Contemporary Chromatography - Analytical Chemistry (ACS

Contemporary Capillary Gas

Chromatography

Twenty years ago a new era in sepa ration science was begun with the in vention of the capillary column. Al though the development of capillary gas chromatography in subsequent years was much slower than many re searchers had predicted, the method continued to be practiced in some lab oratories. Since the beginning of the 1970's, capillary gas chromatography has been undergoing its "renaissance". This undoubtedly has been caused by the recent emphasis on analyzing very complex mixtures and an increased concern in our society about ecological problems, effective utilization of ener gy resources, and better under standing of the many aspects of human health. It is increasingly real ized that efficient multicomponent analyses often may be a fundamental key to the solution of such problems. At present the potential of capillary gas chromatography in contemporary science and technology cannot be overemphasized. History of the Method It is often said that important scien tific advances occur at appropriate times, and important ideas on the same subject are born in different lab oratories for different reasons. While addressing the Symposium on Vapour Phase Chromatography held in 1956 in London (1), the Nobel Laureate for discovery of partition chromatogra phy, A.J.P. Martin, predicted that " . . . we should be able to work from the milligramme down to the microgramme scale. Of course, that will imply that we decrease the diameter of our column correspondingly. We shall have columns only two tenths of a millimetre in diameter, and these

will carry, I believe, advantages of their own . . . ." M.J.E. Golay conducted his column studies at approximately the same time. To explain the discrepancy be tween the plate heights measured with packed columns and the particle size, he substituted the conventional col umn for a length of Tygon tubing (2) and found the nonretained air peak to be much narrower than in the previ ous experiments. Subsequent coating of this tubing resulted in preparation of the first capillary column. Thus, it should be emphasized that this impor tant discovery resulted from an entire ly theoretical study. Figure 1 shows one of the earlier Golay chromatograms (2), detected then with a ther mal conductivity cell. Although this chromatogram is not quite up to the

efficiency standards of today's capil lary GC, it was convincing enough for many researchers that a new impor tant analytical tool was born. It is now hard to imagine the spirit of the 1958 Gas Chromatography Symposium held in Amsterdam, where Golay pre sented (2) his theory of capillary GC and where also some of the most im portant advances in the field were dis cussed for the first time; besides capil lary columns the highly sensitive ion ization detectors simultaneously ar rived on the scene. In subsequent months many researchers could ap preciate the importance of such tim ing: capillary GC could not be well de veloped without ionization detectors that provided sensitivity compatible with the sample size requirements and the internal volume of detector cells

Figure 1 . O n e of the earlier capillary chromatograms obtained by M.J.E. Golay, originator of the method ( 2 , ρ 54) Sample, mixed C6; 150 ft X 0.010 in. capillary; 1 % didecyl phthalate; 20 lb/in. 2 ; temp, 40 °C; flow, 0.96 mL/min

16 A · ANALYTICAL CHEMISTRY, VOL. 50, NO. 1, JANUARY 1978

0003-2700/78/0350-016A$01.00/0 © 1977 American Chemical Society

Report

Milos Novotny Department of Chemistry Indiana University Bloomington, Ind. 47401

that minimize band broadening. Addi tional problems with introduction of small samples into capillary columns had to be overcome soon afterward. Shortly after the presentation of Golay's first results, several research ers in both Europe and the United States demonstrated that unbeliev able column efficiencies of the order of 10 5 or 10 6 theoretical plates were in deed feasible. Desty et al. (3) solved the initial problem of sample injection using a simple inlet splitter that un equally divided the introduced sample between the capillary inlet and a vent. Optimized column, sample injection, and use of the flame ionization detec tor in Desty's laboratory resulted in chromatograms of impressive com plexity obtained from various light pe troleum fractions. Hundreds of com ponents were resolved, revealing for the first time the real complexity of such mixtures. One of these chromato grams obtained by Desty and Goldup (4) is shown in Figure 2, demonstrat ing the excellent results attainable al most two decades ago, but primarily confined to light hydrocarbon sam ples. A combination of the speed of analysis and efficiency also was dem onstrated by the same laboratory. Fig ure 3 shows that nearly 18 000 theo retical plates can be achieved in less than 1 min with a small-bore capillary column. This separation required ex perimental conditions that were some what unusual by the standards of con ventional chromatography. Additional excellent results ob tained in the early days of capillary gas chromatography include the hy drocarbon separations on nylon capil laries by Scott and Hazeldean (5) and the famous one-mile-long capillary of

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Figure 2. Resolution of octane isomers (16 out of 17 components resolved) (4) Column, glass, 600 ft, 0.010 in. bore; stationary phase, squalane; film thickness, ca. 0.3 μ; eluent gas, nitrogen; inlet pressure, 23 psi; temp, 50 °C; efficiency, ca. 300 000 plates

Figure 3. Fast analysis of heptane isomers (4) Column, 50 ft, 0.005 in. i.d.; stationary phase, squalane; temp, 20 °C; earner gas, hydrogen; inlet pres sure, 57 lb/in. 2 ; linear gas velocity, 95 cm/s; efficiency, 17 600 (n-heptane). 1: CH 4 ; 2: 2,2-di Me C 5 ; 3: 2,4-di Me C 5 ; 4: 2,2,3-tri Me C„; 5: 3,3-di Me C 5 ; 6: 2-Me C 6 ; 7: 2,3-di Me C 5 ; 8: 3-Me C 6 ; 9: 3-Et C 5 ; 10: n-heptane

A N A L Y T I C A L CHEMISTRY, V O L . 5 0 , NO. 1, J A N U A R Y

1978 ·

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Figure 4. Resolution of benzene-deuterobenzene pair by capillary gas-adsorption chromatography (9)

Zlatkis and Kaufmann (6). Fatty acid esters separated by Lipsky et al. (7) indicated certain possibilities of capil lary GC outside the hydrocarbon field. Less known, but equally impressive, are the results obtained in the early 1960's in the area of capillary gas-solid chromatography. Working at the col umn temperature of liquid nitrogen and using neon as the carrier gas, Mohnke and Saffert (8) demonstrated the feasibility of resolving hydrogen isotopes and their nuclear spin iso mers. A glass capillary with a layer of silica generated inside the column through the action of ammonium hy droxide was used in their work. Capil lary separations of various isotopic species were further developed by Italian workers (9, 10). As an example, separation of normal and deuterated benzene is shown in Figure 4. In spite of the described excellent work clearly demonstrating the poten tial of capillary GC, progress in the following decade was relatively slow. Although the 1965 monograph by Ettre (11) on the method shows sepa rations of compounds other than hy drocarbons, the overall number of capillary GC applications in chemistry and related fields at that time was rel atively low. This situation can be at tributed to problems in column tech nology, limited capabilities of the method in trace analysis and quantita tion, low sensitivity of ancillary tech niques, and the state of instrumenta tion in general. Support-coated open tubular col umns were later developed by Halasz and Horvath (12, 13). At that time these columns conveniently provided a needed compromise between separa tion efficiency and sample capacity. Other developments of that period in-

elude the preparation of high-efficien cy packed columns and packed capil laries (14). However, high inlet pres sure requirements with these columns make the "open-tube approach" more attractive, as theoretically justified al ready by Golay (2). Many present-day capabilities of capillary GC have their roots in the pi oneering work of Golay, Desty, and others in the late 1950's and early 1960's as well as certain advances of more recent years. The latter achieve ments include primarily the develop ment of stable, efficient, and inert glass capillary columns (15-17); im proved techniques of trace analysis (18-23); and the increased sensitivity of combined G C - M S techniques. Most importantly, the current need for improved understanding of com plex mixtures and the trace analysis of organic compounds below ppb levels has provided the additional stimulus for further development in this field. Glass Capillary Columns

Much current interest in capillary GC stems from the availability of glass columns that combine high separation

efficiency and inertness toward chromatographed samples. In spite of pre viously expressed fears about the fra gility of these columns and their impracticality in a routine environment, glass capillary columns have gained increasing popularity in a number of laboratories. From various tubing materials that are available for preparation of capil lary columns (plastics, glass, copper, nickel, stainless steel), glass has the most desirable features. The most dis tinct advantage of glass is its low cata lytic activity. This property is becom ing increasingly important in the anal ysis of labile substances in complex mixtures. The preparation of glass capillary tubing for chromatography has its ori gin in the work of Desty et al. (24) who designed the first glass drawing machine (Figure 5). Its principle is quite simple. Two pairs of rollers are moved by a synchronous motor, and a desired ratio of their speeds is adjust able. A glass tube is forced by the first pair of rollers inside the heated zone, while the second pair draws out a cap illary that is further passed through a heated bending tube to form a helix. Thus, capillaries of a desired length or diameter can easily be drawn. Where as drawing long glass capillaries pre sents no particular problems, poor wettability of glass surface with organ ic liquids complicated successful prep aration of adequate and stable col umns for a long time. Because a great majority of chromatographic station ary liquids exhibit a large contact angle on the high-energy surface of unmodified glass, uniformly coated films cannot be produced on the capil lary inner wall. However, film thick ness and uniformity along the entire column length are the principal fac tors affecting efficiency of mass-trans fer processes during the chromato graphic separation. Since the time when problems of glass wettability became widely appre ciated, our knowledge of the surface chemistry of glass has been extended significantly. Several practical solu tions to wettability have been sug gested. They include surface carbon-

Figure 5. Apparatus for drawing glass capillaries (20)

18 A · ANALYTICAL CHEMISTRY, VOL. 50, NO. 1, JANUARY

1978

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ization (15), surface corrosion with hydrogen chloride or fluoride (16, 17), and deposition of solid particles (25, 26). All these processes involve an in crease of the surface roughness that results in an easier spreading of organ ic films. In addition, wettability also can be induced by an introduction into the surface of a selective mono layer that interacts with the molecules of a spreading liquid and thus changes the wettability parameters (27). Although some of these column treatment methods have been empiri cally known for some time, more re cent surface chemistry studies that in volve contact angle measurements (28, 29) and electron scanning microscopic observations (29, 30) now shed more light into the wettability problems. As an example of surface geometrical modification, Figure 6 demonstrates the growth of crystalline aggregates, following high-temperature treat ment with dry hydrogen chloride gas according to Novotny and Tesarik (16, 17). In addition, formation of silica "whiskers" recently has been shown (31-33) when hydrogen fluoride-based treatment methods (16, 17) are ap plied. Even when the condition of com plete surface wettability is adequately met, glass capillary columns still may not be acceptable for good analytical work. A? a next step, reduction of the surface residual activity must be en sured. More specifically, insufficiently deactivated columns will effectively separate nonpolar solutes such as hy drocarbons, while giving unsatisfacto ry results (e.g., tailing peaks, sample losses, or decomposition) with more polar substances. Causes of residual activity can be traced to the reactivity of free surface silanol groups and the glass ingredi ents. The exact nature of these unde sirable interactions is presently un

known, but both chemical blocking of the free silanol groups and leaching the ingredients from glass (34) have been observed to improve analytical results. With the exception of a few specific column types, glass surface preferably should be neutral (35). Ad ditional approaches to surface deacti vation include addition of a surfaceactive substance (36-38) or a complete coverage of the capillary wall with thin films of polar polymers (39-41). It is important that both column ef ficiency and inertness toward the ana lyzed samples are considered together. Some criteria for the evaluation of glass capillary columns have been sug gested (37, 42). Column inertness be comes critically important when ana lyzing trace amounts of relatively la bile samples. Although the film uniformity is greatly affected by the chemical na ture of the capillary inner wall, me chanical aspects of the stationary phase application cannot be over looked. Typical film thicknesses are up to only a few tenths of a micron. Coating procedures play an important role in producing efficient and durable capillary columns. Both static and dynamic coating procedures are widely used in prepa ration of capillary columns. If careful ly executed, both procedures yield sat isfactory results. Factors affecting the film thickness and homogeneity dur ing the dynamic coating process were extensively studied (43, 44), and fur ther improvement in the quality of coated columns appears to be caused by a recently described "mercury-plug method" (45). Two versions of the static procedure use low pressure (46) or heat (47) for solvent removal. Sampling Techniques

Sample capacity with capillary col umns is considerably less than with conventional packed columns. Ap proximately 500 ng per component typically represent the upper limit; these or smaller amounts are difficult to introduce into capillary columns in a direct way. An earlier and only solution to these problems was the injection of a larger amount of sample into a heated injec tor, with the subsequent unequal split of a homogeneously evaporated medi um. In this fashion, only a small frac tion of a total sample is allowed to enter the capillary inlet, while the larger volume is vented to the atmo sphere. Although smaller or larger split ratios also are feasible, typical values range from 1:100 to 1:500. Reproducible introduction onto the capillary column of a sample that is representative of an analyzed mixture is the first requisite of any quantita tive evaluation. Due to the noninstan-


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Figure 7. Comparison of (A) direct in jection technique and (B) regular injec tion with sample splitting (1:200). Sol vent, n-dodecane at 170 °C (20)

taneous evaporation and dynamic pro cess of sample splitting, sample com ponents with widely differing boiling points frequently may not be intro duced into the column in a uniform fashion. In quantitative analysis this presents certain problems that are not easily correctable. Various splitter de signs were developed in the past to re duce such problems. Although splitting injectors may be adequate for work with concentrates of complex mixtures, the obvious problems are encountered in trace analysis. Specifically, when the total amount of sample is severely limited, the analyst cannot afford to waste most of his sample through the vent. Direct injection of samples was con sidered impossible in the past. Among several investigators who studied this important aspect of capillary GC, Grob and Grob (18-20) succeeded in demonstrating the feasibility of a di rect injection of dilute solutions. In this injection technique the less vola tile trace sample components are "condensed" at the column inlet with minimum band spreading, while the more volatile large solvent peak passes through the column with little reten tion. In this manner the whole sample is utilized for analysis. Following the solvent tail, the trace components of interest are eluted after a sudden or gradual elevation of the column tem perature. Direct injection procedures are now widely utilized in the trace analysis of environmental pollutants and compo nents of physiological fluids. Experi ence has shown that little damage is done to the thin-film capillary col umns even when several microliters of ordinary solvents are frequently in jected. An interesting phenomenon is further observed (20): under the cir cumstances of a proper choice of sol vent and column temperature, the trace sample components actually are concentrated in an effective manner

behind the condensed solvent band. Thus, such sample introduction ensures minimum peak broadening. Figure 7 obtained with the standard solutes actually demonstrates that the direct sample injection can be at least as effective as the splitting technique in maintaining column efficiency. When more dilute solutions are to be analyzed, the use of a concentration precolumn (23) may be found effective. Quantitative transfer of up to 20-ML samples is feasible with this technique, with subsequent solvent removal and thermal desorption of the sample into the first section of a capillary column held at low temperature. The precolumn also prevents migration of nonvolatile impurities into the analytical column. Determination of trace organics present in air or other gaseous media is of considerable importance. Direct injection of the air or headspace sample into capillary columns is not feasible since mixture components with relatively low vapor pressures are not detectable; and with the present sensitivity of GC detectors, the sample volumes necessarily would exceed the total volumes of capillary columns. Sample preconcentration prior to GC analysis is a mandatory step. Although sample trapping on the conventional absorbents and elution of the trapped materials by a small amount of suitable solvent can be employed, the use of porous polymers for trapping organics and their subsequent thermal desorption have become more popular. Among such materials, Tenax GC (a 2,6-diphenyl-p-phenylene oxide porous polymer) proved to be an unusually effective concentrating medium (21, 22). In this concentration/sampling procedure, a small precolumn first is purged with the analyzed headspace medium and then heated in the injection port of a gas chromatograph to desorb the trapped sample into the first (cooled) portion of a capillary column. Some quantitative aspects of this precolumn procedure already have been studied (22, 48). Detectors and Ancillary Techniques

Many types of detectors were described for GC, but only a few of them are applicable to work with capillary columns. First, it is essential that such detectors have a sensitivity compatible with the optimum operating conditions of high-efficiency columns. If a detector is not sensitive enough, the column must be overloaded, and the primary advantages of high-resolution analysis are lost. Second, small detection cell volumes are mandatory in view of the small internal diameters of capillary columns and correspondingly low flow rates. Detector volume prob-

Figure 8. Chromatograms of volatiles from a 24-h urine of normal maie (49) A, flame ionization detector; B, nitrogen-sensitive detector

lems often can be overcome through the use of an extra carrier gas at the column exit. The almost simultaneous development of the capillary column and ionization detectors was indeed a lucky coincidence. For many years the flame ionization detector has proved to be the most useful, universal detector for capillary GC. Other types of flame detectors (e.g., thermionic or flame-photometric detectors) also have been recognized as powerful selective monitors. Since the column outlet can be flushed effectively with any component of the combustion mixture, dead volume problems have been practically eliminated. Selective detectors have played a progressively important role in capillary GC. In complex mixtures of different origins, these detectors provide chromatographic profiles complementary to the universal recordings by means of the flame ionization detector, and thus a greater amount of analytical information. An example of such a situation is demonstrated in Figure 8, where the complex sample of a human physiological fluid is recorded by a conventional detector vs. one selective for nitrogen compounds (49). Detector selectivity in this case is at least three orders of magnitude higher. An effective utilization of detector selectivity can be of importance to the analytical chemist in several respects. The information that a particular chromatographic peak is due to a sub-


stance containing sulfur, nitrogen, phosphorus, a halogen, etc., can be quite useful. Even when a mass spectroscopic identification is subsequently attempted, this information could be of importance. Also, many selective detectors are more sensitive than the flame ionization detector. Many compounds frequently can be traced that otherwise would remain unnoticed with a less sensitive conventional detector. In the analytical chemistry of complex mixtures, the minor mixture components are often the important ones. Alternatively, selective sample derivatization can be performed, enhancing utility of specific detectors. This principle is now trivial in high-sensitivity biomedical analysis where compounds like steroids, biological amines, amino acids, and various drug metabolites are converted to perfluoroderivatives prior to electron capture detection. Also, ketonic steroids can be derivatized to contain nitrogen (as methoxime derivatives) and subsequently detected (50) by the thermionic detector. It is now technically feasible to place two or more detectors in parallel, while splitting a certain portion of the capillary column effluent (51, 52). Such combinations of the powerful flame-type selective detectors, or a small-volume electron capture detector (53), with the conventional flame ionization detector should find increasing utilization in analyses of complex mixtures.

Development of gas-phase spectro scopic detectors and their increasing use in capillary GC also can be pre dicted. A recent report by Hausdorff (54) exemplifies this trend. With these detectors, selectivity requirements can be adjusted, as shown in Figure 9, where the compounds with hydroxy and carbonyl groups are recorded se lectively in the presence of other con stituents of peppermint oil. Although only support-coated open tubular col umns currently are in use, optimiza tions on both column and detector de sign are feasible. Today it can be safely stated that the wider application of capillary col umns in GC-MS has drastically changed the overall analytical role of this tool. First, the identification power of low-resolution mass spec trometers has been enhanced signifi cantly by improved chromatographic separating capability. The power of combining the two methods has been particularly obvious for the unambig uous identification of isomeric com pounds. Specifically, whereas these compounds may yield similar mass spectra, their chromatographic mobili ty is quite different. As sensitivity of the method further improves and new mass spectroscopic techniques arrive, we may see even further utilization of this powerful an alytical tool. The present-day avail ability for mass spectroscopy of com puter evaluation methods creates a most favorable atmosphere for further advances and applications of capillary GC-MS and multicomponent analyti cal chemistry in general. Also, the combination of capillary GC with high-resolution MS has lately scored impressive gains in sensitivity.

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Figure 9. C h r o m a t o g r a m s of p e p p e r m i n t oil d e t e c t e d by f l a m e ionization detector and IR detector selectively tuned for (A) carbonyl and (B) hydroxy c o m p o u n d s (54)

Applications

Many areas of scientific research and chemical technology have been aided significantly by the availability of capillary GC and GC-MS. In par ticular, an extensive utilization of these analytical methods has been characteristic of the last several years. This trend is understandable in view of the many emerging analytical prob lems in which both reliable multicom ponent determinations and high col umn performance are crucial. The four major areas of analytical activity where capillary columns have been particularly useful (resolution of iso mers, analysis of complex mixtures, chromatographic "fingerprinting", and fast separations) are briefly dis cussed below. Resolution of Isomers. Chromato graphic separation and quantitative determination of isomeric molecules can be of some importance in bio chemistry, toxicology, aroma analysis, or even synthetic chemistry. Here

Figure 10. Separation of / V - t r i f l u o r o a c e t y l - ( ± ) - a - a m i n o acid isopropyl esters on glass capillary c o l u m n c o a t e d with optically a c t i v e stationary phase (57)

belong, for instance, cis-trans isomer ism, position of the double bond or a functional group within a molecule, and optical activity, all of which can be crucial to the biological activity of these molecules. Many isomer resolution problems can be greatly reduced by the future development of highly selective col umn substrates, as already indicated in some recent work on chiral station ary phases (55) and liquid crystals (56). Frequently, a high number of theoretical plates alone can accom plish the necessary degree of resolu tion. Thus, the application of capillary


GC in this area represents the "brute force" approach that often is the most straightforward solution. In some instances, a proper combi nation of column efficiency and selec tivity provides good solution. This is exemplified in Figure 10, which shows the separation of derivatized optically active amino acids on the glass capil lary column coated with an optically active stationary phase (57). Multicomponent Analyses. In the task of resolving mixtures with hun dreds of components present, efficient capillary columns may well have no substitute. Frequently, adequate reso-

lution of complex mixtures is the absolute prerequisite of identification, quantification, and routine monitoring of the individual components. The recent advent of highly efficient glass capillary columns already is having a major impact in this area. The following illustration of capillary GC applications is intended for demonstration of selected features of the method in the major areas of interest and is by no means exhaustive or representative. The present main areas of application are fuel analysis and determination of combustion products, environmental sciences, food and aroma analysis, and biochemical or biomedical investigations. Complexity of various petroleum products and other fuel materials was already demonstrated in much pioneering work on capillary GC in the late 1950's. Whereas chromatography of common petroleum mixtures may be considered the least difficult task for capillary GC, trace analytical techniques for minor components will require further development. Methodology for lighter petroleum fractions currently is well developed, but existing techniques for characterization of high-boiling fuel-related materials are not adequate. Separations up to n-Cr,e hydrocarbon were reported (58). Detection and characterization of carcinogens from various combustion mixtures and atmospheric sources have been an important subject of research for a long time. Use of glass capillary columns for resolution of the mixtures of polynuclear aromatic hydrocarbons and combined GC-MS permits identification of well over a hundred such compounds (59-62). Figure 11 shows that a short efficient glass capillary column allows chromatography of polycyclics up to coronene (7-ring compound) at a relatively low temperature. The possibilities of trace pollutant analysis and resolution of complex environmental matrices also have been revolutionized by this method. Development of sampling techniques for trace volatile organics from atmospheric sources (21, 22, 63, 64) complements the recent advances in column technology and selective detection. Water contamination problems have lately received a tremendous amount of attention. Indeed, water pollution phenomena result in some of the most methodologically challenging problems in any area of organic analysis. Although still in a very early developing stage of application, capillary GC and its ancillary techniques may well be the only effective solution to such problems. Whereas capillary GC has been used on occasions in various applications, covering such diverse 26 A ·

Figure 11. Capillary column gas chromatogram of total polynuclear aromatic hydrocarbon fraction of air particulate matter (62)

cases as "fingerprinting" oil spills (65), detection of pesticides in river water (66*, 67), identification of chlorinated substances in a city water supply (68), and sewage (69), the series of papers by Grob and Grob (70-72) can be considered the most systematic contribution to the analytical methodology of organic water pollutants. This work includes treatment of problems associated with preconcentration techniques, routine multicomponent analyses of several hundred sample components, and their identification at concentrations down to a few nanograms per liter. The present-day methodology of capillary GC for environmental sam-

3-MCG

ples is confined to relatively volatile sample components. The general aspects and potential of this method are discussed by this author in a recent review article (73). Analysis of biological specimens always has been a most inviting area for high-resolution chromatographic methods due to the extraordinary complexity of biological fluids and tissues. Here, capillary GC has an almost limitless scope for application. After a series of isolated successes in this area in the late 1960's (for a review of this period, see ref. 74), many new applications have been reported. Again, much recent success clearly can be attributed to the emergence of glass

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1JUUAJ,UI]LI\xJw Figure 12. Organic acid profiles of normal urine vs. urine of patient with hereditary progressive deafness (84) Both subjects given 20 g of leucine per os; urine collected during subsequent 3 h. 3-HIVA = 3-hydroxyisovaleric acid; 3-MCG = methylerotonylglycine

A N A L Y T I C A L CHEMISTRY, V O L . 5 0 , NO. 1, J A N U A R Y

1978

techniques (88, 89), method automation (51), and data treatment (90) as applied to such samples is likely to contribute significantly to these efforts. A recent report by Jellum et al. (84) exemplifies tbe value of capillary GC in metabolic profile work. Biochemical investigations were carried out on a family with an unusual case of hereditary progressive loss of hearing. Preliminary investigations of the organic acid fraction of the urine of these patients showed an occurrence of two abnormal metabolites, identified as 3hydroxyisovaleric acid and 3-methylcrotonylglycine, that possibly are metabolic intermediates of leucine. Thus, metabolic differences between normal individuals and those suffering from the above disorder could be amplified after the administration of this amino acid. Figure 12 demonstrates this to be

the case. This finding now opens up a whole series of important biomedical investigations to be carried out on the condition of hereditary deafness. High sensitivity and resolution are often simultaneous requirements in biomedical chromatography. An example of meeting such requirements is shown in Figure 13. By use of an effective sample fractionation method, preconcentration prior to capillary GC, samples of plasma as small as 1-5 mL are sufficient for simultaneous determination of the three most important conjugate fractions of blood steroidal hormones and their metabolites (50). In addition, a one-tenth sample aliquot still can be used in determining picogram levels of testosterone with an electron capture detector. Fingerprinting Analyses. In the past "fingerprinting" analysis (linking a particular sample to its origin) em-

Figure 13. Plasma steroid profiles of normal male recorded with glass capillary column (50) A, free-glucuronide fraction; B, monosulfate fraction; C, disulfate fraction

capillary columns, improved sensitivity and reliability of GC-MS and other ancillary techniques, as well as the availability of novel sample fractionation, concentration, and derivatization approaches. Several selected examples that should be cited include separations of urinary (75-77) and blood steroids (50), fatty acid esters (78), amino acids (57), polyols (79, 80), volatile constituents of body fluids (81, 82), and other physiological fluid and tissue components (83, 84). The combination of resolution and high sensitivity of the method now permits quantitative multicomponent analyses to be carried out with small experimental animals (85, 86). Examples of biomedical applications will briefly be described. Multicomponent analyses of human or animal samples, generally now referred to as "metabolic profiles" (87), have received attention as a powerful means for identification of biochemical differences between normal and pathological metabolism and possible clinical diagnostic aids; there are some indications of success in this field. The recent development of novel GC-MS

Figure 14. Capillary gas chromatogram of nitromethane extract from three different engine oils, detected by nitrogen-sensitive detector (60)


ployed b o t h c h r o m a t o g r a p h i c a n d spectroscopic t e c h n i q u e s . T h e best " f i n g e r p r i n t i n g " m e t h o d s are those t h a t provide a m a x i m u m a m o u n t of specific d a t a on a given s a m p l e , pref erably while using t h e s a m e analytical t e c h n i q u e . T h e utility of "fingerprint i n g " m a y include forensic applica tions, technological p r o b l e m s , identifi cation of microorganisms, a n d pollu tion sources. W h e r e a s t h e "finger p r i n t i n g " strategy m a y vary with a t y p e of s a m p l e , capillary G C h a s a n e x t r a o r d i n a r i l y high diagnostic power d u e t o t h e very fine details it can p r o vide. An e x a m p l e of t h e p o t e n t i a l use of forensic " f i n g e r p r i n t i n g " (60) is shown in Figure 14. Profiles of n i t r o gen-containing c o m p o n e n t s (mostly aza-arenes) of t h r e e different engine oils are recorded w i t h t h e glass capil lary column a n d t h e t h e r m i o n i c detec tor. A n objective of s u c h specific pro file d e t e r m i n a t i o n s in forensic investi gations m a y be t o c o m p a r e a s u s p e c t ' s car engine oil with t h a t spilled a t t h e scene of t h e crime. O t h e r "fingerprint i n g " applications m a y include identifi cation of oil spills (65), air pollution sources (91), a n d t h e origin of c a n n a bis p l a n t m a t e r i a l (92). Fast Separations. A frequent a b u n d a n c e of theoretical p l a t e s in less difficult s e p a r a t i o n s by capillary GC m a k e s it feasible t o c o m p r o m i s e be tween t h e s e p a r a t i o n efficiency a n d analysis t i m e . Capillary columns have been shown on n u m e r o u s occasions t o be ideally suited for such a case (11). E x t r e m e l y fast s e p a r a t i o n s were shown (4) in Desty's pioneering work (see also Figure 3 of this article). W h e r e a s t h e a d v a n t a g e s of fast sep a r a t i o n s have n o t been particularly at tractive in research applications of capillary G C , r o u t i n e analyses will need different considerations. A re c e n t work (93) d e m o n s t r a t e s uses of glass capillary c o l u m n s as a p a r t of t h e process analyzer. I t is a n t i c i p a t e d t h a t shorter c o l u m n s (10 m or less) will be increasingly used in r o u t i n e a p plications.

chemical n a t u r e containing h u n d r e d s of c o m p o n e n t s are c u r r e n t l y feasible. Although combined G C - M S has scored impressive gains in b o t h sensi tivity a n d t h e a d v e n t of specialized m a s s spectroscopic t e c h n i q u e s , fur t h e r d e v e l o p m e n t s of ancillary t e c h niques for positive identification of t h e s e p a r a t e d solutes are desirable. F r e q u e n t l y , h u n d r e d s of p e a k s m a y be resolved from a s a m p l e of biological or e n v i r o n m e n t a l origin. Once t h e s a m p l e composition is known, t h e m e t h o d can b e routinely u s e d t o ob serve q u a l i t a t i v e a n d q u a n t i t a t i v e changes u n d e r different circum stances. I n s o m e cases, t h e m e t h o d ac tually m a y be revealing m o r e details on t h e composition t h a n t h e a n a l y s t can or is willing t o utilize. D u e t o t h e u n e x p e c t e d complexity of t h e s e mix t u r e s , a qualitatively new s i t u a t i o n often arises, a n d t h e analytical c h e m ist m u s t adjust his a t t i t u d e according ly. T h u s , it will become progressively i m p o r t a n t t o seek a d d i t i o n a l a p p r o a c h e s t o d a t a simplification a n d in t e r p r e t a t i o n . W h i l e seeking i n t e r p r e t a t i o n of complex c h r o m a t o g r a m s t o a readily u n d e r s t a n d a b l e level, appli cation of c o m p u t e r t e c h n i q u e s will be come m a n d a t o r y . W e are now only a t t h e beginning of t h e s e efforts. F o r t u n a t e l y , t h e designs of c o m m e r cial gas c h r o m a t o g r a p h s a n d G C - M S u n i t s have rapidly r e s p o n d e d to t h e n e e d s of m o d e r n capillary G C . T h e system i n e r t n e s s has b e e n stressed with new i n s t r u m e n t s , p e r m i t t i n g t h e use of glass capillary c o l u m n s . Finally, since m i x t u r e s of high-boil ing a n d highly polar c o m p o u n d s are likely t o b e even m o r e complex t h a n t h o s e now e n c o u n t e r e d in capillary GC, a n i m p r o v e m e n t of LC m e t h o d s in t e r m s of resolution would be q u i t e desirable. A l t h o u g h f u n d a m e n t a l limi t a t i o n s i n h e r e n t t o solute diffusivity a n d m o b i l e - p h a s e viscosity exist, t h e theoretical p o t e n t i a l of L c h a s n o t been utilized (94, 95). R e c e n t results suggest t h a t L C in p a c k e d microcapillaries (96) or coiled o p e n t u b e s (97) m a y be feasible, a n d t h a t we c a n learn a g r e a t deal from t h e available tech nology of capillary G C .

Conclusions T w e n t y years of capillary GC h a v e been m a r k e d with n u m e r o u s advances in column technology, s a m p l e intro d u c t i o n t e c h n i q u e s , a n d t h e develop m e n t of sensitive ancillary tools. High ly efficient, inert, a n d s t a b l e glass cap illary columns presently available a p proach t h e theoretical limit of gas c h r o m a t o g r a p h y . T r a c e analysis capa bilities have been improved drastically by t h e d e v e l o p m e n t of direct s a m p l i n g m e t h o d s as well as t h e availability of highly sensitive detectors. S e p a r a t i o n s of very complex m i x t u r e s of a diverse

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TOTAL GLASS CAPILLARY COLUMN SYSTEM

(81) A. Zlatkis, W. Bertsch, H. A. Lichtenstein, A. Tishbee, F. Shunbo, H. Liebich, A. Coscia, and N. Fleischer, Anal. Chem., 45,763(1973). (82) M. Novotny, M. L. McConnell, M. L. Lee, and R. Farlow, Clin. Chem., 20, 1105(1974). (83) C. Jacobs, E. Solem, J. Ek, K. Halvorsen, and E. Jellum, J. Chromatogr., 143, 31 (1977). (84) E. Jellum, P. Storseth, J. Alexander, P. Helland, O. Stokke, and E. Teig, ibid., 126,487 (1976). (85) M. P. Maskarinec, G. Shipley, M. No votny, D. J. Brown, and R. B. Forney, Experientia, in press. (86) M. P. Maskarinec, G. Shipley, M. No votny, D. J. Brown, and R. B. Forney, submitted for publication. (87) E. C. Horning and M. G. Horning, J. Chromatogr. Sci., 9, 129 (1971). (88) C. C. Sweeley, N. D. Young, J. F. Hol land, and S. C. Gates, J. Chromatogr., 99, 507 (1974). (89) E. Jellum, P. Helland, L. Eldjarn, U. Markwardt, and J. Marhoefer, ibid., 112, 573 (1975). (90) M. L. McConnell, G. Rhodes, U. Wat son, and M. Novotny, submitted for pub lication. (91) K. D. Bartle, M. L. Lee, and M. No votny, Int. J. Environ. Anal. Chem., 3, 349(1974). (92) M. Novotny, M. L. Lee, C.-E. Low, and A. Raymond, Anal. Chem., 48, 24 (1976). (93) F. Mueller and M. Oreans, Chromato graphia, 10, 473 (1977). (94) J. C. Giddings, Anal. Chem., 36, 1890 (1964). (95) M.J.E. Golay, Chromatographia, 6, 242 (1973). (96) T. Tsuda and M. Novotny, Anal. Chem., in press. (97) T. Tsuda and M. Novotny, submitted for publication. Presented at the Division of Analytical Chemis try, 174th Meeting, ACS, Chicago, 111., August 28-September 2, 1977.

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M i l o s N o v o t n y is c u r r e n t l y a n asso ciate professor of c h e m i s t r y a t I n d i a n a University. His research i n t e r e s t s in clude gas c h r o m a t o g r a p h y , gas chro m a t o g r a p h y / m a s s s p e c t r o m e t r y , highperformance liquid c h r o m a t o g r a p h y , a n d applications of t h e s e m e t h o d s to biomedical a n d e n v i r o n m e n t a l p r o b lems.

Contemporary Chromatography - Analytical Chemistry (ACS

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