. V. Ph.D. Thesis, The Pennsylvania State Unlverslty, University Park, PA (1983). (67) Romanl, G. L.; Wllliamson, S. J.; Kauffman, L. Rev. Sci. Instr. 53, 1815 (1982). (67a) Roy. Ind. J . Cryog. 7 , 11 (1982). (68) Rossat-Mignod, J.; Burlet, P.; Quesel, S.;Effantin, J. M.; Delcote, D.; Bartholrn, H.; Ravot, D. J. Magn. Magn.. Materials 31, 398 (1983). (69) Schardt, B. C.; Hollander, F. J.; HIII, C. J. J . Am. Chem. SOC. 104, 3964 (1982). (a) Schilling, J. S.“Phys. Solids under Hlgh Pressure”; Proc. Imt. Conf. on High Pressure; North-Holland: Amsterdam, Netherlands. (70) Schultz, J.; Gulllkson Rev. Sci. Instr. 54, 1383 (1983). (71) Senftle, F. E.; Thorpe, A. N.; Alexander, C. C.; Finkleman, R. E. Fuel61 81 (1982). (72) Smith, J. P.; Mark, H. E.; OrmeJohnson, W. H. J . Biol. Chem. 257, 2310 (1982). (73) Sobry, R.; Van Den Bossche, G. Acta. Crystaiiogr.;Sec. A (French A38, 288 (1982). (74) Stroink, G.; Duniap, R. A,; Hutt, D. Can. Mineral. 29, 519 (1981). (75 Sumlmoto Metal Ind. Ltd. Japan, Kokai Tokyo Koho J.P. 57140806 182 1408061. (76) Tlmkovich, R.; Cork, Margaret S . Biochem. Biophys. Acta. 742, 162 (1983). (77) Toshlru, S.;Hiroshi, M.; Mitsuhlro, M. I n ref 18. (a) Tyagl, A,; Lord, A. E., Jr.; Koerner, R. M. J . Hazard. Mat. 8, 11 (1983). (78) Vasak, M.; Kaegi, J. H. R. Proc. Natl. Acad. Sci. U . S . A . 78, 6709 (1981). (79) Vllenchik, M. M. Biofizika (Russ.) 27, 31 (1982). (80) Weltner, W. “Magnetic Atoms and Molecules”; Van Nostrand Rhelnhold: New York. (81) Wohlfarth, E. P., Ed. “Ferro-Magnetlc Materials; A Handbook on the Properties of Magnetlcally Ordered Substances”; North-Holland Publlshing: AmsterdamlNew YorklOxford, 1982; Vol. 111. 852 pp. (82) Zieba, A.; Foner, S.Rev. Sci. Instr. 53, 1344 (1982). (83) Zleba, A,; Foner, S. Rev. Sci. Instr. 54, 137 (1983).
Column Liquid Chromatography Ronald E. Majors* Varian Associates, Walnut Creek Instrument Division, 2700 Mitchell Drive, Walnut Creek, California 94598
Howard G. Barth Analytical Division, Hercules, Incorporated, Research Center, Wilmington, Delaware 19894
Charles H. Lochmuller Department of Chemistry, Duke University, Durham, North Carolina 27705
INTRODUCTION This review covers the fundamental developments in the field of column liquid chromatography (LC) during the period of 1982-1983. Earlier significant articles which were published in foreign or less available journals, the patent literature, and other sources which were not available at the time of the previous review (2a) are also included. References to oral presentations, conferences, and other unpublished works have been excluded. This review does not attempt to be a comprehensive coverage of all LC literature or merely a recital of LC deveopments. The authors have attempted to be critical in their selection of those references which do the most in extending the fundamental development in theory, methodology, and instrumentation. Applications were selected which provided a better understanding of the fundamentals of LC, gave some insight into future trends, or represented a significant advance in using LC to solve difficult separations. Unlike the earlier review (2a),the current review now includes size exclusion chromatography. 300 R
0003-2700/84/0356-30OR$06.50/0
The emphasis of this review will be on high-performance liquid chromatography (HPLC) since most of the current research and development in the LC technique has been directed toward this “modern” approach. However, any significant development in HPLC is bound to carry over to all forms of liquid column chromatography, be it high; medium; or low-pressure work. For this reason, the term LC will be used throughout the text. For any particular citation which pertains only to work involving small particles packed into columns which require high pressure to provide flow, the term HPLC will be used. Assignment of literature references to a given section was made on the basis of emphasis of the particular citation. The Lockheed computer-based library files, Preston Technical Abstracts (LC), CA Selects (HPLC, GPC), Liquid Chromatography Abstracts (Chromatography Discussion Group, United Kingsom), and the primary chromatographic literature were used to locate the references cited in this work. A recent market survey by Centom (4a) indicated that column LC (consisting of HPLC, column chromatography, amino acid analysis, and ion chromatography) was in 1983, 0 1984 American Chemical Society
COLUMN LIQUID CHROMATWRAPHY
I
I
I
p a r s WMVarian. he has had several a b si~nmentsIn research. applications. and marketing In the U.S. and E w w . He k me a01 60 publications in HPLC. OC. and surlaca chemkby. He has served as Special Ednor 01 me Jamel of chometogephic Sciencs tar iswas devoted to LC columns and cdwnn t s c h d q y and b menw ednw tar a m n m y featwe. "~oiumn a w Is a -tar 01 me Amrkan Chemical Watd" in LC Msga2hs. Dr. M Society. Analytical DMsbn. Caliimia Seclkm. the ChrDmatograDhv Okcussion &ow (LoK(0n). and me Mmputers In Chemkby Mvision. Wowud 0. B.m k mbr m r c h daw1 rrtm me analyHcal dlvkbn 01 HerNles Resard? Csnter In Wllmlngton. DE. He k wow leader Of me Gas chomalowaphv. lqdd chomatogaphv. pamas size analysk. and potwar anawsis secwls. He received his B.A. (1969) and h . D . (1973) In analyaCBI damistry from Nalheastm Univershy. Eelare joining Hercules. Inc.. in 1974. hs wa8 a poit&claral lelbw In ~llnlcalchewloby at Hahnemann Medical College In Phlladaiphla. HDward span1 me fall 01 1981 811 assistant potsssa of damfstry at hls slma mater. He has tal@ at me universny 01 Delaware and is a freqwnt IBC1uTer SI 9 h M -MS mn-ed bv the Deiaware Vallev " ~, apecialae;include cha'ractcdratlm 01 watsr&bia p o l y m s . sbe exclusion chmtography. and HPLC. and he has p u b W over 20 papars in mesa areas. has sisa edned a boac. adem em ~ e w 01s p s ~ c l esize Analysb. scheduled far publlcatlm In 1964. He has been assistant editor and ednw of me D e e m Bulletin. me miaware ACS senon pubiicatbn. and program chairman and chairman 01 me Delaware ACS analvtical toplcal w w p and served as secretary 01 Delaware ACS section. Lh. Barn Is a me+ 01 Dlvkbn 01 AnaWical Chamistry and me Dlvkbn of Polymsr Chadstry of the ACS. ASTM. AAAS. and Deiawaro Valley Chomatography Frmm.
Charlea n. Losknwr ls ROI~MU 01 Ommkby and Chairman of me Dspmwnl at w e un)vasnv. He mwked Ms B.S. h Ommlsbv from Manhallan C d e m and me M.S. and Ph.0. from FomamimkmW. He was a poam0Ctaal a(uocb1e '4%L. B. Rogers al P u d w far 2 yean p m lo jolnlcg
me w e
Iscunv In 1969.
by plasma spectrometers, a much smaller market. Another more detailed study on the HPLC market from a survey of Analytical Chemistry readers, also conducted by Centcom (5d, provided information on types of instruments and acceasories purchased, field and type of work of scientists using the technique, solvents used,and other market-oriented information. Interestingly, 53% of the respondees indicated that they planned to buy an HPLC unit within the next year. Clearly the increase in literature citations has echoed these market surveys. Over 8ooo references occurred in LC in this review period, the overwhelming number being applications-oriented. There has been a great increase in applications of LC techniques, particularly HPLC, into the Life Sciences. The major application areas were proteins, peptides, amino acids, and nucleic acid constituents. Applications in the areas of lipids, hospholipids, and glycerides lagged well behind, probably &e to the lack of suitable, selective, routine detection techniques. Pharmaceutical applications are a strong second with formulations, dosage forms, and drugs and drug metabolites in body fluids the main topics of interest. The applications of ion chromatography to anion analyses, and to a lesser extent cations, in a variety of matrices has also significantly increased. Of the more fundamental papers, efforts have concentrated on understanding the mechanisms of retention in chromato raphy, especially reversed phases, the most widely used L 8 mode. Studies on reversed-phase ion pair chromatography have resulted in a better prediction of retention behavior of ionic and ionizable compounds. The use of mobile phase additives such as chiral compounds and complexing species in reversed phase has seen greater application. Micro-LC and microbore columns are attracting greater interest as the price of solvents goes up and trace analysis limits go down. The investigation of new detection principles and improvements or extension of existing detection techniques continue to dominate the literature. An increase in the development and a significant increase in the use of electrochemical detecton and the continued work on the interfacing of liquid chromatography m a s spectrometry (LC/MS) have occurred. Pre- and p t c o umn derivatization has seen a more widespread application.
BOOKS AND REVIEWS With the continued ra id growth in HPLC, review articles,
books, and monographs aLund. The two newest general basic
E.
.a
He teaches
despite a slow worldwide economic recovery,the single largeat analytical instrument market ($568 million) and should grow to $866 million in 1986,about 15% growth per year. The HPLC market is now the single largest cate ory with 1983 worldwide sales of $365 million, with the U d . counting for about half of the total. By 1986 in the US.alone the sales should exceed gas chromatography by $100 million. The above study was briefly reviewed by Borman (la). Another recent survery (30)also indicated that LC, despite its already large user base, will maintain its rapid growth rate, only surpassed
books have been published by authors from the United Kingdom (4b, 1Ob). Additional HPLC-related books can he found in the 'Books and Reviews" reference section. The technique of reversed-phase chromatography is by far the most widely used today. The hook by Krstulovic and Brown (Ilb) coven the theory and practice, with about 30% of the text being devoted to applications in the life sciences where both authors have their expertise. More advanced monographs have appeared on the scene. These volumes are multiauthored and edited by a noted chromatographer and usually cover advanced topics or have an in-depth coverage of topics which only receive cursory coverage in basic texts. For example, the third volume of the 'High Performance Liquid Chromatography: Advances and Perspectives", edited by C. Horvath (96) covers four topics: reversed-phase chromatography of nucleic acid fragments (Scoble, Brown), the separation of proteins (Hancock, Sparrow), the separation of peptides (Hearn) and mobile phase effects (Snyder). The Chromatographic Sciences Series has added two new volumes (166,226): Volume 23 is devoted to liquid chromatography detectors with detailed chapters on UV-Vis absorption, fluorescence, electrochemical, refractive index, mass spectrometric detectors. An interesting chapter on less popular detectors (e&, infrared, transport radioactivity, etc.) and a useful chapter with coverage on data handling in LC round-out the hook. Volume 24 covers HPLC in forensic chemistry with applications in diverse topics such as explosives, toxicology, and writing inks. Two volumes have also been added to the "Advances in Chromatography" Series (Zb, 36). The LC topics covered in Volume 20 (26) are proteins, vitamin D3 and metabolites, applications in Children's hospital, the silica gel surface and interactions with solvents and solutes in LC, and a treatise ANALYTICAL CHEMISTRY. VOL. 56. NO. 5. APRIL 1984
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on analysis of the fundamental obstacles to steric exclusion chromatography of polymers of ultrahigh molecular weight. Volume 21 (3b) is devoted mostly to LC topics which include LC MS, high-performance liquid affinity, dynamic anion exc ange, droplet-countercurrent chromatography, chromatographic determination of copolymer composition, HPLC of K vitamins and their antagonists, and capillary columns in LC. The latter chapter of Ishii reviews his many contributions to this important research area which appears to have great analytical potential. Texts on more specialized topics provided in-depth coverage on topics of limited general interest. The CRC Press series “Handbook of Chromatography’’ has volumes which cover phenols and organic acids (5b)and polymers (21b). In addition to general discussions, these volumes tabulate LC retention and other chromatographic data. For example, the text devoted to polymers (21b) compiles data for over 1 2 000 compounds, including polymers, monomers, additivies, plasticizers, antioxidants, and pyrolysis products. In addition, it briefly covers theory and practice of steric exclusion chromatography, solvents for polymers, sample preparation, and techniques of extraction of additives from the polymer matrix. Lawrence has been particularly prolific in this review period, having edited or written three texts on HPLC application to trace analysis (12b, 13b) and pesticides and plant growth regulators (14b). In the book on organic trace analysis (12b), he devoted about half of the book to basic LC and the rest to chemical derivatization, sample extraction and cleanup, approaches to methods development, and routine analysis. About 40 pages were on applications. His second volume (13b) covered trace vitamins, LC/MS, size exclusion chromatography in trace analysis, trace enrichment for organic trace analysis, and polar substances on unmodified silica. The pesticide and plant growth regulator text (14b)covered HPLC methods for insecticides, acaricides, fungicides, herbicides, plant growth regulators, and rodenticides. Methods for both formulations and residues were given. The general format of each compound type (e.g., carbamates) was compound name, molecular strucutre, general information, and analysis method. The analysis section contained reviews of published methods and then recommended an appropriate detailed method including the extraction procedure, column, mobile phase, and chromatographic conditions. Other texts with LC coverage were noted. A book with specialized coverage on HPLC in food analysis was edited by McCrae (18b). Although not devoted to HPLC, a book on polycyclic aromatic hydrocarbons (PAH) in water systems had particularly good coverage on LC methods ( I b ) . The availability of high sensitivity fluorescence and reversed-phase packings were the main reasons of LC’s success for PAH separation and analysis. A section on coupled LC columns for sampling and concentration was of interest. Retention indexes of PAH and comparison of four types of LC detectors (fixed wavelength detection at 254 nm, variable wavelength detection, fiiter fluorometry and spectrofluorescencedetection) were other noteworthy sections of this book. A book of “The Best of American Laboratory” excerpted the more important papers on separation techniques, including LC, from the magazine ( I 7b). Symposia on HPLC in the life sciences have generated several noteworthy texts. The subject of HPLC of proteins and peptides has been a popular one. One book resulted from the first “International Symposium on HPLC of Proteins and Peptides” held in 1981 and was edited by Hearn, Regnier, and Wehr (8b). The text had 28 selected papers from the lectures and poster papers. The topics ranged from applications of ion exchange, reversed-phase, and size exclusion chromatography to protein separations to preparative fractionation and purification of polypeptides. The second Symposium, held a year later, added polynucleotides to the program and the proceedings were published as a single issue of the J. Chromatogr. (7b). This expanded volume had 58 papers which included the earlier topics plus detection methods and applications on PTH-amino acids, hemoglobins, enzymes, transfer RNA, and DNA fragments among others. Another volume on biological and biomedical applications of LC was the end product of a Fourth Symposium on this topic (6b). Besides proteins, peptides, and nucleic acids, use of HPLC in clinical, therapeutic drug monitoring, pharmaceutical, toxins, and synthetic organic chemistry was covered. Finally,
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a meeting held at the Max Planck Institut fur Biochemistry in 1981 also resulted in a volume devoted to proteins and peptides (15b). A number of general review articles on basic LC or recent developments in HPLC have been published in and are covered in the “General Reviews” references (“c” references). These are covered in the “General Reviews” references (“c” references). Although applications papers are not the thrust of this current review an alphabetical listing of these specialty reviews is given in Table I as a convenient, time-saving device for interested readers.
GENERAL THEORY A great deal of emphasis on the development of a theoretical basis for chromatographic separation and efficiency has been devoted to reversed-phase systems and is dealt with in that section. This section deals with work that is more general in its significance although the question of what constitutes “theory” is far from settled. Theory, in this review section includes optimization strategies as well as the more obvious questions of retention and performance. Studies which provide insight into the nature of chemically bonded phases are also found here. Schoenmakers, DeGalan, and Billiet ( I 7e) presented an informative discussion on the modified, Hildebrand solubility parameter as a tool for understanding liquid chromatography. Solute-solvent localization effects and their influence on mobile phase selectivity was the topic of an important paper by Snyder, Glajch, and Kirkland (21e). The contributions of Golay and of Golay and Atwood to a theoretical basis for band dispersion in chromatography are monumental. The question of peak dispersion in short, straight tubes was discussed by these authors ( l e ) . The general question of the nature of the bonded phase formed by the silylation of silica continues to be of interest. Nonreversed phase studies include those of Lochmuller, Marshall, and Wilder ( I l e ) using fluorescent probes in studies of solvation of the bonded phase. The discovery of evidence for microheterogeneity in the distribution of the bound molecules (12e) and its further confirmation (13e) suggests that much more needs to be determined about the reaction chemistry of bonded-phase manufacture. “End-capping” has become a popular method to remove residual chemically accessible silanols in bonded-phase materials. The choice of end-capping reagent can have significant effect on column performance especially for acidic and basic solutes (14e). Optimization in chromatgraphy continues to be an area of active interest. A very good review of the optimization of selectivity is provided by Glajch and Kirkland (5e). A need to balance and use such important parameters as mobile and stationary phase, pH, and other ionic effects is presented. The use of mixture-design statistical technique is the object of two reports (6e, 7e)by the same authors and Snyder. A contrasting approach is put forward by Schoenmakers and DeGalan (18e, 19e). Statistical methods for optimization which neither require nor consider the physicochemical basis for separation and are often successful. Wegscheider and Matthias used a multifactor model to optimize selectivity in the chromatography of peptides, acids, and amino acids (15e). Deming (16e) has also utilized this approach in reversed-phase problems. For some problems where the number of components is small and the number of factors limited, the SIMPLEX approach is useful (2e). The use of SIMPLEX in multicomponent, multifactor separations is greatly complicated by (a) the sluggishness of the high-order SIMPLEX approach and (b) the existence of many local optima. Fast, Culbreth, and Sampson applied multivariate (and univariate) methods to reversed-phase chromatography of steroids (4e). If one constrains the number of factors (e.g., mobile phase components), the discovery of optima (which are unlikely to be global optima) is of course easier. An example of such a case is the “graphical optimization” approach for binary mobile phases (9e). Of interest is the use of molecular structure-property relationships for the prediction of retention indices of polpuclear aromatic compounds by Jurs (8e). Pattern recognition methods were applied to both GC and LC analysis of oly chlorinated biphenyls (10e). Juvet has reexaminecf the problem of quantitative resolution of severely overlapping
COLUMN L I Q U I D CHROMATOGRAPHY
Table I. Speciality Reviews references
pharmaceuticals
39d, 47d, 130d 4d, 48d, 60d, 61d, 72d, 104d 20d, 24d, 27d, 84d, 116d 6d 30d 26d 23d, 121d 6 5d 81d 89d, 123d 13d, 24d 99d 37d 31d, 54d, 64d, 83d, 96d, 120d 92d, 124d, 132d 102d 35d, 97d 63d 2d, 12d 115d 10d, 101d, 119d, 136d 138d 5d, 43d, 46d, 75d, 82d (in brain), 103d (polyhormones), 108d, 114d (neuro-), 128d, 135d 14d (clinical chem), 17d, 28d (human interferon), 38d (serum lipoproteins), 42d, 44d, 49d (reversed phase) , 74d (and polynucleotides), 76d (glycohemoglobins), 85d, 86d, 90d, 91d, 94d, 109d (neurochemical interest), 110d, 112d (metalloproteins), 134d 87d, 88d 107d 125d 95d
pharmacokinetics therapeutic drug monitoring vitamins B, C
life science amines, biogenic catecol amino acids, free
PTH using chiral eluents B-blockers bile acids bile pigments bilirubin metabolism biopolymers, general carbohydrates carboxylic acids cholesterol biosynthesis clinical, general microbiology glycosides, cardiac hemoglobin variants isoenzymes lipids lysozyme (in egg white) nucleic acid constituents nucleosides only peptides only
peptides and proteins
ion exchange steroids, adrenocortico conjugates estrogen pharmaceuticals
references
antibiotics, general peptide p-lactam ant iconvulsants antifungal drugs antimicrobial agents drugs, general antineoplastic p-adrenoceptor blocking
77d 56d 71d 57d 129d 131d 50d, 52d, 55d, 78d, 106d llld 70d
peaks by computer methods (3e). Another approach to resolution and classification is to use a multidetector approach with detectors whose responses are as orthogonal as possible. The power of such an approach in the classification of coal was provided by Sepaniak and Yeung (20e).
COLUMNS The major advances in column technology took place during the 1970s. The development of the microparticulates with average particle diamters 10 pm and under and optimum methods to pack them occurred in the early 1970s. Many studies on the technology of bonding a variety of stationary phases appeared in the mid-to-late 1970s. The 1980s have seen a refinement in bonding procedures, especially with regard to phase reproducibility, the development of column packings optimized for specific applications (e.g., oligonucleotide and ion chromatography),the more widespread use of microbore columns (0.5-2 mm internal diameters, i.d.), and
D n
retinol isomers water soluble
references 59d 4 Od 80d 7d 126d 105d 137d 93d
foods, agriculture, natural products bitter acids (in hops, beer) brewing technology enology flavonoids flavor compounds foods, general nutritional analysis (foods) plant, phenolics foods, agriculture, natural products pigments sugars vitamins (in food)
references 19d 58d 25d 16d 62d 8d, 18d, 21d, 34d, 67d, 6 8d 122d 118d references 98d 51d, 127d 45d
industrial
references
coal carbonization cosmetics oligomers polyurethanes surfactants, general nonionic
33d 73d 15d, 79d 3d 41d 29d references
environmental general my cot oxins pesticides polynuclear aromatic hydrocarbons trace organics, general water pollution miscellaneous organometallic/metallic coordination compounds
Id lOOd 36d 22d 9d, 32d, 113d 53d references 133d
further research studies on open tubular and packed capillary (C0.3 mm i.d.) columns and related instrumentation. A number of more convenient column hardware designs have appeared commercially, ranging from finger tight end fittings to integrated guard-analytical column assemblies. According to a recent survey (5a),the purchase of prepacked microparticulate columns is favored over “pack your own” by a margin of 81. Prepacked columns are available for aJl modes of HPLC and are usually tested and of guaranteed performance (435). Two general reviews of columns were published (13f, 695).
The trend toward the use of shorter conventional columns (4.0-4.6 mm i.d.) for fast separations (4f, gf-llf, 24f, 595) and narrow microbore columns (32f, 53f, 56f, 715) places additional constraints on instrumentation and column design as was pointed out by several workers (29f, 33f, 40f, 585). A simple comparison of conventional vs. micro-LC columns was provided by Majors (415). The advantages of microcolumns are (1)increased sensitivity, especially for sample limited situaANALYTICAL CHEMISTRY, VOL. 56, NO. 5, APRIL 1984
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Table 11. LC Column/Mode Usage column usage (19821983)
%
chromatographic mode reversed phase (total) C18 C8
c2 phenyl other normal bonded phase (Le., -CH, -NH,) liquid solid (adsorption) ion exchange cation anion size exclusion organic aqueous total papers surveyed: 369 the journals surveyed were:
72
(54)
(9.6) (2.5) (2.1)
(3.2) 5.9 10
6.3 (2.3) (4.0)
5.5 (1.9) (3.6)
%
column usage
3
(1981-
5
1982)
7.5 10 > 10
61 (48) (8.7) (1.0)
a
1.4 35 7.5 54 2.6
Total papers and journals survey: same as Table 11.
(1.1) (1.7) 6.8 14 12
(5.7) (6.5) 6.0 (2.2)
(3.8)
Clin. Chem. J. Chromatogr. Sei. Anal. Chem. Chroma tographia J. A m . Assoc. Anal. Chem. LC Mag. HPLC J. Chromatogr. J. L iq. Chroma togr. J. Chromatogr. (Biomed. Appln.) Anal. Biochem.
tions, (2) reduced solvent consumption, and (3) enhanced detector capabilities, especially with LC MS, electrochemical detectors, and possibly flame-based G detectors. A review of the so-called “fast LC” columns was covered by DiCesare (7f). The main advantage of short, larger i.d. (0.6 cm) columns packed with 3-pm particles is that conventional equipment can be used since extra column effects contributed from connecting tubings, injectors, end fittings, detector flow cells, etc. of many commercial chromatographs are of lesser importance (62f). The main disadvantage is that higher flow rates (i.e., increased solvent use) are required to keep separation times reasonable. Other comparisons of the 3-pm to 5-pm coluns have been published (Sf, l l f , 24f). Basically, an optimized system which has an extra column variance of less than 0.2 pL2 (57f) is deemed suitable for the short 3-pm columns or microbore columns (1m X 0.5 mm i.d.). Microbore columns are reviewed elsewhere in this article. Table I1 presents data representative of the published use of the different LC modes and types of columns in the review period 1982-1983. The data were compiled from a cross section of journals which deal with LC applications. The 369 surveyed articles were randomly selected. The relative changes since the previous review (2a) are also tabulated. It is no surprise that reversed-phase chromatography (RPC) still dominates the applications literature. In fact, compared to the 1980-1981 timeframe, the RPC technique has even grown relative to the other modes, now representing about 72% of all publications. This rise coupled to the marked decrease in ion exchange applications leads one to believe that the use of reversed-phase ion pairing techniques is beginning to take a strong hold in the separation of ionic and ionizable compounds. The use of C18-columns still represents over half of all LC applications. The 20% drop was noted in the relative use of normal phase for both adsorption and bonded phase columns. In normal-phase work, however, use of adsorption (mainly silica gel) columns still dominates bonded phases by 21. Bonded phases have not displaced silica gel as the favored packing in this mode. In Table I1 the use of size exclusion columns are undoubtedly slightly low since no polymer journals were included in the survey. The average particle diameter (d,,) of the microparticulates used in packing HPLC columns varies from 3 pm up to around 15 pm. Table I11 summarizes the distribution of d used in applications papers published in the surveyed articles. The 10-pm columns have been the “workhorse”columns but clearly 5-pm packings are gaining in popularity. The newest 3-pm packings have not made an impact in the applications literature. The reason may be that most liquid chromatographs in use are not optimized for the greatly decreased extra column
(4
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Table 111. Particle Sizes of LC Columnsa av particle size, pm % use
ANALYTICAL CHEMISTRY, VOL. 56, NO. 5, APRIL 1984
band broadening requirements of the shorter columns. Furthermore, the shorter columns do require more care in their operation than do the larger d, columns. Although improvements have been made in the batch-tobatch reproducibility and performance of commercial bonded reversed-phase columns, when attempts are made to switch an analytical method from one manufacturer’s column to another, solute retentivities are significantly different as well as performance and numbers of residual silanol groups (14f). Column Packing Materlals
Overall, the use of silica gel-based packings for all LC modes still dominates. The major limitation of silica gel is its upper pH limit, about 7 to 7.5. This limitation has encouraged the development of alternatives. In reversed phase, carbon-based and resin-based packings have received some attention. Knox (28f) and Unger (640 have reviewed the propects of carbon as a packing material in HPLC. Advantages of carbon are wide pH range, good retention for polynuclear aromatic hydrocarbons, different selectivity from reversed-phase silica, and relative inertness; the major disadvantages are lack of rigidity of microparticulate, adequate surface area, and poor efficiency for strongly retained compounds. At this time, no commercial carbon-based packings are available. The polystyrene-divinylbenzene (PS-DVB) packings permit operation over a wide pH range of 1-13 as discussed in a review article (36f) but give somewhat poorer efficiency than silica-based packings. The separation of chloriated phenols at pH values of 7.4-12 (450 and pyrrolizidine alkaloids using acetonitrile-ammonia gradients (51f) illustrated the applicability of PS-DVB-based packings. Alumina has also been advocated as a replacement for silica since it, too, has greater stability at higher pH values (34f). However, when siloxane phases are bonded to alumina, it is the phase itself, not the base alumina, which is unstable. Alumina apparently has more potential as an amphoteric ion-exchange packing (35f). Unlike alkyl bonded silica, the alkyl bonded zirconium phosphate Dackinm have no Dolar residual sites to interact with basic compobds (440. An interesting phase consisting of acriflavin bonded to silica exhibited not only charge transfer properties but electrostatic and hvdroDhobic interactions (60fl. It was used for the separatiin of !ow molecular weight biochemical substances such as purine and pyrimidines as well as nucleosides and nucleotides. For normal-phase work, several new types of phases have been prepared and/or evaluated: pellicular polyamide (63f); tetrachlorophthimidopropyl silica recommended for aromatic hydrocrbons (22f); and nitro aromatic bonded phases prepared by derivatizing amine bonded phase (18fl. Hara (19f,200 has provided useful comparisons of cyano and amino phases bonded to silica gel for steroids. Characterlzatlon
Verzele and co-workers (Sf, 48f, 65f, 66f, 67f, 68f) have carried on numerous studies in the comparison and characterization of silica-based column packing materials. One study on the influence of particle shape has shown that columns packed with spherical silica gel particles provide 1.5 to 2 times the theoretical plates than irre ularly shaped particles, provided optimum packing procejures are used for each (65f). Issaq and Gourley (24f) compared 3-pm and 5-pm ODS spherical particles to irregular ones of similar size and noted differences in separations when using conventional, unmodified instrumentation. However, the packings were obtained
COLUMN LIQUID CHROMATOGRAPHY
from different sources and packed into columns of different lengths. An investigation of the influence of particle size distribution (Ad,) of packing materials used in reversed-phase chromatography was carried out by Dewaele and Verzele (Sf). Mixtures of packings with mean particle sizes (d,) of 3 and 3-10 pm were prepared with different compositions and column efficiency and back pressure measurements made. The study concluded that a large particle size distribution has no influence on efficiency if the flow velocity is near the optimum and only a small negative effect at higher velocities. However, at all velocities a large particle size distribution gave higher column back pressure. The recommended maximum practical distribution of A d , / d , is approximately 1.5-2. Of course, the presence of “fines” or dust in all column packing materials is undesirable. Most polymeric phases in reversed-phase chromatography were found to be actually monomeric phases (66f). Pyrolysis (48f) or NMR (Zf) studies of reversed-phase materials can yield information about the alkyl side chains of the bonding phase as well as determine if tri-, di-, or monofunctional reagents were used to prepare the packing. The effect of trace metals present in silica gel can have an undesirable effect for classes of compounds which can form complexes. Trace metal probes have been proposed to uncover the presence of these active sites (67f, 68f). In order to determine the pore size and distribution of ion exchange polymers which swell in water, Crispin and Halasz (37f) used the partial exclusion of sugars and dextrans from the pores of a cation exchange resin with a given counterion. With different counterions the pore volume changed due to Gibbs-Donnan effects. Column Packlng Technlques
Studies on the optimization of high pressure slurry packing techniques continue to be carried out. A review of many of these procedures has been published (55f). Methods to pack high efficiency columns with commercially available packings and equipment was reported (46f). Halasz and Maldener (17f) described another new method to pack macrobore columns (>3 mm i.d.) with silica gel and, contrary to some earlier works, demonstrated that one can couple columns with no loss in efficiency. A single slurry packed wide-bore (8 mm i.d.) column of 50 cm length gave more theoretical plates than two narrower bore (5 mm i.d.) columns in series (38f). However, the total plate count of the two narrower bore columns connected in series was the same as the sum of the individual columns provided a short 5 cm piece of 0.15 mm or 0.5 mm i.d. tubing was used for the connection. In selection of slurry packing solvents, agglomeration caused by coulombic attraction forces between particles must be minimized and wettability achieved. The use of organic acids to protonate the silica gel surface active sites (37f) and imparting an electrostatic potential by application of a pH gradient ( I f ) were two recommended procedures. The effect of the quality of the column inner wall was investigated (26f). Polished and nonpolished tubes were packed and investigated by either central injection (A) or distributed injection (B). For procedure A no difference in performance was observed while for procedure B polished tubes gave higher efficiency since the packed bed near the walls of nonpolished tubes are disturbed resulting in uneven flow profiles. Fused silica capillary columns with internal diameters less than 0.5 mm require special packing techniques. Novotny et al. (150 have developed optimum packing conditions and have evaluated the performance of capillary columns slurry packed with 3- and 5-pm spherical particles. Column Hardware
Stainless steel columns are still the most widely used in HPLC. Nickel tubing has been proposed as an alternative (49f). Being softer it is easier to machine, and the authors claim the acked column can be cut with a tubing cutter with no disturfance of the packed bed. Since the column end fittings usually represent a substantial portion of its cost and the compression type of fittings inconvenient when connecting
and disconnecting columns to the chromatograph, attempts have been made to do away with end fittings. Commercially serveral companies have developed holders which permit column cartridges to be used. The most popular are the radially compressed plastic cartridges (12f). A critical evaluator (31f) of stainless steel and the radially compressed plastic columns found that for flow velocities around the optimum value radial compression does appear to improve bed homogeneity as evidence by a low “A” term. However, at higher linear velocities the resistance-to-masstransfer dominates and any improvement of radical compression is lost. Representative applications of radially compressed columns are for the separation of peptides ( Z I f ) , glycoalkaloids (47f), and antiepileptic drugs in serum (27f). Column Performance and Evaluation
A popular controversial topic during the review period was techniques to determine the void volume, Vo (or to),in HPLC. For rigid packings which do not shrink or swell in the presence of solvent, the void volume (i.e., total volume occupied by solvent in the packed column) should be a measurable, unchanging quantity. Yet, for supposedly nonretained (inert) species such as a deuterated mobile phase, Vois often found to be solvent dependent. For example, in a dry dichloromethane eluent, Halasz and Groh ( I 6 f )found that C6 cyclic hydrocarbons eluted and actually could be separated prior to the inert peak, a deuterated dichloromethane. The “negative” adsorption was attributed to a blockage of pore volume by adsorbed eluent when dry. As trace amounts of water were added to the dichloromethane,the adsorbed eluent was displaced with an adsorbed monolayer of water and the to value changed. The conclusion of the study was that for routine LC, isotopically labeled eluents should be used as the inert peak. For reversed-phase chromatography, Guiochon et al. (300 compared methods for measuring V,. The methods compared were injection of deuterated forms of mobile phase components, injection of NaN03 and DzO,linearization of retention data for a homologous series, and a new proposed technique which measured the convergence of several homologous series. When only the latter technique was used, the same value of Vowas found. Unfortunately the approach is time consuming but gave a good estimate of V,,. Another later study compared a wide variety of solutes for use on C8, C18 and PS-DVB reversed phase columns when using aqueous methanol or acetonitrile eluents (Wf).The authors concluded that NaNOz or NaN03 was suitable for Vomeasurements but DzO gave two peaks on a C18 column. A method on the dependence of retention times with temperature (2‘) with equidistant 1 / T intervals was proposed (500. Additional discusion on Vo determination can be found in Reversed-Phase Chromatography: Theory. A universal retention index scale for reversed-phase chromatography was proposed by Smith (61f). By use of a series of alkyl aryl ketones, this author obtained a linear plot of log k’and carbon number. These compounds absorb at 254 nm and were unaffected by change in the ionic strength, pH, and buffer type. Such a system would offer an easily selectable series of compounds as potential internal standards in reversed-phase method development. Column Maintenance and Troubleshooting
Since modern LC columns are expensive and somewhat fragile, methods for increasing their useful lifetime are of great interest. Column protection devices range from simple in-line filters with 0.25-hm sintered metals frits to elaborate low volume guard/analytical column combinations. By far the most popular column protection device is the guard column which is placed between the injector and the analytical column. Of the 369 applications papers surveyed for the data presented in Table 11, approximately 27% reported the use of guard columns. Some case histories on the successful use of guard columns have been published (42f). Ideally, the packing used in the guard column should exactly match that of the analytical column, although if judicious care in selection is used, different packing materials are possible (Sf). The effect of guard columns on reversed-phase column ANALYTICAL CHEMISTRY, VOL. 56, NO. 5, APRIL 1984
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efficiency has been the object of a study by Lundanes et al. (39f). They compared the use of porous to pellicular packings as guard column packing materials. They concluded that pellicular packings were advantageous over porous packings unless the size of the guard column was reduced (presumably to have equal stationary phase volume) or the guard column was packed with a less retentive stationary phase. Another investigation (520 recommends the efficient use of microparticulate packed guard columns and considered connecting tube dimensions, particle size of guard column packings, and use of low or zero dead volume end fittings. Some columns may be re aired by merely cleaning or replacing the end frit, backdshing to remove accumulated materials on the frit or strongly sorbed compounds at the head of the column, or washing the column with a series of strong solvents as reported by Wehr (70f).A void or channel created in the inlet of the column bed can sometimes be repaired by “topping off“. Some columns can be occasionally reversed to avoid buildup of undesirable materials (230.
INSTRUMENTATION General
Improvements continue to be made in LC instrumentation, especially in the areas of decreasing extra column effects, new detector developments, and increased automation capabilities. Most of the modern LC instruments are microprocessor controlled, which provides more programmability, system integration, and (hopefully)ease of use. Instrument controllers can generally gradient control pumps, injectors, detectors, column heaters, data systems, and provide external events to time program other peripheral devices such as column switching valves or postcolumn reagents pumps. The need for lower instrument band dispersion contributions is driven by the shorter, 3- and 5-pm conventional microparticulate columns and microbore columns (1and 2 mm i.d.). Scott and Simpson (58f) have developed procedures to determine extra column dispersion occurring in different components of an LC system. Using a conductance detector with cell volume of 80 nL, connecting tubing of 0.36 pL total volume, they evaluated the extra column dispersion at 20 pL/min for the 1mm i.d. microbore columns. Brinkmann et al. (29f) also developed a simple procedure to investigate extra-column band dispersion and showed that conventional LC instrumentation can be modified to accommodate the newer microbore columns. A new equation which described the interdependence of instrument band spreading, injection volume, and in ut profile was developed (33f).Scott and Katz (59f) discussefthe instrument design characteristics for the successful application of short, conventional LC columns. Besides several new books with chapters on instrumentation (covered in Books section), several general reviews have been published (34f,36g, 40g). The general one by McNair (34g) reviews the newest commercial instrumentation while articles on specific instruments have been covered (12, . - 72. - 1 7- ~2. 2 ~ . 25g,-27g, 30g). One apparent trend in HPLC instrumentation is the increased application of general purpose chromatographs to specific analyses once dominated by dedicated analyzers, such as amino acid analyzers and ion chromatographs. A commercial amino acid analyzer was compared to a high-performance liquid chromatograph and good agreement between the two was found for the analysis of plasma amino acids (18g). Amino acid analysis using conventional HPLC equipment is also the subject of other investigations (19g,20g). Girard and co-workers (llg-13g) and others (6g, 9g, l o g , 21g) have performed single column, ion chromatographic analysis with conventional HPLC instrumentation. I
I
I
I I
Pumps, Hydraullcs, and Gradlent Devices
Improvements in conventional commercial pumping systems have been in the area of multiple solvent single pump devices, with ternary (17g,22g, 25g) and quaternary solvents (7g,27g, 30g) with low pressure proportioning leading the way. Multiple solvent pumping systems are most useful for automatic methods development (22g,25g), optimization of separations (30g, 31g), and isocratic proportioning. The latter technique refers to the use of a gradient system to provide isocratic mobile phase compositions and is convenient from an operational standpoint. Here pure solvents can be pro306R * ANALYTICAL CHEMISTRY, VOL. 56,NO. 5, APRIL 1984
portioned with the pump(s) to generate solvent mixtures without the need for the chromatographer externally manually preparing isocratic mobile phases. Noncommercial programmers have also been constructed. One system is based on a PDP11/03 microcomputer with controls which synchronize two two-way switching valves connected to the solvent reservoirs for low pressure gradient formation (29g). The valves provide the proportioned solvents to a high-pressure dual piston reciprocating pump equipped with displacement transducers. The computer software corrects for solvent compressibility and generates gradients of virtually any shape from 0 to 100% of strong solvent. A similar computerized system controls the flows of two individual pumps and permits an infinite set of gradient shapes, preprogrammed column regeneration, and control of column switching valves (2g). An HPLC system controller whose control is based on detector threshold monitoring has been described (3g). For gradient formation at the low flow rates (99% removed at the ppm level. Uranium(V1) was determined in groundwater and urine using a bonded- hase cation exchanger with an a-hydroxyisobutyrateeluent (68. By in-line trace enrichment, concentrations from 0.5 to 50 ng/mL from groundwater and 25 to 400 ng/mL from artificial urine were measured. Sixteen rare earth elements along with SCand Y were determined in 75 min by win a strong cation exchanger. An a-hydroxybutyric acid and pggradient was required; the metal ion was detected coulometrically. Peptldes and Protelns
The literature now abounds with applications of both cation and anion exchange chromatography to the separation of proteins and peptides. In the Reviews and Books section, a number of these contributions were cited. Studies on the retention model for high-performance ion exchange chromatography were carried out by Regnier (18t). The retention of a protein on an ionic surface results from the charge of 322R
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protein, the surface charge, and the charge characteristics of the surrounding medium. The previously accepted net charge model was used to explain protein retention, but retention mapping studies on strong ion exchange columns showed it to be inadequate. Instead, deviations may result from charge asymmetry since it appears that only a fraction of the protein surface interacts with stationary phase. Retention is also altered by the type of displacing salt. Further recommendations on mobile-phase selection were provided (18t). A study of experimental conditions of the effect of gradient conditions, flow rate, column length, and sample loading on resolution and retention were carried out on TSK gel IEX-545 DEAE SIL (15t). Column length was relatively unimportant in the gradient elution experiments since only a portion of the sample uses the column exchange capacity at any iven time in the gradient, in agreement with the earlier finings. Macroporous supports with lo00 and 4000 A pores with heavy layers of poly(ethy1eneimine) and cross-linked with 1,4-butanediole diglycidyl ether have enhanced anion exchange capacity and resolution for proteins as well as small molecules (33t). Cation exchange has also proved to be a useful technique for basic peptides (16g) and proteins (5g,44g). Variables in the cation exchange of proteins on silica-based cation exchanger were studied (9t). An increase in temperature decreased retention and resolution but increased recovery. A decrease in pH in the final eluent or increase in pH in the initial eluant caused an increase in retention time. For some proteins, the decreases in pH resulted in a greater total recovery. Improvement in resolution of hem0 lobin variants, important in the diagnosis of certain metaiolic disorders, continues to be made (134 32t). Nonlonlc Separatlons on Ion Exchangers
Many separations reported to be ion exchange often involve mixed mechanisms where other sorption effects contribute to chromatographic separation. The use of ion exchange packings for the separation of nonionic compounds, such as the earlier cited examples of the separations of oligosaccharides (28t)and lipids (30t)on Ag+ loaded cation resin represent an extreme in this phenomenon. The separation of organic acids on cation resins in the H-form represent the ion exclusion mechanism based on the repulsion of a completely ionized solute from the fixed hydro en ions in the resin surrounded by the Donnan membrane b 6 t , 36t). Counterion effects in ion exchange partition chromatography were studied by Dieter and Walton (34th For the polar aromatic compounds studied, retention volumes and band width were affected by the counterions Li, Na, K, Mg, and Ca, with Li generally giving the best chromatography. In addition to the water of hydration, the "free" water in the resins also had an effect on retention by interacting with solute by hydrogen bonding.
ION CHROMATOGRAPHY The last 2 years has seen a tremendous growth in the applications of ion chromatography (IC). The latest Centcom survey (4a) shows that IC is one of the fastest growing markets with a growth rate even above HPLC itself. The IC technique is the preferred method for the determination of low concentrations of anions in aqueous solution and the applications of the technique are broad and far reaching. The published use of the nonsuppressed (or single column) technique has seen a most rapid growth since special suppression columns are not required and conventional LC equipment can be used. However, newer suppression techniques, such as packed hollow-fiber suppressors (64u),improved columns ( 6 3 ~and )~ instrumentation have maintained interest in the suppressed IC approach as well. At least one new book devoted to IC appeared during the review period (60u).A large number of general reviews of IC have been published (5u,6u,9u,28u, 31u, 38u, 61u, 74u, 75u) as well as reviews in more specific applications areas such as ion analysis in high purity water (46u),water quality studies (27u),pulping and bleaching process liquors (24u),process IC ( ~ O U )trace , metals ( 4 9 4 , wastewater and power plant water (47u), general water analysis (23u, 55u), foods (Zlu),environmental (58u),electroplating industry (29u),bombing investigations (48u),and the iron and steel industry ( 1 6 ~ )AS .
COLUMN L I Q U I D CHROMATOGRAPHY
Table VII. Applications of Ion Chromatography by Ion Type reference ion acetat e arsenate bicarbonate boric acid bromate bromide
5 7U 35u, 84u 2u 7 9u 131.1,M u , 42u, 73u 18u, 43u, 53u, 57u, 621.1, 65u, 6711, 71u, 72u,81u
cations chloride
l u , 8u, 56u, 66u
chromate cyanate cyanide dichloroacetate fluoride formate glycolate iodate molybdate nitrate
84u
nitrite organosulfur oxalate oxyselenium ions phosphate sulfate sulfide sulfite thiocyanate thiosulfate tungstate
2u, 7u, 4 3 4 5 3 4 59u, 62u, 67u, 71u, 72u, 81u 57u 20u, 50u 13u 43u, 45u, 68u,69u, 721.1, 80u 13u 13u 13u, 18u, 57u 2 2 4 84u 2u, 13u, 14u, 18u, 25u, 261.1, 43u, 5311, 57u, 67u, 71u, 82u 13u, 17u, 18u, l u , 36u,43u, 57u, 82u 77U 44u, 57u
83u, 84u 17u, 35u, 67u 41u, 53u, 59u, 65u,67u, 71u 5Ou Tu, 15u, 3611,391.1, 65u 13u, 3 4 4 361.1 3611,651~ 22u, 84u
can be seen in Table VII, the application of IC to the analysis of common ions abound. Columns other than ion exchange can also be used for the practice of IC. Skelly (57u)used a reversed-phase column and added octylamine salts of mineral acids but attributed the mechanism to ion exchange rather than ion pair partitioning. Dynamically coated columns using hydrophobic molecule containing ionic functional groups in the eluant to separate different metal cations or anions have also been used (IOU). For cations, clzor czo sulfates and c6or c8 sulfonates were used while for anions (28, Cl6, cZ5, c32,or Cs9quaternary ammonium salts were dynamically coated. A similar study by Barber and Carr (3u) used a UV-absorbing quaternary ammonium compound as the ion-interaction reagent. Using both permanently and dynamically coated columns, Cassidy ( I Iu) was able to separate a wide variety of anions. With the Hamilton PRP-1 polystyrene-divinylbenzene resin packing, tetrabutylammonium or tetramethylammonium salicylates were used in the eluent forming dynamically coated columns. Permanently coated columns were prepared by coating with cetylpyridinium salicylate. An amino column was used with a phosphoric acid eluent to determine anions in 95% sulfuric acid and 20% sodium chloride solution ( 1 3 ~ )Microparti. culate anion changes (1000-50 000-8,diameter) were agglomerated onto microparticles of cation exchangers (5-100-pm diameter) to produce a low capacity pellicular anion exchanger packing but produced a low efficiency column (63u). However, the microparticles when agglomerated onto an efficiently packed 15-pm cation exchanger gave 3 times the number of plates. A silica column modified to display strong anion exchange properties was used for IC (76u). Weak acid anions were determined by ion exclusion by Pohlant ( 4 5 ~ )With . use of a dilute HC1 eluant, a Ag-loaded cation exchange column was used downstream to precipitate eluant anions, thereby allowing determination of low concentrations of fluoride in tap water. Studies of factors affecting the resolution and detectability of inorganic anions by nonsuppressed IC were conducted by several workers (214 37u, 53u, 54u). In addition to improvements in analytical precision, temperature control of the column and detector can increase sensitivity by an order of magnitude (3724). A dual differential conductance monitor
cancelled out temperature variations, allowing detection of about mol quantities of anions (53u). Step gradient elution has been used to get better sensitivity and shorter analysis times for sulfur oxyanions (39u). Preconcentration by Donnan dialysis prior to IC also gave enhanced sensitivity (19u). In suppressed IC, band broadening and the need for periodic suppressor column regeneration are problems which can be overcome by use of a packed hollow fiber suppressor (64u). The cation exchange membrane hollow fiber is packed with small plastic beads which generated mixing inside the tube and presents ions to the membrane at a faster rate. Use of unpacked hollow fiber suppressors was also reported (4u,32u, 5Iu, 52u). A particularly useful study which has potential for increasing IC sensitivity was a micro LC experiment where a 0.2-mm i.d. sulfonated hollow fiber tube was used with a 0.19-mm i.d. fused silica column packed with a newly developed anion exchange resin with low capacity (5Iu). Detection in the classical practice of IC has been use of conductance. However, other commonly used LC detection principles can also apply. The direct measurement of absorbance in the low UV was used to detect a variety of anions in nonabsorbing eluants (13u, 18u, 76u, 78u). A variablewavelength UV detector used in series with conductance can aid in the identification of unknown peaks, help resolve overlapping peaks, help to eliminate problems observed with “carbonate dip” in suppressed IC, and provide the ability to detect anions not normally determined by conductance detection such as sulfide and arsenite (78u). Applying the practice of vacancy chromatography, indirect photometric detection using a light absorbing ion in the mobile phase has been used to detect transparent or poorly absorbing sample , 59u, 76u). For example, dilute potassium ions ( ~ O U I, ~ u 334 hydrogen phthalate aqueous mobile phase was used with a 254-nm UV detector and the response from conductance and . UV deUV detectors were compared for anions ( 1 2 ~ )The tector gave 5-30 times greater response and showed better linearity. By using ion-interactive agents and indirect photometry, a 0.5 nmol anion detection limit was claimed (3u). Indirect refractive index detection was also used for nonUV-absorbing inorganic and organic anions and cations (30u). Decrease in the refractive index detector response of an eluant containing an aromatic counterion was compared to indirect UV absorbance and direct conductance detection. Other detection principles as applied to IC were streaming current ( ~ O U ) amperometry , (73u),and coulometry ( 6 9 ~ )The . use of potentiometry with a silver wire electrode coated with a sparingly soluble Ag salt was carried out (34u). This detection technique was claimed to be more sensitive to halides and thiocyanate than the more commonly used conductance detection.
SIZE-EXCLUS I ON CHROMATOGRAPHY Introduction This section reviews fundamental papers related to SEC (steric or size exclusion chromatography). In order to maintain a consistent nomenclature, SEC rather than GPC (gel permeation chromatography) will be used throughout this section. 1982-1983 have been rather lean years for books on SEC. CRC Press has published a handbook of chromatography of polymers ( 2 9 8 ~ )Although . it touts SEC coverage, very little information is presented in this area. The brief reviews of SEC and other chromatographic techniques are surprisingly poor. The book on HPLC of proteins and peptides, edited by Hearn, Regnier, and Wehr (87u), contains about five research articles on SEC. However, these papers originally appeared in Analytical Biochemistry. An excellent chapter on SEC was written by Bywater and Marsden (2%) which reviews in depth conventional packings, applications, and mechanisms of separation. Unfortunately very little information is presented on high-performance SEC and applications are limited mainly to separations on crosslinked gels. Several recently published books on polymer characterization contain a number of chapters of SEC interest: “Polymer Characterization”, edited by C. L). Craver (38u) (aqueous SEC, SEC of copolymers, and orthogonal chromatography), and “Developments in Polymer Characterization”, edited by J. V. Dawkins, Volume 3 (49u) (characterization of copolymers) and volume 4 (50u) (long-chain branching). ANALYTICAL CHEMISTRY, VOL. 56, NO. 5, APRIL 1984
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Finally, by the time this review a pears an ACS Symposium series on Recent Advances in SEE, edited by T. Provder and C. Kuo, should be out, which contains many pertinent papers. TheoryIBand Broadening
Giddings ( 7 6 ~reviewed ) the theory of SEC with emphasis on the potential limitations of this technique. Included in this comprehensive study were discussions of flow suppression in pores, relationship between particle diameter and analysis time for high molecular weight polymers, shear degradation, concentration polarization effects on the packing, and other secondary mechanisms. Aubert and Tinnell(9u) reviewed flow rate dependence of elution volumes, Flow rate dependence may occur if the distribution coefficient is concentration dependent, if viscous fingering occurs, or if molecular diffusion phenomena are important. In addition, instrument anomalies may result in apparent flow rate dependent elution volumes if the instrument performance depends upon flow rate. Ujimoto et al. ( 2 1 4 ~established ) a procedure for determining distribution coefficients of inorganic ions on Sephadex columns. They found that valid distribution coefficients could be obtained by using radioactive tracers (22Na,36Cl, and tritiated water) rather than with refractometry and potentiometric detection of “cold” ions. El’tekov ( 6 4 ~studied ) the dependency of the distribution coefficient on the physical characteristics of the packing, chemical structure and molecular weights of the solute, and temperature. Dawkins (46u, 48u) presented an excellent review of peak dispersion in high performance SEC and nonsize exclusion effects. Solute dispersion mechanisms were described including the influence of particle size of the packing, flow rate, and the solution diffusion coefficient. SEC separation mechanism was also discussed in detail. Mobile-phase optimization for proteins was given. Janca ( 1 1 3 ~obtained ) relationships between SEC column efficiency and peak asymmetry by using statistical moments. In a subsequent paper Janca ( 1 1 4 ~studied ) dispersion of polymer solutions flowing in capillaries. Variables investigated were polymer molecular weight, specific viscosity of the injected polymer solution, and flow rate. Gao et al. (714 72u) studied flow rate dependence on separation and band broadening using glass beads and porous silica as packings. Dispersion in the mobile phase was obtained by a reverse-flow method. This group of investigators also presented a computer program for correcting band broadening effects for molecular weight calculations. Netopilik ( 1 6 3 ~presented ) a procedure for correcting axial dispersion for viscometer or light-scattering detectors. Duobinis et al. ( 6 2 ~described ) an integral equation for correcting for instrumental broadening which was simpler than the iterative approximation method. Mori et al. ( 1 5 5 ~reported ) on a simplified solution of Tung’s instrumental spreading equation which can be done on a small computer. Busnel and Bruneau (24u) demonstrated that the errors introduced by axial diffusion were negligible for columns designed for low molecular weight polymers. For linear calibration curves, two correction constants, for number and weight average molecular weights, were used even for nonGaussian axial diffusion. For nonlinear calibration curves, the effect of axial dispersion became significant and a simple correction was impossible. Chiantore and Guaita ( 3 3 ~studied ) the effects of flow rate, solute mass transfer, and sample polydispersity on peak broadening. In properly packed columns, the eddy diffusion term was of minor importance; whereas, solute mass transfer could be minimized only at extremely low flow rates. Johnson et al. ( 3 7 ~investigated ) the effect of flow rate on efficiency and found that efficiency increased at mobile-phase velocities below 0.02 cm/s. From plate height measurements of nonpermeating polymers, Dawkins and Yeadon ( 4 7 ~found ) that the contribution to broadening from solute dispersion in the mobile phase was influenced very little by eluant flow rate. In view of this, a simplified plate height expression was developed for permeating polymers which permitted the evaluation of the broadening contribution from solute mass transfer in the pores of the packing. From the experimental dependence of plate height on flow rate, the mass transfer contribution was de324R
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termined. With this approach, the polymer diffusion coefficient in the pores of the packing was estimated. Rand and Mukherji (1724 evaluated several high-performance silica packings in terms of plate height measurements, polymer polydispersity, and specific resolution as a function of flow rate. The influence of injection volume and slice width on these measurements was also presented. Kat0 and Hashimoto ( 1 2 3 ~ studied ) the influence of injection volume and sample loading on resolution. Holding et al. (102~) investigated concentration and temperature effects for SEC of dextrans. In these experiments, mobile phases containing 10-20% dextran were employed. The presence of these high polymer concentrations had a dramatic effect on the elution volume af aextran test solutes. Several explanations to describe this effect were given. Ring et al. (17th) applied a computerized calibration technique capable of calculating a calibration equation that was a function of the true modal positions of Gaussian monodisperse polymer chromatograms and the appropriate dispersion correction term. Also presented was a dispersionally correct universal calibration derived from the dispersioncompensated equations. Busnel (23u) reviewed the major factors concerning data handling in SEC. Included was a simple method for taking into account axial dispersion using a non-Gaussian spreading function. Callbration
Universal Calibration. Lecacheux et al. ( 1 3 4 ~examined ) the applicability of the universal calibration technique by using polydisperse polymers. Also discussed was the characterization of heterogeneous polymers such as branched polymers or copolymers and problems caused by instrumental peak dispersion. Zhao (229~)reviewed the use of the universal Calibration procedure and the determination and applicability of Mark-Houwink constants. Wen (220~)studied the influence of different mobile phases on the universal calibration of polystyrene and silanized silica as the packing. For low polarity solvents (benzene and toluene), the universal calibration was valid. For polar solvents (methyl ethyl ketone and tetrahydrofuran), solvent molecules adsorbed onto the packing to form a restrictive layer. It was speculated that this caused a reduction in pore dimensions of the packing. Samay et al. (190~) compared average molecular dimensions of rigid molecules obtained by SEC and X-ray diffraction methods. Based on this, it appeared that SEC yielded the same dimensions as X-ray diffraction of the solid materials. In the case of very rigid molecules, the use of the universal calibration procedure appeared valid. Kat0 et al. ( 1 2 1 ~investigated ) the use of poly(ethy1ene oxide), pullulan and dextran as aqueous SEC standards with TSK-PW packings and 0.1 M aqueous sodium chloride as the mobile phase. The universal calibration curve was valid for these standards. See the references in Table VI11 under cationic polymers and polyelectrolytes for additional studies on the validity of the universal calibration procedure for aqueous SEC. Broad Molecular Weight Standards. Bauer and Raubach (15u) presented a method of calibrating columns with a broad molecular weight standard. A calibration function calculated from a theoretical molecular weight distribution for alternating copolymers was employed. Kalfus ( 1 1 9 ~de) scribed an iterative method for broad molecular weight standards. Kubin (129~) developed a general calibration method using polymer standards having arbitrary shaped molecular weight distributions with known number and weight average molecular weights. The method gave both the dependence of molecular weight on elution volume as a polynomial of statistically correct degree and the dependence of the spreading factor on elution volume which could be used in subsequent axial dispersion corrections of unknown samples. In a subsequent paper, Kubin ( 1 3 0 ~applied ) this technique to polystyrene, poly(methy1 methacrylates), and dextrans. Using two broad molecular weight standards, Rudin et al. ( 5 5 ~demonstrated ) that Mark-Houwink constants in solvents other than the mobile phase used for the analysis could be determined.
COLUMN L I Q U I D CHROMATOGRAPHY
Table VIII. Selected SEC Applications compound acrylonitrile-styrene copolymers bisphenol A based epoxy resin bkDhenol A-pcresol-divinslbenzene copolymer butadiene-styrene copolymers cationic polymers cellulose coal/petroleum products epoxy resins fulvic acid humic substances isoprene-styrene copolymers lignin metal p-diketone complexes nitrocellulose nylon 12 oligomers/small molecules oligosaccharides organotin copolymers phenolic resins phosphorylated poly(ethy1ene oxide) polyacrylamides pol yacrylates /polymethacrylates polyamides polyelectrolytes/water-soluble polymers polyesters poly(ethy1ene glycol) poly(ethylene glycol dimethacrylate) poly(1actic acid) polymer additives polyolefins ethylene-propylene copolymers polybutadiene polyisobutene polyethylene polypropylene poly( phenylquinoxaline) polyphosphates polysaccharides cellulosics (water soluble) cyclodextrins dextran gum acacia heparin xanthan polyst yrem polyvinyl copolymers proteogl ycans proteins/peptides
RNA
silicone elastomers styrene-methylmethacrylate copolymers
reference 73v, 74v, 75v, 213v, lOOm 4l v 61v 70v 58v, 59v, 68v, 79v, 81v, 122v, 137v, 142v, 143v, 173v 22m l v , 20v, 92v, 117v, 125v, 136v, 168v, 223v, 133m 209v 227v, 228v 146v 226v 94v, 161v, 170v, 184v, 73m 186v 27v, 139v, 140v 189v 40v, l l O v , 165v, 166v, 187v, 210v 1 1 5 ~128v, ) 169v, 178v, 179v, 211v, 224v 107m 132v, 182v, 194v, 210v, 143m 6 7v 162v, 71m 28v, 129v, 131v, 201v, 202v, 200v 108m 4v, 5v, 18v, 54v, 61v, 58v, 121v, 167v, 177v, 180v, 66m 91V) l l l v , 1918 127v, 60m, 63m 133v 215v 196v 218v 16v 3l v 19v, 21v, 51v, 63v, lOlv, 216v, 84m 101v, 216v 15v 152v, 153v, 154v 120v 207v 6v, 13v, 69v, 130v, 200v, 71m 158v 84v, 116v 8l m 47v, 130v, 82m, 112m 31v 171v 3v, 4v, 2v, 14v, 26v, 42v, 44v, 46v, 52v, 65v, 66v, 69v, 85v, 86v, 8817, ~ O V 93v, , 105v, 124v, 123v, 126v, 144v, 145v, 175v, 18iv, 185v, 192v, 195v, 205v, 208v, 219v, 9m, 10m, l l m , 65m, 89m, 103m, 131m, 132m 80v 89v. 147v. 150v 33m, loom
Hoechst ( 1 0 0 ~showed ) that if the mean elution volume (or center of gravity) of the elution profile of monodisperse standards is used for calibration, the mean elution volume of a polydisperse sample is a weight average. Thus, a broadstandard calibration can be used without interference from column dispersion if a log-linear calibration curve is obtained. The potential application for nonlinear calibration curves was discussed as well as the use of the universal calibration technique employing polydisperse standards. Stokes Radius. Horiike et al. ( 1 0 5 ~established ) protein calibration curves by plotting the SEC distribution coefficient vs. Stokes radius using high-performance columns. In a subsequent paper, these authors (106~) found that the distribution coefficient of proteins was more closely correlated with the intrinsic viscosity-based Stokes radius rather than the translational frictional coefficient based Stokes radius. Frigon et al. (69u) studied the discrepancies between calculated hydrodynamic radii of dextrans and their radii estimated by using globular protein standards. Under ionic conditions suitable for SEC of native proteins, globular proteins proved to be unsatisfactory as size calibration standards
for dextrans due principally to a lack of correspondence between retention volume and the Stokes radii for dextrans and proteins, respectively. However, both proteins and dextrans fell fairly close on the same curve of a universal calibration plot. Davis ( 4 4 ~proposed ) that Stokes radius rather than the molecular weight of a protein was the more fundamental parameter measured by SEC. A simplified calibration procedure was described in which the reciprocal of elution volume vs. Stokes radius for standard proteins was plotted. Only two standards were needed to provide a calibration independent of the void and interstitial volumes of a column. In addition, various plotting methods of calibratin columns were given. Miscellaneous. Shioya et al. (147~7related the flow rate needed to obtain the optimum height equivalent to a theoretical plate (HETP) to molecular weight of peptides (200-10000 daltons). With this approach, a linear relation was obtained between log molecular weight and the flow rate required for minimum HETP values. Mitchell ( 1 5 1 ~de) veloped a new calibration technique based on the fundamental relationship between the mean effective pore diameter of the ANALYTICAL CHEMISTRY, VOL. 56, NO. 5, APRIL 1984
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COLUMN L I Q U I D CHROMATOGRAPHY
packing and the apparent hydrodynamic diameter of the polymer. A linear relationship was obtained between the natural logarithm of the SEC distribution coefficient and the effective hydrodynamic volume of the polymer. Prelovskaya et al. (170~) described a method based on linear combination of Gaussian functions for calculating average molecular weights. Hintzsche et al. (Xu) presented a method for evaluating nonlinear calibration curves. Gloor (771~) extended his generalized exponential (Gex) distribution theory for describing molecular weight distributions. Included was a discussion on the limits of applicability of the Gex molecular weight distribution analysis as a mathematical model for linear homopolymers. Accuracy/Preclslon
Since the accuracy of the calculated molecular weight at a given elution volume is roughly 10-20 times the precision of the corresponding volume measurement, Chamberlin and Tuinstra ( 2 9 ~evaluated ) a previously developed thermal pulse time-of-flight liquid flow meter ( 1 4 8 ~for ) use in SEC. With this detector, a heating thermistor imparts a thermal pulse into the flowing eluent which is then detected via a sensing thermistor downstream. The time of flight of the warmed zone is inversely related to the liquid flow rate. Use of such a flow meter allowed for both minor and major flow variations to be accomodated. Precision of molecular weight calculations based on the use of this flow meter was within 4% under all flow conditions studied. Rudin et al. ( 2 1 2 ~investigated ) factors affecting accuracy and precision of average molecular weight determinations. Parameters studied were data sampling frequency, data precision, noise, base-line selection, and axial dispersion. Janca and Kleparnik ( 1 1 2 ~discussed ) factors affecting accuracy and precision associated with molecular weight distribution measurements of polymers with narrow and ultranarrow distributions. Parameters that were considered included flow rate and precision of detector response. From theoretical studies using model log-normal and Poisson molecular weight distributions, molecular parameters and polydispersity of polymers could be determined by SEC with high reliability. Lecacheux and Lesec ( 1 3 5 ~reported ) a method for determining the dead volume between SEC detectors used in series. In general, the geometric determination of connecting dead volume leads to unsatisfactory results because of changes in peak shape. The method described by these authors was applied to a continuous viscometer connected in series to a differential refractometer. Computer Processlng
Malawar et al. ( 1 3 8 ~developed ) a Sigma Basic program to generate a calibration curve and molecular weight distribution data from a broad molecular weight standard. Navas ( 1 6 0 ~ ) wrote a short program for a programmable calculator for calculating average molecular weights and molecular weight distribution. Rand and Mukherji (174~) described a curve fitting program to compensate for calibration curve variations caused by day-to-day changes in a standard curve and to correct for differences in response that arise from changing columns and pumping systems. Narasimhan et al. (159~) interfaced an Apple I1 Plus microcomputer to an SEC system with a differential refractometer and UV detector. Hardware design and software were given. Hutchens and Hester (108~) constructed an inexpensive system for interfacing an SEC system with a computer for data logging and analysis. Two programs were also written for data acquisition, real-time smoothing, and data reduction by boxcar averaging. In addition, an electronic balance was used to continuously monitor the flow rate. Miller ( 1 4 8 ~described ) the features of a commercial automated data reduction SEC system. Broyer and Abbott ( 1 9 ~wrote ) a computer program based on multicomponent models for calculating the weight fraction of each component in a polydisperse polymer. This method was used to analyze SEC profiles of high-density polyethylene. 326R
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Mobile Phases
Aqueous. Kat0 and Hashimoto ( 1 2 4 ~investigated ) the effects of eluant pH and ionic strength on the elution properties of proteins by using a modified silica packing (TSK-GEL SW). The acidic nature of the packing was evaluated by titration with base. Silanol groups began to dissociate at a pH value slightly below 5 and reached 0.013 mequiv/mL packing material at pH 8. At eluant pH values of 5 or above, the elution volumes of proteins with isoelectric points below the eluant pH increased with increasing ionic strength signifying the presence of ion exclusion. For those proteins with isoelectric points above the eluant pH, elution volumes decreased with increasing ionic strength suggesting cation exchange. For proteins that had isoelectric points that matched that of the mobile phase, their elution volumes were independent of ionic stren h (
