Instrumentation Edward S. Yeung Robert E. Synovec Ames Laboratory-USDOE and Department of Chemistry Iowa State University, Ames, Iowa 50011
DETECT Liquid chromatography (LC) is, for all practical purposes, a hyphenated technique. Physical separation of components is only one part of LC. The other goals, quantitation and identification, are conveniently achieved by putting appropriate detectors after the chromatographic column. Even in preparative applications, it is necessary to monitor the effluent to synchronize fraction collection. In principle, any measurement method can be used. In practice, there are several constraints when a measurement method, that is, a detector, is interfaced to LC. First, good sensitivity is needed to deal with the low concentrations of typical analytes, which are often diluted substantially during the separation. This is not simply because of the general rule that the better the detectability, the broader the scope of application but also because the injected quantity must be kept below the point of overloading the stationary phase. Second, the volume of the detector must be small to avoid additional band broadening due to extracolumn effects, to preserve the quality of the separation. This volume includes, for example, the connecting tubings to the end of the column, in addition to the actual volume active in producing the signal. Recent developments in microcolumn LC and supercritical fluid chromatography (SFC) make this problem even more severe. Volumes below 1 (itL for 1-mm i.d. (commercial packed microbore) columns and below 1 nL for open microtubular columns of 10 Mm i.d. are needed. Third, the detector must be able to function in the presence of a large background—that of the eluent molecules. Unlike gas chromatography (GC), where the mobile phase can be 0003-2700/86/A358-1237$01.50/0 © 1986 American Chemical Society
for Liquid
Chromatography
an inert material like He, the LC mobile phase often affects the actual detector signal. It is necessary to be able to null out this background signal and to maintain it at a stable level to reduce noise. Fourth, the time response of the detector must be compatible with the chromatographic event. This is even more important for the newer high-speed columns. If multidimensional information is to be obtained, for example recording entire spectra, the detector must cycle rapidly to be useful. Indirectly, time response also affects detectability, because long time constants cannot be used to average out noise. Fifth, the shape of the detector cell is important. It turns out that the physical lengths (peak volumes/column cross-sectional area) of the eluted chromatographic peaks are not too different from one type of column to the next. They are all in the 10-cm range for a capacity factor (k') of one, if the detector i.d. is identical to that of the corresponding column. This means that detector cell lengths up to 1 cm can be used. In fact, it is usually desirable to use the full 1-cm path length to maximize concentration detectability. Last, detector selectivity is much more important in LC compared with GC. Current technology provides column efficiencies (theoretical plates) in LC that are inferior to those in GC. Chances of peaks overlapping in LC are then much higher. A selective detector can be used to monitor a subset of all analytes. Effectively, components can thus be resolved without physical separation. Detection methods are not just ancillary techniques for LC. In many situations, analytical measurements can
RS
be greatly improved by interfacing the transducer to an LC. What is gained is essentially a simple procedure for sample preparation and cleanup before the measurement. Interferences can then be reduced or eliminated. It is true that one has to worry about additional problems due to dilution and the presence of the eluent. However, for analytes in solution, LC allows one to measure in a well-prescribed environment—the eluent—rather than the original, often less predictable, matrix. The presence of the eluent can also be turned into an advantage. Because some species will always be present at the detector, one can produce a signal in certain detectors even when analytes are absent. When analytes that give no response at the detector are eluted, displacement of the eluent occurs at the detector cell. A negative signal can thus be generated to provide a means for quantifying the "inactive" analytes. This indirect mode of detection substantially increases the scope of a given measurement technique. Another application of the signal dependency on both the eluent and the analyte is that of quantitation without standards (1). If two chromatograms of a sample are obtained by using two eluents with different detector responses (for example, an optically active solvent and its racemic counterpart), the pairs of peak areas for each component can be used to calculate its concentration. Finally, the time dependence in LC can be used to improve the accuracy and detectabilities of many analytical measurements. This can be called sample modulation. As long as the noise associated with the analytical measurement is not on the same time scale as the chro-
ANALYTICAL CHEMISTRY, VOL. 58, NO. 12, OCTOBER 1986 · 1237 A
Table I. Performances of LC detectors matographic event, one can reduce the "noise" compared with static mea surements by proper signal averaging or baseline correction. It is beyond the scope of this article to give a comprehensive overview of LC detectors. The reader is referred to the many fine references that have ap peared (2, 3). A summary of "typical" capabilities of detectors is given in Ta ble I. In what follows, we will present some of the more recent developments in detection methods for LC. Many of these are still in the early stages of de velopment; others are already avail able commercially. With the growth of LC instrumentation in recent years, it is quite likely that new LC detectors will become vital tools in the analyti cal laboratory.
LC detector
Commercially available
Mass LOD (commercial detectors) 3
Mass LOD (state of the art)»
Absorbance Fluorescence Electrochemical Refractive index Conductivity Mass spectrometry FT-IR Light scattering6 Optical activity Element selective Photoionization
Yesc Yesc Yesc Yes Yes Yes" Yes" Yes No No No
100 pg-1 ng 1-10 pg 10 pg-1 ng 100 ng-1 μg 500 pg-1 ng 100 pg-1 ng
1 pg 10 fg 100 fg 10 ng 500 pg 1 pg 100 ng 500 ng 1 ng 10 ng 1 pg-1 ng
1 M9 10 jug
— — —
1
Mass LOD is calculated for injected mass that yields a signal equal to five times the σ noise, using a mol wt of 200 g/mol, 10 μί. injected for conventional or 1 μL injected for microbore LC. ' Same definition as a, above, but the injected volume is generally smaller. : Commercially available for microbore LC also. 'Commercially available, yet costly. 5 Including low-angle light scattering and nephelometry.
Detection methods Refractive index. The refractive index (RI) detector has a unique place in LC. It is one of very few universal detectors available. Unless the analyte happens to have exactly the same RI as the solvent, a signal will be ob served. So, for the initial survey of samples, when the physical and chem ical properties of the analytes are not known, the RI detector can provide useful information. The most severe limitation is the poor limits of detec tion (LOD) that can be achieved corn-
pared with other detectors. Also for complex samples, when components co-elute, there is a possibility of signal cancellation as both positive and nega tive RI changes can occur. The common RI detectors are based on refraction, reflection, or interfer ence of light beams. The third ap proach has provided the best concen tration LOD in commercial instru ments and in research prototypes. This is because a longer path length
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can be used to compensate for lower concentrations. However, this implies that interference-type refractometers cannot be scaled down to function in small volumes, which in turn is advan tageous for optimizing mass LOD. Ul timately, concentration LOD of any universal detector is limited by fluctu ations in the RI of the LC effluent. Solvent delivery systems must be pulse free to avoid pressure effects, and thermostated cells must be used
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CIRCLE 126 ON READER SERVICE CARD 1238 A · ANALYTICAL CHEMISTRY, VOL. 58, NO. 12, OCTOBER 1986
Zip. AL
to avoid temperature effects. It seems that going beyond the present LOD of 4 X 10~9 RI units (4) will be difficult. Yet, there are some new approaches to RI detection that address mass LOD through miniaturization. Refraction-type RI detectors rely on Snell's law at the interface between the cell wall (glass) and the flowing liquid to deflect a light beam. Changes in RI are monitored at far field by a position sensor or as an intensity change on a small-area photodiode. Because the effect is generated at the interface, very small volumes are pos sible if the proper cells can be made. An interesting geometry has been sug gested recently (5), in which a laser beam propagates along the edge of the cylindrical liquid cell, as shown in Fig ure la. As long as propagation is not normal to the glass-liquid interface, deflection will occur. Because the laser beam can be focused to a narrow beam waist (~10 μαι), only a short length of the capillary flow cell is needed. The cylindrical geometry is ideal for oncolumn detection in open-tubular cap illary LC and in SFC or for postcolumn detection in other LC systems by connecting an appropriate flow cell. The total detector volume (cell area X laser beam width) is 4 nL, with a noise level equivalent to 3 X 10~7 RI units. Reflection-type RI detectors rely on
Fresnel's laws at the interface between the cell (glass) and the liquid, which has a smaller RI. Sensitivity increases as the incidence angle approaches the critical angle. Actually, the transmit ted light is monitored to reduce noise. A modification of the commercial ver sion (6) is shown in Figure lb. A sec ond prism is used to couple the light out to preserve the optical quality and to double the effect. The liquid inlets and outlets are almost tangent to the interface to minimize flow turbu lences. By using an 80-Mm-thick gas ket in a channel that is 1 mm wide, the cell volume is 0.8 /iL for an optical path of 1 cm. Naturally, still thinner gaskets and shorter optical paths can be used to reduce the volume further. The reason for the 1-cm path is to al low simultaneous monitoring of ab sorption and fluorescence in the same optical region. These two signals are proportional to path length, and 1 cm seems to be a good compromise. Fluo rescence is detected by a phototube placed above the prisms. Absorption is monitored in the same light beam that probes RI changes. It is of interest that absorption attenuates the light intensity whereas an increase in RI in creases the light intensity. The two types of signals can thus be distin guished if a solvent of low RI is used. With this arrangement, the three most
common detection modes in LC—RI, absorption, and fluorescence—are achieved simultaneously. For the test compounds, the noise levels in the three modes are equivalent to 2 ng, 16 pg, and 300 fg of injected analyte, respectively. Absorption. UV-vis absorbance de tection is by far the most widely used. Detectors are available spanning the full range from single-wavelength to simultaneous multiwavelength detec tion capabilities. Wavelength selectiv ity and often excellent analyte sensi tivity in the absorption process are two key advantages of this mode of detection. For conventional LC condi tions, typical detectability with com mercial absorbance detectors is 1 ng of injected analyte or an injected concen tration of 5 X 10~7 M. The former val ue is improved by a factor of 10 with commercially available cells (1-cm path, 0.5 μΐ,) adapted for microbore LC. Peak volume is the primary factor in comparing mass LOD in conven tional and microbore LC, with some additional gain in detectability attrib utable to the (typically) slightly higher separation efficiency with microbore LC. Apart from being quite sensitive, se lectivity in absorbance detection is quite useful. Unresolved peaks in a chromatographic analysis can be diag-
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Stopped Flow Rapid Scan Spectrophotometer • Minimum dead time — 500 ^sec • 16 spectra measured every 1 msec sequentially •Simple and robust mixing system without syringe • Fluorescence, T-jump and flash accessories. CIRCLE 1 ON READER SERVICE CARD 1240 A · ANALYTICAL CHEMISTRY, VOL. 58, NO. 12, OCTOBER 1986
Please write to: ATAGO B U S S A N C O . , L T D . 7-23, 5-chome Shimbashi, M i n a t o - k u , T o k y o 105 T e l e x : 2 8 4 2 1 Phone: (03) 4 3 2 - 8 7 4 1
(a)
4 θ
h
Cylindrical flow œil
Φ)
Gasket Tubing for chromatographic effluent
Prism
X *- h
k
/«
Figure 1. New designs for Rl detection (a) Deflection type; (b) reflection (transmission) type. I& incident intensity; fe, reflected intensity; lT, trans mitted intensity
Wavelength (nm)
0.15
0.12
8 c (Β
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Figure 2. Four chromatographic signals acquired at four wavelengths overlaid for comparison as a means of peak purity determination Shifting retention times in the second and fourth peaks suggest coeluting compounds. Reproduced with permission from Reference 7, copyright 1982 by the AAAS, and through the courtesy of Hewlett-Packard Co., Scientific Instruments Division, Palo Alto, Calif.
1242 A · ANALYTICAL CHEMISTRY, VOL. 58, NO. 12, OCTOBER 1986
nosed using simultaneous multiwavelength detection. This concept is dem onstrated (7) in Figure 2, with simul taneous absorbance detection at four wavelengths. The presence of coelut ing peaks is diagnosed from the shift ing of retention times as a function of detection wavelength. Purity determi nation within a chromatographic peak is often quite easily performed even with dual-wavelength absorbance techniques, for example, by plotting the ratio. At the other extreme, absorb ance detection at a single wavelength in the range of 185-210 nm can pro vide sensitive and almost universal de tection of analytes, with proper choice of eluents. Another feature of absor bance detection is the compatability with most gradient elution schemes. The usefulness of spectral informa tion has led to new commercial instru mentation based on diode arrays, rapid-scan spectrometers, and even Fourier transform (visible) interfer ometers. Still, UV-vis bands are broad and cannot compete with infrared bands in information content. Com mercial accessories for Fourier trans form infrared (FT-IR) detection fol lowing LC are available either as thin flow cells or off-line collection devices based on solvent removal. This is be cause unlike GC, the LC eluent ab sorbs strongly in the IR to obscure analyte signals. Both of these approaches benefit from the development of mi crocolumns and SFC. In microcol umns, the smaller amounts of solvent required allow the use of exotic sol vents, such as deuterated or fluorinated species, to create spectral windows in on-line IR detectors, and allow more efficient solvent removal in off line IR detectors. In SFC, the fluids themselves are relatively transparent in the IR region, especially for fluids like Xe, and are readily vaporized af ter the column to facilitate solute col lection. Photothermal effects. There have been many recent publications on thermal lens detection in LC. This is because the effect is very easy to ob serve with moderate laser intensities and moderate absorptions (8). When light is absorbed by the analyte, heat is eventually produced in the system. The temperature of the eluent in creases, leading to an RI change, that is, a density change. When the excita tion is attributable to a laser beam with a Gaussian cross-sectional inten sity distribution, the center part of the optical region will be heated more than the sides. This typically results in an RI distribution that resembles a diverging lens. The experimental ar rangement calls for a flow cell placed beyond the focal point of the laser beam and an aperture at far field in front of a photodiode. Without the ap-
erture, one basically has a normal transmission type of absorption mea surement. With the aperture, one lim its the spatial region through which the beam can pass. As the beam di verges because of the thermal lens in the cell, a smaller fraction of the total beam is able to pass the aperture, so more light is lost to the detector com pared with normal absorption. This enhancement essentially gives a larger signal at the detector than simple transmission. In fact, for a given absorbance, the lens increases in strength with laser power. Enhance ments of several hundred have been observed, and absorption detectability is improved. However, because an intensity is be ing measured, some of the gain is ne gated by the intensity instability of the laser. Continuous lasers with suffi cient powers for thermal lens spec trometry have inherent stabilities of 1%. This is about 2 orders of magni tude worse than well-regulated con ventional light sources. Power stabili zation or electronic compensation cir cuits can reduce fluctuations to 0.1%. A second laser with better intensity stability can also be used to probe the thermal lens generated by a more powerful, but less stable, pump laser. The main difficulty is the alignment and the matching of the beams in the
Bragg cell
Argon ion laser
Lens
Chromatographic flow cell
Driver
Aperture
Beam splitter
Photodiode Lock-in amplifier
Square-wave generator
Chart recorder
Figure 3. Experimental arrangement for high-frequency modulation in thermal lens measurements
probe region, especially when small sample volumes are of interest. The optical arrangement that solves many of these problems {9) is shown in Figure 3. An argon ion laser is sent into an acousto-optical modulator driven by a square wave from a signal generator. About 60% of the beam is modulated into the first-order Bragg component at 150 kHz. A lens is
placed immediately after the Bragg cell to focus the laser beam into the microcell and to further separate the deflected beams. The flow cell for the LC effluent is simply an aluminum disk containing a 1-cm-long 0.6-mm i.d. optical region, except that a pieshaped wedge is cut on one side to al low the passage of the beams deflected by the Bragg cell. An aperture serves
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CHROMPACK FROM CHROMATOGRAPHERS FOR CHROMATOGRAPHERS CHROMPACK INTERNATIONAL B.V. P.O. Box 8033, 4330 Ε A Middelburg.The Netherlands Tel:01180-27352 Telex: 37852 CIRCLE 34 ON READER SERVICE CARD 1244 A · ANALYTICAL CHEMISTRY, VOL. 58, NO. 12, OCTOBER 1986
CIRCLE 95 ON READER SERVICE CARD
to analyze the thermal lens signal. The active area of the photodiode is large enough (1 cm2) to receive both the zeroth-order (sample) beam and the firstorder (reference) beam. Output from the photodiode is sent directly to a lock-in amplifer. The use of high-frequency modulation and a reference beam allows for baseline stabilities of the order of 10~4. So, with the aperture removed (normal transmission measurement), the system is at least as good as conventional absorption detectors. With the aperture in place, the thermal lens enhancement allows absorption measurements at a noise level of 1.3 X 10~6 au, which is equivalent to 1 pg of benzopurpurin dye injected into the micro-LC system. There are several related photothermal effects suitable for LC detection. One of these is photothermal deflection (10), which relies on the RI gradient produced by the absorption process to deflect a second laser beam. A signal is then generated in a position sensor some distance away. Another approach is to monitor the bulk RI change and not the RI gradients. Thus, the change in density along the optical path can be converted into a mode shift in an interferometer (4). Similarly, the density change can be measured as a decrease in solvent optical rotation when a polarimeter is
used as a detector (11) (see below). Finally, in photoacoustic detection, the density change is monitored as a pressure wave via a piezoelectric transducer. It turns out that most of these photothermal techniques are limited by absorption in the eluent, which produces a background signal. What are normally considered transparent solvents in the near-UV and visible spectral regions actually have inherent absorptions on the order of 10~3 to 10~4 c m - 1 as a result of vibrational overtone transitions. Fluorescence. Fluorescence detection is more selective than absorption detection, because all species that absorb light do not necessarily fluoresce. Detection of fluorescence can be made even more selective by adjusting both the excitation and emission wavelengths. There are many examples where such three-dimensional (excitation, emission, and retention time) chromatograms have been used to distinguish species that are not chromatographically resolved. Fluorescence generally provides better detectabilities than absorption. Commercially available fluorescence detectors for LC produce LOD about 100 times better than those provided by commercial absorbance detectors. Fluorescence intensity generally increases with excitation intensity. The
first impression is that the higher the excitation intensity, for example, by using lasers, the better the detectability. In practice, fluorescence detection is limited by the presence of background light, which includes various types of light scattering, luminescence from the flow cell walls, and emission from impurities in the solvent. All of these increase with excitation intensity to produce no net gain. When lasers are used, the situation is even worse because laser intensities are inherently much less stable than conventional light sources. It is the fluctuations in the background that limit detection. It is not surprising that the development of laser fluorometric detection is really attributable to new designs of flow cells and optics that reduce stray light (12). Even though fluorescence intensity increases with path length, high-power lasers can be used to provide sufficient signal levels over short path lengths so that once again background (solvent fluorescence and solvent Raman scattering) is the limiting factor. This is the easiest way to achieve small detector volumes. The advantage of laser fluorometry is thus the mass detectability, which can be 100 times lower than that obtained from conventional excitation sources. When sample sizes are limited, for example, in some bio-
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CIRCLE 202 ON READER SERVICE CARD 1246 A · ANALYTICAL CHEMISTRY, VOL. 58, NO. 12, OCTOBER 1986
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Figure 4. Chromatogram of solvolyzed plasma steroids derivatized with bromomethyl coumarin using fluorescence detection Column, 2.25-m X 220-/im i.d., 5-μπι ODS; eluent, continuous gradient 75-100% acetonitrile in H 20; flow rate, 1.5 μί/Γηίη. Approximately 50 pg of each steroid was injected. Tentatively identified compo nents: 1 = 5«-androstan-3a, 110-diol-17-one; 2 = 5/3-androstan-3a, 110-diol-17-one; 3 = 5/3-pregnane3a,110,17a,21-tetrol-2O-one; 4 = 5/3-pregnane-3a,17a,2O0,21-tetrol-11-one; 5 = 5/3-pregnane3a,11/3,17a-20/3,21-pentol; 6 = 5|3-pregnane-3a, 17α, 20α, 21-tetrol-11-one; 7 = 5/3-pregnane3a,11/3,17a,20a:,21-pentol; 8 = 5a-androstan-3a-ol-17-one; 9 = 5-androstene-3£i-ol-17-one; 10 = 5(3-pregnane-3a,20a,21-triol; 11 = 5/3-androstan-3a,17/J-diol. Reproduced with permission from Reference 13
logical applications, laser fluorometry may be the only technique suitable for LC quantitation. There have been many recent studies on derivatization schemes to take advantage of the HeCd laser, which emits at 325 nm and is relatively reliable in its opera tion. For example (13), the analysis of a complex mixture of steroids (which exhibit only moderate fluorescence) with detectabilities on the order of 1 pg is shown in Figure 4. With high light intensities, it is pos sible to observe fluorescence derived from two-photon excitation (12). This process reaches a different set of elec tronic states, is polarization depen dent, is more restrictive, and can be resonantly enhanced, so complemen tary information is obtained compared with normal fluorescence. A continu ous laser provides detectabilities in the nanogram range. Because the twophoton process is enhanced by inci dent intensity (peak power), we re cently (14) redesigned the detector based on a copper-vapor laser. The
CIRCLE 167 ON READER SERVICE CARD 1248 A · ANALYTICAL CHEMISTRY, VOL. 58, NO. 12, OCTOBER 1986
noise equivalent is estimated to be 100 fg of 2-(4-biphenyl)-5-phenyl1,3,4-oxadiazole injected. With the im proved detectabilities, two-photon ex cited fluorescence is competitive with most other LC detectors in sensitivity. Normal fluorescence will still be slightly better in ideal cases where emission is spectrally far away from solvent Raman bands. The fact that there is so little background from the solvent here implies that solvent puri ty requirements can be relaxed. In fact, most biological matrices show very little two-photon fluorescence background, so the unique selectivity is valuable. The transverse excitation geometry and small optical volume make this detector suitable even for on-column measurements in capillary LC, SFC, or capillary zone electropho resis. Best of all, the same laser can excite both normal and two-photon fluorescence in the same optical region to maximize the information content. Optical rotation. Optical activity is a particularly interesting molecular
Plant Proteins
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Robert L. Ory, Editor U.S. Department of Agriculture Examines the major role plant proteins play in both human and animal diets. Discusses the use of plant proteins in both Western and traditional foods of developing countries; plant and meat protein blended foods; effects on skeletal integrity, trace mineral utilization, atherosclerosis, and cancer incidence; effects of processing on proteins; and novel, lesser known proteins. Of interest to nutritionists, food scientists, and clinicians concerned about nutritional aspects of plant proteins for humans. Also helpful to food chemists and biochemists, dieticians, and medicinal chemists concerned with nutrition. CONTENTS Plant Proteins: The ABCs of Peanut and Cowpea Flours · Field-Pea Flours as Protein Supplements · Vegetable Food Proteins in Traditional Foods · Uses of Soybeans as Foods · Incorporation of Cottonseed into Foods · Soy Proteins in Meat Products · Soy Protein Products in Whole Muscle Meats · Effects of Protein on Skeletal Integrity · Utilization of Trace Minerals in Humans · Nutritional Quality of Dry Beans · CornGlandless Cottonseed Blended Food · Influence of Protein on Serum Cholesterol · Mortality of Adventists in Relation to Diet · Protein, Starch, and Fiber from Grain Legumes · Processing and Use of Bean Flours · Winged Bean as a Source of Protein · Tropical Plants as Sources of Protein · Protein of the Sweet Potato · Cucurbit Seed Protein and Oil · Protein-Nitrogen Conservation in Yams Developed from a symposium sponsored by the Division of Agricultural and Food Chemistry of the American Chemical Society ACS Symposium Series No. 312 296 pages (1986) Clothbound LC 86-10848 ISBN 0-8412-0976-6 US & Canada $59.95 Export $71.95 Order From: American Chemical Society Distribution Office Oept. 19 1155 Sixteenth St., N.W. Washington, DC 20036 or CALL TOLL FREE 800-424-6747 and use your credit card!
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John F. Scamehorn, Editor University of Oklahoma Demonstrates the wide range of physical phenomena in which surfactant mixtures are important. Describes current thought and techniques for modeling surfactant interactions. Discusses micelle formation, surfactant adsorption on solids, surfactant adsorption at fluidfluid interfaces, solubilization, liquid crystal formation, and emulsification. Of interest to colloid and surface chemists, surface scientists, interface chemists, and anyone studying surfactant systems. CONTENTS Overview of Phenomena · Nonideal Mixed Micelles · New Mathematical Models of Mixed Micellization · Solutions of Hydrocarbon and Fluorocarbon Surfactants · Solution Properties of Mixed Surfactant Systems · Sodium Decanoate-2-Butoxyethanol in Water • Inverted Micelles of Calcium Alkarylsulfonates · Nonideal Mixed Monolayer Model · Monolayer Properties · The Penetration of Monolayers by Surfactants · Synergism in Binary Mixtures of Surfactants · Surface Adsorption and Micelle Formation · Effect of Alkyl Alcohols on Surfactants · Fluorocarbon and Hydrocarbon Surfactants · Anionic Surfactants on Alumina · Properties of Minerals Above The Critical Micelle Concentration · Competitive Adsorption of Polystyrene Latex · Vegetable Oil-Nonionic Surfactant Mixtures · The Mesophase Formation · Molecular Assemblies in Mild Surfactant Solutions · Micellar Solublization of Methanol and Triglycerides · Interfacial Properties of Nonaethoxylated Fatty Alcohol · 1-Octadecanol and Dodecylammonium Chloride · Future Perspectives Developed from a symposium sponsored by the Division of Colloid and Surface Chemistry of the American Chemical Society and the 5th International Conference on Surface and Colloid Science ACS Symposium Series No. 311 349 pages (1986) Clothbound LC 86-8062 ISBN 0-8412-0975-8 US & Canada $66.95 Export $80.95 Order From: American Chemical Society Distribution Office Dept. 18 1155 Sixteenth St., N.W. Washington, DC 20036 or CALL TOLL FREE 800-424-6747 and use your credit card!
1250 A · ANALYTICAL CHEMISTRY, VOL. 58, NO. 12, OCTOBER 1986
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Figure 5. Separation of cholesterol and cholesterol esters in human serum using optical rotation as a detection system Peaks: 1 = cholesterol and cholestanol; 2 = cholesteryl linolenate and arachindonate; 3 = cholesteryl palmitoleate and linoleate; 4 = cholesteryl palmitate and oleate; 5 = cholesteryl stéarate. Mobile phase, tetrahydrofuran:water (76:24, v/v); flow rate, 0.5 mL/min.; column, 10-μΓη C1e- Re produced with permission from Reference 16
property because it is generally associ ated with biological activity. Tradi tional polarimeters are not sensitive enough for LC detection. The instru ment has recently been improved sub stantially with a new design based on a laser (15). Currently, rotations of the order of 1 microdegree (1 ng of a mate rial injected with [a] = 100°, a path length of 1 cm, cell volume of 1 μL, and a peak volume of 10 μϋ.) can be detected. Demonstrations have been successful for the quantitation of cho lesterol in blood, sugars in urine, com ponents in shale oil, extracts of coal, and enantiomers of amino acids. The first of these applications (16) is shown in Figure 5. The laser-based polarimeter can also be used in a nonselective mode (11) (indirect polarimetry) much like the RI detector, which is limited by its poor detectability and the relatively large volumes in commercial units. If an optically active solvent is used for LC, there will be a large constant background rotation in the absence of any analyte. This background can be
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again a low light intensity reaches the phototube. Thus, the noise level in the polarimeter remains low. When an opcompensated for by physically rotat ing the second polarizer so that once tically inactive analyte elutes from the column, it displaces an equal amount of the solvent in the optical region. Fewer solvent molecules will be present in the optical region, and a de crease in optical rotation will result. It can be seen that unless the analyte has exactly the same specific rotation as the solvent, a signal will be observed. Detectability was found to be 4 ng of injected material. The success of indi rect polarimetry is a direct result of the low solvent consumption in microbore LC, making the cost low enough for routine use. Circular dichroism (CD) also probes optical activity in molecules. However, this technique measures the difference in absorption of left (LCPL) and right circularly polarized light (RCPL) and is closely related to normal absorp tion. This then allows the "local" chirality to be investigated—that is, the presence of both asymmetry and an absorbing chromophore. Commercial CD spectrometers are not sensitive enough for the low concentration lev els in LC. Because this is a signal cor responding to the difference between two large intensities, stability of the light source is the key. This is empha sized by the fact that CD is a very small (10 _3 -10 -4 ) effect on top of nor mal absorption. It turns out that one needs a moderate intensity (~10 mW) to stay above the shot noise limit, thus requiring a laser. To overcome the sta bility problem in laser sources, one can incorporate modulation at high frequencies. With proper operation {17), noise levels equivalent to ΔΑ = 3 Χ 10 - 7 au or 3 ng of injected materi al in microbore LC can be achieved. CD is a difficult measurement to make. When the intensities of RCPL and LCPL are not identical, a false signal is produced by the background absorption (17). The system must be checked with highly absorbing but op tically inactive species. To improve detection, one can use fluorescencedetected CD; that is, the difference in fluorescence intensities when the LC effluent is excited alternately by RCPL and LCPL can be monitored. For microcolumns, a 10-fold gain in detectability for fluorescence vs. ab sorption can be realized. Also, the same small volume advantage as in standard laser fluorometry can be maximized. Electrochemical detectors. Elec trochemical (EC) detectors offer selec tivity capabilities analogous to absorbance and fluorescence detection. Sen sitive detection is possible for species exhibiting a reversible electron trans
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Figure 6. Separation of phenols by capillary LC using electrochemical detection Peaks: 1 = hydroquinone (fR); 2 = 4-methylphenol; 3 = 2-methylphenol; 4 = 3,4-dimethylphenol; 5 = 3,5-dimethylphenol; 6 = 2,3-dimethylphenol; 7 = 2,4-dimethylphenol; 8 = 2,6-dimethylphenol; 9 = 2-methyl-4-ethylphenol; 10 = 2-isopropylphenol; 11 = 2,4,6-trimethylphenol. Mobile phase, 10~3 M HCI04 in distilled water; flow rate 1.7 nL/s; pressure, 2.5 MPa; column, 2.8-m Χ 16-μΓΤΐ i.d.; stationary phase, OV-101. Flow through the splitter: 12 μ ί / s . Reproduced with permission from Reference 18
fer for a particular functional group. Thus, EC detection is particularly useful for the analysis of aromatic amines and phenols. Detectabilities for conventional LC are quite favor able for these types of species, ranging from about 10 pg to 1 ng injected ma terial. Because EC detection is a sur face phenomenon and not a solution phenomenon, in contrast to the other detectors, it is uniquely well suited to be scaled down for micro LC. State-ofthe art EC detection volumes have been reported in the subnanoliter range. LOD of typical, electroactive species is on the order of 100 fg for open-tubular LC. An excellent exam ple of EC detection (18) for the sepa ration of phenols is shown in Figure 6. Rapid-scanning and dual-electrode techniques (19) have provided a means to further enhance the selectiv ity already implicit in EC/LC detec tion. However, EC can also be used to detect electrochemically inactive spe cies by monitoring adsorption and competition at the electrode surface (20). This is accomplished by a triplestep potential waveform for pulsed amperometric detection on, for exam
1252 A · ANALYTICAL CHEMISTRY, VOL. 58, NO. 12, OCTOBER 1986
ple, noble-metal electrodes. After cy cling through a "clean" and a "re duce" potential, the analytical signal that is derived from an added elec troactive species is "measured" at a third potential. During the reduce pe riod, the analyte adsorbs on the elec trode to decrease the subsequently measured current. Applications to the determination of carbohydrates, carboxylic acids, and amino acids are par ticularly interesting. Mass spectrometry (MS). LC/MS instruments are commercially avail able, although they are somewhat costly to be considered bench-top in struments. This should not detract from the recognition that MS is an ex cellent tool for analyte identification and structural confirmation in chemi cal analysis. The union of GC with MS detection was fairly easy as compared with LC because the density of the carrier stream is much lower. Thus, much of the original work in combin ing MS with LC has dealt with remov ing the solvent without affecting the analyte. Various LC/MS interfaces have been developed, such as the mov ing-belt interface and the thermo-
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analytical chemistry
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Figure 7. Separation of a terpene mixture (total ion chromatogram) and correspond ing mass spectra Column, 300 X 0.22 mm, 3-μΓη ODS; eluent, methanol:water (80:20). Reproduced with permission from Reference 23
spray interface (21). The advent of capillary and open-tubular LC col umns has allowed direct liquid injec tion (22) (DLI) to be more readily de veloped. Essentially, the flux of mate rial due to the eluent flow for these chromatographic systems is found to be small enough to allow for DLL LC/MS in such systems is quite favor able for thermally labile species that could not survive a GC/MS analysis. If total ion current is measured, LC/MS offers universal detection at subnanogram levels. DLI from a capillary LC system produced the separation of a terpene mixture (23) shown in Figure 7. Also, the real-time mass spectra of a-terpinene and /3-pinene are provided to show the system's ability to provide infor mation for definitive molecular identi fication. Detectabilities for state-ofthe-art LC/MS systems are on the or der of 1-10 pg for open-tubular LC with DLI. Summary It is apparent that substantial ad vances in detection methods for LC have been made in recent years. The use of lasers to reduce detector vol umes and to provide added selectivity and sensitivity and the development of small columns to minimize contri butions from the solvent and to allow cost-effective use of exotic mobile and stationary phases are two important
1254 A · ANALYTICAL CHEMISTRY, VOL. 58, NO. 12, OCTOBER 1986
reasons. The development of separa tion processes has in turn benefited from this new generation of detectors. This trend is expected to continue. It is up to our imaginations to seek out new frontiers for the next generation of detection methods. The key, per haps, is to consider LC in the broader context of measurement science and not simply to try to adapt known mea surement principles to known LC ef fluents. Acknowledgment The authors thank the many co workers who have contributed to vari ous parts of this work, particularly M. J. Sepaniak, W. D. Pfeffer, S. D. Woodruff, J. C. Kuo, D. R. Bobbitt, B. H. Reitsma, K. J. Skogerboe, and S. A. Wilson. The Ames Laboratory is operated for the U.S. Department of Energy by Iowa State University un der Contract No. W-7405-eng-82. This work was supported by the Office of Basic Energy Sciences. References (1) Synovec, R. E.; Yeung, E. S. Anal. Cham. 1983,54, 1599-1603. (2) Scott, R.P.W. Liquid Chromatography Detectors, 2nd éd.; Elsevier: Amsterdam, 1986. (3) Liquid Chromatography Detectors; Vickrey, T. M., Ed.; Marcel Dekker: New York, 1983. (4) Woodruff, S. D.; Yeung, E. S. Anal. Chem. 1982,54, 2124-25. (continued)
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(5) Bornhop, D. J.; Dovichi, N. J. Anal. Chem. 1986,58,504-5. (6) Wilson, S. Α.; Yeung, E. S. Anal. Chem. 1985,57,2611-14. (7) Miller, J. C; George, S. Α.; Willis, B. G. Science 1982,218,241-46. (8) Harris, J. M.; Dovichi, N. J. Anal. Chem. 1980,52,695-706 A. (9) Skogerboe, K. J.; Yeung, E. S. Anal. Chem. 1986,58,1014-18. (10) Boccara, A. C.; Fournier, D.; Jackson, W.; Amer, N. M. Optics Lett. 1980,5, 377-79. (11) Bobbitt, D. R.; Yeung, E. S. Anal. Chem. 1985,57,271-74. (12) Yeung, E. S.; Sepaniak, M. J. Anal. Chem. 1980,52,1465-81 A. (13) Gluckman, J.; Shelly, D.; Novotny, M. J. Chromatogr. 1984,317,443-53. (14) Pfeffer, W. D.; Yeung, E. S. Anal. Chem. 1986,58,2103-5. (15) Yeung, E. S.; Steehoek, L. E.; Wood ruff, S. D.; Kuo, J. C. Anal. Chem. 1980, 52,1399-1402. (16) Kuo, J. C.; Yeung, E. S. J. Chroma togr. 1982,229, 293-300. (17) Synovec, R. E.; Yeung, E. S. Anal. Chem. 1985,57, 2606-10. (18) Krejci, M.; Slais, K. J. Chromatogr. 1982,235,21-29. (19) Roston, D. Α.; Shoup, R. E.; Kissinger, P. T. Anal. Chem. 1982,54,1417-34 A. (20) Hughes, S.; Meschi, P. L.; Johnson, D. C. Anal. Chim. Acta 1981,132,1-10. (21) Hardin, E. D.; Fan, T. P.; Blakley, C. R.; Vestal, M. L. Anal. Chem. 1984, 56,2-7. (22) Covey, T.; Henion, J. Anal. Chem. 1983,55,2275-80. (23) Alborn, H.; Stenhagen, G. J. Chroma togr. 1985,323,47-66.
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Edward Yeung (right) received his A.B. degree, magna cum laude in chemistry, from Cornell University, Ithaca, N.Y., in 1968. In 1972he re ceived a Ph.D. in physical chemistry from the University of California, Berkeley. That same year, he joined the faculty at Iowa State University, where he is now professor of chemis try. His research interests span the areas of fundamental and applied la ser spectroscopy. Robert Synovec (left) received his B.A. degree, summa cum laude, from Bethel College, St. Paul, Minn., in 1981. He then studied under Edward Yeung at Iowa State University and received a Ph.D. in analytical chemis try in 1986. He is interested in vari ous aspects of laser spectroscopy and chromatography. In September of 1986, he joined the University of Washington, Seattle, as assistant professor of chemistry.
