Recent Advances in New and Potentially Novel Detectors in High

9 Recent Advances in New and Potentially Novel Detectors in High-Performance Liquid Chromatography and Flow Injection Analysis

Downloaded by TUFTS UNIV on November 22, 2015 | http://pubs.acs.org Publication Date: January 27, 1986 | doi: 10.1021/bk-1986-0297.ch009

Ira S. Krull Department of Chemistry, The Barnett Institute, Northeastern University, Boston, M A 02115

A review is provided of some of the latest advances in new and potentially novel, selective detectors for high perfor mance liquid chromatography and flow injection analysis. A number of very useful and practical element selective de tectors are covered, as these have already been interfaced with both HPLC and/or FIA for trace metal analysis and speciation. Some approaches to metal speciation discussed here include: HPLC-inductively coupled plasma emission, HPLC -direct current plasma emission, and HPLC-microwave induced plasma emission spectroscopy. Most of the remaining detec tion devices and approaches covered utilize light as part of the overall detection process. Usually, a distinct de rivative of the starting analyte is generated, and that new derivative is then detected in a variety of ways. These include: HPLC-photoionization detection, HPLC-photoelectrochemical detection, HPLC-photoconductivity detection, and HPLC-photolysis-electrochemical detection. Mechanisms, instrumentation, details of interfacing with HPLC, detec tor operations, as well as specific applications for each HPLC-detector case are presented and discussed. Finally, some suggestions are provided for possible future develop ments and advances in detection methods and instrumenta tion for both HPLC and FIA. For a t l e a s t the p a s t twenty y e a r s o r more, tremendous p r o g r e s s has been r e a l i z e d i n t h e g e n e r a l f i e l d o f h i g h performance l i q u i d chromatography (HPLC). S i g n i f i c a n t p r o g r e s s has been r e p o r t e d i n many a r e a s of i n s t r u m e n t a t i o n , i n t e r f a c i n g , automation, computer/microprocessor c o n t r o l , d e s i g n , and even r o b o t i c s c o n t r o l . I t has become v e r y c l e a r t h a t HPLC i s a dominant a n a l y t i c a l a r e a , whose growth c o n t i n u e s t o d a y . Though chromatographic s e p a r a t i o n s have p r o g r e s s e d v e r y r a p i d l y o v e r the p a s t two decades i n h i g h performance l i q u i d chromatography, t o the c u r r e n t s i t u a t i o n where p r o t e i n s , enzymes, p o l y n u c l e o t i d e s , and much s m a l l e r m o l e c u l e s can now be r e a d i l y r e s o l v e d and r e c o v e r e d , t h e 0097-6156/86/0297-0137S08.50/0 © 1986 American Chemical Society

In Chromatography and Separation Chemistry; Ahuja, S.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.


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f i n a l detection of analytes has progressed somewhat more slowly (1-14)· Any number of support materials, bonded phases, ion exchange packings, o p t i c a l l y active phases, normal bonded phases, etc. have been made, and these were evaluated for the separation of inorganic and organic species during at least the past twenty years. Significant advances have also been made i n the uniformity of p a r t i c l e sizes, narrow p a r t i cle diameters, uniform p a r t i c l e d i s t r i b u t i o n s , and related parameters of importance related to the stationary phase. A large number of mo b i l e phases have also been developed for HPLC separations, modifiers for these mobile phases, metal additives for the mobile phase, binary and ternary solvent mixtures, gradient e l u t i o n , and combinations there of. I n i t i a l l y , detection involved the r e f r a c t i v e index (RI) approach, and though t h i s crude method i s s t i l l used for polymer and preparative separations, i t has f a l l e n by the wayside i n just about a l l low-level a n a l y t i c a l determinations. U l t r a v i o l e t - v i s i b l e (UV-VIS) detection a l so developed quite rapidly from the inception of HPLC, and today there are commercially a v a i l a b l e fixed wavelength, v a r i a b l e wavelength, multiwavelength, l i n e a r diode array spectrometers, fast scanning spectrometers, and other approaches, a l l of which are general, non selective methods of analyte i d e n t i f i c a t i o n . In most HPLC labs today, there are a number of UV-VIS detectors, and perhaps one fluorescence (FL) and one electrochemical (EC) detector for HPLC, FL monitors are quite u s e f u l , i n that they are very sensitive, routinely operate at the trace and u l t r a t r a c e l e v e l s , and are r e l a t i v e l y selective i n that few compounds exhibit inherent or native fluorescence. However, neither UV-VIS nor FL detectors r e a l l y provide any compound i d e n t i f i c a t i o n , other than the presence of a chromophore and/or fluorophore. EC detectors have appeared more recently, but they have caught on very quickly, and appear to o f f e r s i g n i f i c a n t promise both with regard to s e l e c t i v i t y and s e n s i t i v i t y of analysis. E s p e c i a l l y with dual elec trode approaches, already commercially available from several firms, EC i s able to provide good to excellent analyte i d e n t i f i c a t i o n or s e l e c t i v i t y , together with very good s e n s i t i v i t y and detection l i m i t s . It would appear that LCEC w i l l continue to develop quite r a p i d l y , es p e c i a l l y with regard to other applications, both organic and inorganic. But, i t w i l l always be limited to those compounds that have inherent electrochemical properties. Of course, a l l detection methods become suitable for any analyte when o f f - l i n e or on-line d e r i v a t i z a t i o n s are employed, but there are some serious disadvantages i n using homo geneous, solution d e r i v a t i z a t i o n s i n HPLC (15-17). Ideally, i n the future, a l l d e r i v a t i z a t i o n s w i l l be performed on-line, pre- or postcolumn, using reagents that do not get mixed with the HPLC eluent v i a two flowing streams. Solid phase reagents i n HPLC would avoid the need for extra pumps, mixing chambers, reaction chambers, dead volume, etc. Perhaps UV-VIS, FL, or EC detectors can a l l be considered some what s e l e c t i v e , i n that they respond only to those compounds that are chromophoric, fluorophoric, and/or electrophoric. None of them respond to a l l compounds passing through, as with the RI, but other than for wavelength r a t i o i n g or dual electrode response r a t i o i n g , they cannot provide true analyte i d e n t i f i c a t i o n or detector s e l e c t i v i t y f o r a p a r t i c u l a r functional group, element, or size of molecule. They are a l l extremely s e n s i t i v e , and indeed any detector today that cannot reach into the low p a r t s - p e r - b i l l i o n (ppb) range doesn't receive much attention i n HPLC. The next generation of detectors w i l l have to be



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more sensitive, and to provide more s e l e c t i v i t y than i s now possible with e x i s t i n g detectors. That i s , such newer detectors w i l l have to be able to discriminate one analyte peak from the next, and to provide some r e l a t i v e l y unambiguous i d e n t i f i c a t i o n or c h a r a c t e r i s t i c f o r each analyte d i f f e r e n t from others present. We want the detector to see very low l e v e l s , and also to t e l l us something about what i t i s seeing at such low l e v e l s . Though the mass spectrometer (MS) provides true analyte s e l e c t i v i t y and i d e n t i f i c a t i o n , i t i s not about to become a very widely used or e a s i l y employed detector i n most HPLC s i t u a t i o n s / applications. Surely HPLC-MS i s here to stay, i t i s developing quite rapidly, but i t may never become as readily used and applied as HPLCUV or LCEC are already. The next generation of detectors should t e l l us something about an analyte or class of analytes that i t detects, and/or i t should respond to very select compounds, even i n the presence of many other similar eluting compounds/analytes. The ultimate selective detector would respond to only one p a r t i c u l a r compound, but i t would not be very p r a c t i c a l or economical, since i t could not also be used f o r any other compounds. Ideally, a selective detector should, at times, be selective f o r one class of analytes, and under other operating conditions, selective f o r another c l a s s , but always provide informa tion f o r each class separately. The word selective i s interpreted i n c o r r e c t l y ; i t may not mean that the detector responds to a unique compound or compounds. Does i t mean that the detector responds to many compounds, but does so d i f f e r e n t l y with d i f f e r e n t information? I f i t responds to a very small number of compounds, i t i s much more s e l e c t i v e in i t s response, but less useful f o r other classes. I f i t responds to many classes, i t i s much less s e l e c t i v e , but more useful and p r a c t i c a l , and could s t i l l provide information that adds to the s e l e c t i v i t y of the f i n a l response. In general, the term s e l e c t i v e has come to mean response to only c e r t a i n classes of compounds, and the term general means that a detector responds to everything that passes through that detector. We are going to consider i n this chapter a very select group of new and p o t e n t i a l l y novel detectors, a l l of which provide f o r greater s e l e c t i v i t y i n t h e i r responses than UV-VIS, FL, and/or EC. Our choice of which detectors to discuss and consider here has been based, i n part, on how we perceive the current trends i n detector development and a p p l i c a t i o n . I t has also been based on our own biased and personal i n t e r e s t s , as dictated by our own recent research and development results with these very same detectors. The requirements f o r a p o t e n t i a l l y novel, selective detector i n HPLC are several, including: 1) high s e n s i t i v i t y for many classes of analytes, perhaps into the low ppb range or below; 2) high degree of s e l e c t i v i t y i n responses as dictated by many experimental v a r i a b l e s ; 3) compatibility with a l l important HPLC mobile phases; 4) q u a l i t a t i v e and quantitative information about a p a r t i c u l a r analyte that d i f f e r e n t i a t e s i t s response from a l l or almost a l l others eluting nearby; 5) r e p r o d u c i b i l i t y of response f o r repeated i n j e c t i o n s ; 6) high degree of accuracy and p r e c i s i o n i n quantitative determinations; and 7) an ease of interfacing with commercial HPLC instrumentation, f i t t i n g s , and equipment. Some of these requirements are met by many new detect ors, but most new detectors do not meet a l l of these suggested needs. Some other, perhaps less important, but s t i l l p r a c t i c a l requirements are that the detector be easy to use, modular i n design, inexpensive,


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f a c i l e to turn on and operate on a d a i l y basis, easy to maintain and repair, immune to changes i n flow rates and back pressures i n the HPLC system, and compatible with multiple detection (series or p a r a l l e l ) approaches. The detector should also contribute r e l a t i v e l y l i t t l e to o v e r a l l band broadening of the HPLC peak, i t should be almost unnoticed in the f i n a l peak shape produced, and i t should be nondestructive by returning most or a l l of the o r i g i n a l l y injected analyte to the ana l y s t f o r further studies or determinations. Table I summarizes most of these i d e a l requirements f o r our new and p o t e n t i a l l y novel, s e l e c t i v e detector for HPLC or FIA. Table I. Ideal Requirements for New and P o t e n t i a l l y Novel, Selective Detectors i n HPLC or FIA


Suggested Detector Requirements

Reasons f o r Suggested Requirements

high s e n s i t i v i t y and low detection juseful f o r trace and u l t r a t r a c e limits analyses high degree of s e l e c t i v i t y

improved analyte i d e n t i f i c a t i o n and q u a l i t a t i v e confirmation

compatible with important HPLC mobile phases

can be used with current separations does not require new methods

q u a l i t a t i v e and quantitative information about analytes

improved analyte i d e n t i f i c a t i o n , improved quantitative analyses

r e p r o d u c i b i l i t y of responses i n peak height and peak area

improved accuracy and p r e c i s i o n i n quantitative determinations

high degree of accuracy and pre c i s i o n i n quantitative analyses

improved o v e r a l l usefulness of f i n a l analysis

ease of i n t e r f a c i n g with current HPLC instrumentation

[widespread acceptance by communityat-large, greater usage and applications, less expenses

easy-to-use, operate, maintain, and/or repair

more up-time, better acceptance by analysts, more u t i l i t y , reduced expenses, faster sample turnout

modular i n design

may be moved from lab-to-lab, easy to transport, interface, arrange

immune to changes i n flow rates, back pressure of column

greater q u a l i t a t i v e and quantita t i v e r e p r o d u c i b i l i t y , more accurate and precise r e s u l t s

compatible with multiple detection useful with other detectors at the same time to improve analyte identification/confirmation l i t t l e additional band spreading

l i t t l e loss of chromatographic resolution, e f f i c i e n c y , shape

nondestructive to analyte

recovery of injected analyte, more studies on same material possible

unreactive and unresponsive to HPLC mobile phases

low background noise l e v e l s , no f a l s e positives due to solvent, no large solvent front present



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We discuss here a select group of sélective detectors, these being: 1) element selective detectors (ESD) that respond s e l e c t i v e l y to one or more element at the same or d i f f e r e n t times, including atomic absorption spectrometers (AAS), flame (FAA) or graphite furnace (GFAA), inductively coupled plasma (ICP) emission spectrometers, direct current plasma (DCP), and only i n c i d e n t a l l y microwave induced plasma (MIP) or atomic fluorescence spectrometer (AFS); 2) photoionization detector (PID) based on a photochemical ionization of the analyte molecule leading to formation and eventual collection/detection of ions and electrons; 3) a post-column, photolytic d e r i v a t i z a t i o n i n LCEC or HPLC-photolysis-EC (HPLC-hv-EC) that uses a discrete photolytic degradation of the o r i g i n a l analyte as the basic detection p r i n c i p l e ; 4) a new photoelectrochemical detector (PED) that again uses l i g h t energy as an on-line, post-column d e r i v a t i z a t i o n step, but now to generate a s h o r t - l i v e d , possibly excited species that has novel EC properties; and f i n a l l y , 5) a possibly novel electrogenerated lumi nescence detector (ELD) that uses electrochemistry to generate new species that combine, s e l f - a n n i h i l a t e , and then together luminesce, t h i s then being detected by a suitable photomultiplier tube or monochromator outside of the ELD c e l l . Some of these newer approaches to detection i n HPLC or FIA are s t i l l i n development stages, indeed perhaps most of them f i t that description, but s t i l l others have a l ready been f u l l y optimized and remain to be more f u l l y applied to actual samples and s p e c i f i c applications. Element Selective Detectors for HPLC and FIA Trace inorganic analysis and speciation have become very popular and accepted only within the past decade or thereabouts, especially i n view of the late r e a l i z a t i o n on the part of many environmentalists and/or t o x i c o l o g i s t s that d i f f e r e n t metal species may have d i f f e r e n t b i o l o g i c a l / t o x i c o l o g i c a l responses or effects i n animals and man (1829). Though a variety of approaches have already been developed to perform inorganic, especially metal, speciation; including anodic stripping voltammetry, d i f f e r e n t i a l pulse voltammetry and amperometry, double d i f f e r e n t i a l pulse amperometry, d i f f e r e n t i a l pulse polarography, etc., many of these suffer from the lack of an i n i t i a l separation or chromatographic step before the selective detection step. Thus, of l a t e , most reports deal with the i n t e r f a c i n g of either gas chroma tography (GC) or high performance l i q u i d chromatography (HPLC) with some type of element selective detection. P r i o r to the current wide spread popularity of various plasma induced emission techniques, this involved both flame and graphite furnace atomic absorption spectrosco pies (FAA/GFAA). This area has most recently been adequately reviewed both by Schwedt and by Jewett and Brinckman (25, 26). I t has long been our b e l i e f that true element selective detection i n HPLC can only be done using some type of atomic absorption or emission spectroscopy (AAS or AES). Others contend that element selective detection can also be r e a l i z e d by various types of post-column d e r i v a t i z a t i o n , chelation, complexation, etc., as well as by electrochemical approaches (30-34). Nevertheless, perhaps some metal and nonmetal speciation studies today involve some type of GC/HPLC-element selective detection using atomic absorption or emission spectroscopy. Though FAA and GFAA have been used for quite some time now, both of these hyphenated methods in HPLC suffer some serious disadvantages and p r a c t i c a l l i m i t a t i o n s



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(35-52). HPLC-FAA approaches generally have impractical detection l i m i t s for many r e a l world applications, although sample preconcentration methods can often get around such l i m i t a t i o n s . Of a l l element s e l e c t i v e detectors f o r HPLC, perhaps FAA suffers from having the highest minimum detection l i m i t s (MDLs). This severely l i m i t s i t s use fulness, except with those samples that already contain very high lev els of metal species, and/or those where analyte (metal species) preconcentration i s an easy task before HPLC-FAA. HPLC-GFAA, on the other hand, offers perhaps the best MDLs of a l l element selective detection approaches, at times reaching into the low ppb or high parts-pert r i l l i o n (ppt) ranges. This i s due to the inherent analyte preconcent r a t i o n step b u i l t into a l l GFAA methods by the use of a graphite furnace that allows for multiple sample introductions with desolvation p r i o r to atomization-detection of a l l preconcentrated metal species. However, a serious disadvantage to HPLC-GFAA i s the fact that some type of merry-go-round or automated sample c o l l e c t o r and injector must be interfaced between the end of the HPLC column and furnace of the GFAA unit (49-52). Brinckman's group at the U.S. National Bureau of Standards has best pioneered and evaluated the HPLC-GFAA i n s t r u mentation, methods, and f i n a l applications. Though extremely useful and p r a c t i c a l f o r many environmental samples, the o v e r a l l system r e quires a modest investment of time, energy, money, and t o t a l i n s t r u mentation before i t i s routinely operative. It also produces f i n a l HPLC-GFAA chromatograms that are i n histogram fashion, because of the nature of the AA data output, and thus i t i s not generally possible to obtain a continuous, smooth chromatogram for t h i s system. The HPLC portion of the system must be f i n e l y tuned so that there are no over lapping peaks due to the same element containing moieties/analytes, otherwise the GFAA detection step might not be able to f u l l y resolve or separate these for f i n a l quantitative measurements. Though quite p r a c t i c a l and useful, r e l a t i v e l y few groups today use HPLC-GFAA approaches, perhaps because of the non-continuous nature of i t s data. Most l i t e r a t u r e reports today seem to u t i l i z e some type of plasma emission detection, be t h i s inductively coupled plasma (ICP) or direct current plasma (DCP). Though microwave induced plasma (MIP) emission spectrometry has been used f o r at least twenty years i n GC, i t does not appear at this writing to be very compatible with most convention a l HPLC flow rate requirements (20, 21). Though there are some reports using the MIP as a low flow-rate (microbore) HPLC detector, these appear to offer l i t t l e promise for more widespread acceptance and p r a c t i c a l applications. We have recently attempted some HPLC-MIP work, but at any flow rate over 100ul/min, depending on the power of the MIP, the plasma immediately becomes extinguished. Some other groups, notably that of Caruso i n Cincinnati, seem to have had more success with a high powered MIP i n combination with low flow-rate microbore HPLC. Much more work seems to have been done i n HPLC-ICP than i n HPLC-DCP (18-22, 53, 54). Though these are very expensive detectors by conventional HPLC-detector standards, they appear to o f f e r some very s i g n i f i c a n t advantages for true element s e l e c t i v e detection and o v e r a l l inorganic, esp. metal, speciation today. I t i s not yet clear which of these two plasmas i s more advantageous i n HPLC, though sure l y more publications appear to use the ICP. This could be simply be cause more researchers have purchased the ICP since i t i s commercially available from many more suppliers than the DCP. Only one commercial DCP i s available today, that being from Smith, Kline, and Beckman.



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Their Spectraspan VI that appeared on the market i n 1984 appears to be an i d e a l element s e l e c t i v e detector for HPLC, though very few papers have thus far u t i l i z e d that s p e c i f i c model i n HPLC-DCP studies. I t i s arguable that MDLs v i a HPLC-DCP must, by the very nature of the DCP, always be i n f e r i o r to the HPLC-ICP system. We have not found this always to be the case, and indeed i n a very recent HPLC-DCP study of various chromium containing species, the HPLC-DCP MDLs appeared some what better (lower) than those reported by HPLC-ICP methods (22). We have already suggested some possible explanations for these observa tions, though nobody has yet made a f u l l , d i r e c t comparison of HPLCDCP vs HPLC-ICP MDLs for even a few metal species. There are s t i l l some s i g n i f i c a n t problems i n both HPLC-ICP and HPLC-DCP i n t e r f a c i n g for p r a c t i c a l applications where low MDLs are generally needed. But for a few miraculous reports using HPLC-ICP that provided MDLs at or below the published MDLs for direct-ICP work, we and others have come to r e a l i z e that the basic nebulizers developed years ago for direct-ICP are less-than-ideal for any HPLC-ICP i n t e r facing today. Such nebulizers, aside from being inherently i n e f f i c i e n t , were never designed from the s t a r t for the constantly changing concen trations of analytes e l u t i n g from an HPLC column. Even with the u l t r a sonic nebulizers that provide for much more e f f i c i e n t sample i n t r o duction into the ICP, the f i n a l MDLs v i a HPLC-ICP with this p a r t i c u l a r nebulizer are s t i l l l e s s than adequate for trace inorganic speciation requirements today. Many have suggested that this i s indeed the f i n a l solution to the HPLC-ICP MDL problem, but we and others are less than convinced based on the evidence at hand. What then i s the r e a l answer to the major obstacle to more widespread acceptance and u t i l i t y of the ICP/DCP systems for HPLC applications, or i s there more than one single answer possible? We tend to believe that there are several possible solutions to t h i s problem, and these could be: 1) e l e c t r o thermal or graphite furnace/cup sample vaporization i n HPLC-ICP; 2) post-column hydride d e r i v a t i z a t i o n i n HPLC-ICP or what i s termed HPLC-HY-ICP; 3) post-column chemical chelation or complexation of metals i n HPLC-ICP; and 4) use of the Vestal thermospray interface for HPLC-ICP/DCP and related approaches. It would be possible to spend the rest of this chapter just discussing these four possible approaches for improved HPLC-ICP/DCP i n t e r f a c i n g , but we w i l l attempt to do so in less than one or two paragraphs. The ICP has some s i g n i f i c a n t advantages for trace metal detection in comparison with both FAA and GFAA, e s p e c i a l l y with regard to m u l t i element c a p a b i l i t i e s and elimination of most matrix e f f e c t s . Perhaps for these very reasons, as well as i t s current popularity, a great amount of work appears today i n the l i t e r a t u r e attempting to interface both GC and HPLC with ICP (18-21, 25, 55-57). Most of t h i s work has involved conventional ICP nebulizer interfaces, and only within the past few years have serious alternative approaches for HPLC-ICP i n t e r facing been evaluated for improved MDLs. Electrothermal vaporization for improved sample introduction into direct-ICP systems has been described, and already some investigators are using HPLC-electrothermal vaporization-ICP approaches for true metal speciation (78-84). However, v i r t u a l l y a l l of these attempts to use electrothermal or heated tantalum ribbon sample introduction methods for HPLC-ICP coup l i n g are non-continuous, operate i n a step-wise manner, require c o l l e c t i o n of HPLC f r a c t i o n s , and cannot function i n a true on-line mode. At times, depending on the p a r t i c u l a r analyte species being



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studied, these methods also require simultaneous UV detection of the HPLC eluents, followed by c o l l e c t i o n of those f r a c t i o n s of interest for manual or automated introduction into the ICP. These approaches are reminiscent of the HPLC-GFAA methods, and though producing semicontinuous HPLC-ICP element s p e c i f i c chromatograms, they are f a r from i d e a l or routine i n operations. If the HPLC eluent can be continuously introduced into a heated graphite rod or tube, with continuous preconcentration and removal of solvent followed by continuous i n t r o duction of the analyte alone, this might be a p r a c t i c a l approach. Post-column, on-line, continuous hydride d e r i v a t i z a t i o n after HPLC separation, with continuous introduction of the now-formed metal hydrides into the ICP appears a t r u l y p r a c t i c a l and on-line approach for various applications (28, 54, 85-87). Figure 1 i l l u s t r a t e s i n schematic fashion the instrumental arrangement for performing on-line, continuous, post-column, real-time hydride d e r i v a t i z a t i o n i n HPLCHydride Generation-ICP approaches (28)· We have now successfully used t h i s system and approach for the trace analysis and speciation of arsenic i n drinking well waters i n the U.S., as well as for the deter mination of methylated organotins i n l i q u i d and s o l i d food samples for the U.S. Food & Drug Administration (FDA)(54). Figure 2 i l l u s t r a t e s a t y p i c a l HPLC-HY-DCP chromatogram for three methylated organotins, now using a paired-ion, reversed phase HPLC separation. MDLs, c a l i b r a t i o n p l o t s , l i n e a r i t i e s , analyte s e l e c t i v i t y , and related a n a l y t i c a l para meters of importance for true trace analysis and speciation of these metals capable of forming hydrides, a l l appear adequate for a d d i t i o n a l applications. We have already used HPLC-Cold Vapor-ICP approaches for organomercury speciation, analogous to what others have done for HPLCCV-GFAA of these same mercury species (88)· These newer approaches for metal speciation also appear quite adequate and appropriate for r e a l applications i n the future. Hydride or cold vapor generation techniques as a separate post-column d e r i v a t i z a t i o n step for select elements thus appears to be a quite p r a c t i c a l and v i a b l e o v e r a l l hyphenated method i n HPLC-ICP/DCP f o r future end uses. Detection l i m i t s are presumably improved over conventional HPLC-ICP/DCP i n the absence of t h i s postcolumn reaction by several orders of magnitude i n a l l cases studied, which thus makes the HPLC-HY-ICP/DCP approach p r a c t i c a l today for at least those metals capable of forming hydrides or a cold vapor. However, that s t i l l leaves a very large number of elements i n capable of forming v o l a t i l e hydrides, for which some a l t e r n a t i v e , p r a c t i c a l , on-line approach remains to be developed for future HPLCICP /DCP i n t e r f a c i n g . Post-column metal/element chelation or complexat i o n has been used somewhat i n the past f o r direct-ICP/DCP sample introduction, but most of these results have been less*-than-promising (89-93). We have also studied t h i s approach, with o f f - l i n e d e r i v a t i z a tion of various metals into known, presumably v o l a t i l e derivatives that have been used i n GC analysis. Use of such pre-formed chelates or complexes i n direct-ICP analysis, using conventional cross-flow nebulizers, has not yielded any s i g n i f i c a n t enhancements i n f i n a l s e n s i t i v i t i e s or MDLs. Even using organic solvents for introduction of these metal chelates has not yet yielded s i g n i f i c a n t lowering of the MDLs i n comparison with conventional metal introduction into direct-FAA or direct-ICP systems (94-96). It does not now seem l i k e l y that either v o l a t i l e metal derivatives and/or the use of more v o l a t i l e organic solvents, such as methyl isobutyl ketone (MIBK), w i l l have pronounced, p o s i t i v e e f f e c t s on f i n a l FAA or ICP/DCP sensitivities/MDLs.


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Acid Mixing Toe Spray Chamber


Reagent Addition Toe X Flow Nebulizer 2 Channel Peristaltic Pump

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Figure 1. Schematic diagram of the t o t a l HPLC-HY-ICP instrumenta t i o n . Reproduced with permission from Ref. 28. Copyright 1984, Marcel Dekker.

s

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Figure 2. HPLC-HY-DCP chromatogram of mono-, d i - , and trimethyltin; column: 25 cm χ 4.1 mm i . d . , lOum, PRP-1; mobile phase: 0.003M PIC B-6/0o003M KF/0.02N H S0 /2.5% HOAc at 2.5 ml/min flow rate; 200ul loop i n j e c t i o n ; chart speed 0.5 inch/min; f u l l scale 5 mV. Repro duced with permission from Ref. 54. Copyright 1985, Williams & Wilkins. 2

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One other possible approach for o v e r a l l improved HPLC-ICP/DCP performance related to lowered MDLs, has to do with the use of a heated spray chamber of even a Vestal-type thermospray introduction system (97-104). The idea of these approaches i s to produce a much f i n e r aerosol-mist or heated spray from the HPLC eluent, which i s then p a r t i a l l y or f u l l y desolvated p r i o r to entering the flame of the FAA or the plume region of the ICP/DCP. In some ways, this i s an extension of the current u l t r a s o n i c nebulizer, which also produces a very fine mist that i s more e f f i c i e n t l y transported into the plasma plume area for f i n a l detection. Though some work has already been done using a heated aerosol or spray chamber, and much work has been done by Browner s group on the effect of aerosol droplet size as these effect f i n a l detection l i m i t s , o v e r a l l results-to-date do not appear very promising for s i g n i f i c a n t l y reduced MDLs. Much less has been done on the possible use of a heated spray chamber/nebulizer f o r HPLC-FAA or HPLC-ICP/DCP, though a great deal of work has already been done i n HPLC-mass spectrometry with such interfaces (101-106). We would expect these newer approaches of sample introduction for HPLC-MS to have some possible applications to HPLC-ICP/DCP, and e f f o r t s are encouraged to explore such newer i n t e r f a c i n g techniques (107). In closing this section, i t should be emphasized that element s e l e c t i v e detection i n HPLC i s an area now undergoing very rapid ex ploration and development, and s i g n i f i c a n t improvements i n HPLC-ICP/ DCP detection l i m i t s with resultant p r a c t i c a l applications should be r e a l i z e d i n the near future. If a then r e l a t i v e l y inexpensive element s e l e c t i v e spectroscopic detector could be developed f o r con ventional or microbore HPLC, with a successful interface providing low MDLs, t h i s could result i n a more widespread acceptance and a p p l i cation of both the o v e r a l l instrumentation and f i n a l techniques.


1

The Photoionization Detector i n HPLC or FIA For many years, the photoionization detector (PID) has been studied and applied i n GC, and i t i s recognized as both a very sensitive and selective approach for trace analysis (108-115). For about the past decade, various people have attempted to interface t h i s same PID with HPLC separations, i n order to provide a f i n a l HPLC-PID system com patible with reversed and normal phase HPLC solvents (116-119). Most recently, D r i s c o l l et_ a l . have described the successful interfacing of reversed and normal phase HPLC with a high temperature PID (PI-52) v i a a heated oven that e f f e c t i v e l y vaporized a l l of the HPLC effluent p r i o r to i t s introduction into the PID unit (116). In some ways, this i s similar to the thermospray interface for HPLC-MS, as already discussed above. Figure 3 i l l u s t r a t e s the o v e r a l l instrumentation schematic for t h i s p a r t i c u l a r HPLC-PID approach, wherein a l l or a f r a c t i o n of the HPLC effluent can be introduced into the high temperature GC oven. Using this approach, the HPLC effluent i s 100% vaporized within the oven, as a function of temperature and residence time therein, dependent on t o t a l flow rate entering the oven, and t h i s vaporized mixture of mobile phase solvents and analyte molecules i s then passed through to the f i n a l PID. The o v e r a l l approach has been optimized with regard to c a r r i e r gas, lamp energies, mobile phase compatibility, flow rate compatibility, and f i n a l MDLs. C a l i b r a tion plots for t y p i c a l analytes have been over several orders of mag nitude s t a r t i n g from the detection l i m i t range. Under the best of



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American Chemical Society Library 1155 16th St., N.W. Washington, D.C. 20036 In Chromatography and Separation Chemistry; Ahuja, S.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.


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conditions yet r e a l i z e d , and for the compounds showing the greatest s e n s i t i v i t y , MDLs can be i n the low ppb range (5-50 ppm) (116). A wide variety of classes of organics have already been studied by t h i s HPLCPID i n t e r f a c e , many of which are summarized i n Table I I , together with MDL (ng) and lamp energy (eV) used for those p a r t i c u l a r compounds. Typical HPLC-PID chromatograms are i l l u s t r a t e d i n Figures 4-5, one for a normal phase separation of N-substituted a n i l i n e s , the second for a reversed phase separation of these very same a n i l i n e s , now e l u t i n g i n the exact opposite (reversed) order. Work i s continuing i n our labs on improvements i n the o v e r a l l HPLC-PID i n t e r f a c e , the use of v o l a t i l e organic and inorganic buffers for reversed phase separations of bioorganics, improvements i n MDLs and s e n s i t i v i t i e s , and extension of HPLC-PID applications to other classes of organics and inorganics. In view of the large number of classes of compounds suitable i n GC-PID, often at the ng detection l i m i t s , i t i s hoped that most of these w i l l be suitable candidates i n HPLC-PID as w e l l . This could suggest the eventual trace determina tion of amines and amino acids, perhaps peptides and proteins, without any p r i o r , o f f - l i n e or on-line d e r i v a t i z a t i o n step before detection by the PID. If t h i s proves f e a s i b l e , i t would be the only s e n s i t i v e and s e l e c t i v e detector available today i n HPLC that could detect such underivatized materials. This may also prove useful i n the eventual detection of peptides, polypeptides, and perhaps even proteins and enzymes, which could o f f e r s i g n i f i c a n t advantages over current methods. The PID works on the p r i n c i p l e of i o n i z i n g an electron from the s t a r t i n g compound, usually t h i s i s a non-bonding electron on nitrogen, s u l f u r , oxygen, or a halogen, though i t could be an aromatic P i - e l e c tron on benzene, and the resultant organic cation and electron are then captured by the electrodes within the PID to produce a resultant, o v e r a l l current. The number of cations and electrons generated per g or millimole of analyte entering the PID then determines that compound's s e n s i t i v i t y and f i n a l MDLs. This i s a function of the ease of photo i o n i z a t i o n or i o n i z a t i o n by photochemical means for that p a r t i c u l a r compound, which electrons are available for photoionization, and the energy and i n t e n s i t y of the l i g h t s t r i k i n g these molecules. Thus, photoionization i s r e a l l y akin to the flame i o n i z a t i o n process, but instead of using a flame, the PID uses l i g h t energy to ionize the o r i g i n a l analyte molecules. Operating the PID at a high temperature of about 275°C ensures that none of the analyte or mobile phase molecules w i l l recondense on the window of the PID. Use of methanol/water mobile phases also maintains a very clean window i n the PID, so that repro d u c i b i l i t y of response i s generally quite high i n HPLC-PID operations, even for extended periods of time. Although there i s one commercial system now on the market (HNU Systems, Inc., Newton Highlands, Mass.), i t has only been available for about one year, and i t has not yet been widely applied to r e a l samples. Such work i s now i n progress, and we hope to report further r e s u l t s with t h i s very HPLC-PID system i n the near future (120). Not a l l normal or reversed phase solvents w i l l prove compatible with the PID requirements, e s p e c i a l l y those solvents that have any photoionization propensity or properties. V i r t u a l l y a l l alcohols and water are f u l l y compatible with the PID, but solvents such as acetone or a c e t o n i t r i l e , and others, are not. This r e a l l y has to do with the i o n i z a t i o n potentials of these solvents and the IP of the lamp being used. Those solvents having IPs lower than the lamp energy w i l l produce very high background noise l e v e l s , making them incompatible with routine, continuous HPLC-PID operation.


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Figure 4o The HPLC-PID chromatogram of N-substituted anilines on a Lichrosorb Si60, 25 cm χ 4.6 mm i . d . column. Mobile phase: 3% IPA/ hexane, flow rate 0.46 ml/min, s p l i t r a t i o 3:7 (PID:waste), 10.2 eV ]-|mp, interface at 230°C, PID at 290°C, attenuation setting 1 χ 10" ^ aufso Reproduced with permission from Ref. 116. Copyright 1984,

Elsevier

Science.


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Figure 5· The HPLC-PID chromatogram of three N-substituted a n i l i n e s : N-methyl, Ν,Ν'-diethylaniline, and NjN'-dimethylaniline. Column: Cg, lOum, 25 cm χ 4.6 mm i . d . ; mobile phase: HOH/ACN (25/75, ν/ν), flow rate 0.8 ml/min, s p l i t r a t i o 7:3 (PID:waste), 9.5 eV lamp, interface oven 240°C, PID at 290°C, attenuation setting 2 χ 1 0 ~ aufs. Reproduced with permission from Ref 116 Copyright 1984, 10

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Table I I . Minimum Detection Limits for Certain Organic Compounds by HPLC-PIDο Organic Compounds Bromobenzene Iodobenzene Phenol Chlorobenzene Ν,Ν'-Dimethylaniline Dioctylphthalate Fluorobenzene N,N -Diethylaniline N-Me thy1ani1ine o-Xylene Anthracene Benzene Naphthalene 2-0ctanone N-MethyIf ormamide Ν, Ν -DimethyIformamide Ν,Ν -DiethyIf ormamide 3-Hexanone


T

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Minimum Detection Limits (ng)

Lamp Energy (eV) 10.2 10.2 10.2 10.2 9.5 10.2 10.2 9.5 9.5 10.2 9.5 10.2 9.5 10.2 10.2 10.2 10.2 10.2

3 4 4 5 5 5 7 20 60 60 75 150 200 125 500 500 600 700

a. HPLC conditions: A l l t e c h Cg column, lOum, 25 cm χ 4 6 mm i . d . ; mobile phase, acetonitrile-water (75:25, ν/ν), 0.8 ml/min, d i r e c t l y to the photoionization detector i n HPLC-PID. 0

Source: Reproduced with permission Elsevier S c i e n t i f i c , Holland.

from Ref. 116. Copyright

1984,

Post-Column, On-Line, Continuous Photolytic Derivatizations i n HPLCElectrochemical Detection (EC) and FIA-EC Liquid chromatography (LC) with electrochemical detection (EC) or LCEC has now been popular for about the past decade, and i t has rapid ly become a widely accepted and r e a d i l y applied method of trace organ i c and inorganic analysis (121-126). Both oxidative and reductive LC EC have been described i n numerous publications and presentations, and several firms now offer commercial instrumentation based on both amperometric and coulometric EC detection f o r HPLC. As with any detection method, only those compounds that have inherent EC proper t i e s , that can actually be electrochemically oxidized/reduced, w i l l prove useful, to varying degrees, i n LCEC. Those compounds that do not have natural, inherent EC properties must then be derivatized, either o f f - l i n e or on-line, pre- or post-column, to form new materials with EC detection properties v a s t l y d i f f e r e n t than the s t a r t i n g , underivatized analyte (127-131). There are some serious disadvantages i n the use of o f f - l i n e , pre-column derivatizations for any type of detection i n HPLC, and i t i s generally accepted that on-line, postcolumn approaches should be used whenever possible and necessary Indeed, various commercial post-column reactors are now on the market for performing automated, on-line, real-time, continuous chemical 0



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derivatizations i n HPLC, especially with UV or FL detection methods (132). Most of the already described d e r i v a t i z a t i o n s for LCEC; however, have u t i l i z e d o f f - l i n e approaches, generally pre-column, and there have been serious disadvantages i n a l l such approaches/schemes. To try and overcome t h i s apparent deficiency i n d e r i v a t i z a t i o n s for LCEC, and r e a l i z i n g that post-column d e r i v a t i z a t i o n methods are d i f f i c u l t i n LCEC using chemical approaches, we have now developed a quite useful and p r a c t i c a l photolysis or photohydrolysis approach i n LCEC (133-136). Figure 6 i l l u s t r a t e s the basic HPLC-hv-EC apparatus i n operation, wherein the analytes eluting from the HPLC column are i n d i v i d u a l l y passed around a medium pressure, broad spectrum mercury discharge lamp immersed i n ice-water or a low temperature water bath. Species that have photolytic or photohydrolytic properties are rapidly and often very e f f i c i e n t l y converted into new products, perhaps stable inorganic or organic anions, such as n i t r i t e , benzoate, etc., and these are then passed into the f i n a l EC detector. Though, i n p r i n c i p l e , i t should now be possible to use both reductive and oxidative EC methods, we have generally found i t more p r a c t i c a l and simpler to use oxidative techniques, as for n i t r i t e or s u l f i d e or benzoate anions. Depending on the p a r t i c u l a r s t a r t i n g analyte, one or more than one photoproduct may be formed, and each of these could have d i f f e r e n t EC detector properties. Optimization of the system involves determining the best (longest) residence time for each analyte i n the i r r a d i a t o r Teflon c o i l or mesh, ideal s a l t and i t s concentration i n the mobile phase, mobile phase solvents compatible with hv-EC requirements and HPLC separations desired for p a r t i c u l a r analytes, and f i n a l l y , EC detector electrode materials, working p o t e n t i a l s , single or dual ( p a r a l l e l or series) arrangements, etc. Once a l l of the various i n s t r u mental requirements are met and compatible, the HPLC-hv-EC system can be operated routinely f o r extended periods of time, often with automated sample i n j e c t i o n and data c o l l e c t i o n . The only l i m i t a t i o n that we have found to date, after several years of operation, has to do with the fact that the Teflon i r r a d i a t o r tubing tends to harden with time and crack. This necessitates i t s replacement, perhaps every s i x months, depending on how long and often i t has been used. We have developed a c e r t a i n woven mesh arrangement for t h i s Teflon i r r a d i a t o r tubing, which removes most e f f e c t i v e dead volume or system variance due to the post-column photolysis system. This i s quite similar i n design to that described e a r l i e r by Halasz, Englehardt and Neue, and others for post-column chemical d e r i v a t i z t i o n s , on-line i n HPLC. Peak shape i s quite similar to any HPLC-UV peak, and o v e r a l l postcolumn variance i s l i t t l e more than without the entire i r r a d i a t o r i n - l i n e between the HPLC column and EC detector entrance (10%). This newer a n a l y t i c a l approach could e f f e c t i v e l y replace any need for reductive LCEC i n the future, since i t appears that most compounds already studied by reductive means can now be done by oxidative HPLC-hv-EC methods as w e l l . This has included compounds such as: 1) organic n i t r o derivatives, C-nitro, 0-nitro, N-nitro, etc.; 2) N-nitroso compounds, such as N-nitrosamines, N-nitroso amino acids, etc.; 3) organothiophosphates, such as malathion, parathion, ethion, etc.; 4) aromatic esters and amides, such as benzamide, vitamin B^, a l k y l benzoates, etc.; 5) beta-lactams, such as p e n i c i l l i n s and cephalosporins; 6) mycotoxins, such as vomitoxin or deoxynivalenol; 8) drugs such as chlordiazepoxide, barbiturates, cocaine, and others.


In Chromatography and Separation Chemistry; Ahuja, S.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986. STEEL TUBING

Figure 6. Schematic diagram of the HPLC-hv-EC detection system operation. Reproduced with permission from Ref. 134. Copyright 1984, American Society for Testing & Materials (ASTM).

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We believe that many other classes of compounds w i l l be shown to be suitable analytes for these approaches, and that MDLs w i l l be, as they have been for most of the above compounds, i n the low ppb ranges (1-50 ppb or below). In those instances where p a r t i c u l a r analytes are already amenable to oxidative LCEC, without post-column photolysis, confirmative evidence for t h e i r presence can be obtained by HPLC-hv-EC. That i s , the dual electrode response r a t i o s for these compounds with and without i r r a d i a t i o n are almost always very d i f f e r e n t , depending on the p a r t i c u l a r s t a r t i n g analyte. Also, the optimum working electrode potentials with and without i r r a d i a t i o n post-column are also very d i f f e r e n t , providing further confirmation p o s s i b i l i t i e s . We have demon strated for many compounds additional confirmation v i a the dual electrode response r a t i o s possible with and without i r r a d i a t i o n , using the same electrode working potentials or d i f f e r e n t sets of these. Though not yet commercially a v a i l a b l e , many suppliers o f f e r i r r a d i a t i o n units suitable for this type of post-column photolysis, which only requires the preparation of the Teflon mesh, but even that i s already commercially available (Kratos Instruments, Ramsey, N.J.). Various publications have described how to weave one's own Teflon i r r a d i a t o r , and we have summarized such approaches i n our own recent publications (133-136). A separate paper w i l l shortly be published dealing with p r e c i s e l y how to weave t h i s type of post-column i r r a d i a tor, and that should provide s u f f i c i e n t d e t a i l s f o r anyone to repro duce what we have already done i n HPLC-hv-EC (137). In the interim, the excellent work of Engelhardt and Neue, as well as that of Kratos Instruments, should be consulted for further d e t a i l s (138). Any commercially available HPLC system and EC detector can, i n p r i n c i p l e , be used i n HPLC-hv-EC with t h i s approach, and the entire system can be constructed within one single day, i f desired. It i s easy to learn, easy to construct, easy to operate, and easy to apply to new samples and a n a l y t i c a l problems. At the moment, we are investigating a wide variety of l i c i t and i l l i c i t drugs, including many barbiturates, and other drugs of abuse. These r e s u l t s are most encouraging and e x c i t i n g (137). S i m i l a r l y , we have evidence-at-hand that many organohalogen compounds, including organobromines, organoiodines, and possibly organochlorines, w i l l a l l be suitable substrates for HPLC-hv-EC methods (139). That could provide the HPLC analyst, at l a s t , with true halogen s e l e c t i v e detection methods, e s p e c i a l l y v i a the use of dual electrode response r a t i o s and d i f f e r e n t working electrode surface materials. This work i s only now i n progress, but already we have s u f f i c i e n t evidence to indicate that a l l organoiodine and organobromine compounds should be suitable substrates i n HPLC-hv-EC. It i s our hope and intention that these newer methods of trace organic and inorganic analysis w i l l be adopted by many other analysts and labs, and that they w i l l become standard a n a l y t i c a l approaches i n the very near future. The Photoconductivity

Detector

i n HPLC (HPLC-PCD)

This i s a detection approach that has been described i n the l i t e r a ture for several years, i s already commercially available from Tracor Instruments Corp. (Austin, Texas), and has been applied to various classes of compounds (140-143). The detection of non-ionic compounds by e l e c t r o l y t i c conductivity depends on the conversion of the o r i g i n a l compound to i o n i c species which, when dissolved i n an aqueous e l e c t r o -



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l y t e , increases the conductivity of that f i n a l e l e c t r o l y t e . It has long been known that halogenated compounds, organohalogens, such as many of the commonly used p e s t i c i d e s , are p h o t o l y t i c a l l y degraded to form halogen acids and other products. The photoconductivity detector (PCD) u t i l i z e s the increased conductance of the mobile phase e l e c t r o l y t e which results from the formation of halogen acids when halogenated compounds are exposed to UV l i g h t . Not only halogenated compounds, but also compounds such as N-nitrosamines, N-nitro d e r i v a t i v e s , organothiophosphates (malathion, parathion, e t c . ) , sulfonamides, and a wide variety of drugs, are a l l suitable analytes i n HPLC-PCD methods. Figure 7 i l l u s t r a t e s a block diagram of the various parts that make-up a commercial photoconductivity detector i n HPLC-PCD operation (142). In many ways, and perhaps t h i s i s why we have included t h i s novel and selective detector i n this review, the PCD resembles the above d i s cussed HPLC-hv-EC approach. The HPLC-hv-EC or photoelectroanalyzer detection scheme u t i l i z e s the exact same photolysis step post-column, continuous, real-time, on-line, and then passes the newly generated anions or other photoproducts to the EC detector for measurements. The PCD does the same thing, but i t uses a much more general, non selective conductivity measurement approach, which perhaps provides much less analyte s e l e c t i v i t y and information than the photoelectro analyzer approach. Nevertheless, the PCD has found r e l a t i v e acceptance and u t i l i t y , although the number of papers describing i t s applications and usefulness are somewhat limited i n number. The HPLC mobile phase requirements for the PCD are probably very similar to those for the HPLC-hv-EC method, i n that mobile phase constituents should not cause excessively high background noise levels on the conductivity detector. These same constituents must also not undergo any photolytic process that could convert them to newer materials then having conductance properties above the o r i g i n a l mobile phase constituents or analytes. V i r t u a l l y any HPLC system i s compatible with the PCD, though as with any detector, pressure fluctuations should always be minimized as much as p r a c t i c a l i n order to reduce o v e r a l l noise l e v e l s . As with the photoelectroanalyzer, the PCD i s not compatible with most normal phase solvents or separations, and thus both methods must be used with aqueous based mobile phases i n reversed phase HPLC. Both are on-line, real-time, and continuous processes, and both can be used for long periods of time with high o v e r a l l r e p r o d u c i b i l i t y , accuracy, and p r e c i s i o n . I t has always appeared strange that the PCD has never become as popular as i t could or should be, and we believe that this has been due to a lack of promotion, publications, presentations, and v i s i b l y good applications of general and widespread i n t e r e s t . Basic a l l y , i t has appeared that the manufacturer has not promoted i t s own product, and though they have often presented papers at s c i e n t i f i c meetings, notably the various Pittsburgh Conferences, formal publica tions i n a large number of d i f f e r e n t journals have been somewhat lack ing. The PCD has always been a p o t e n t i a l l y useful and very p r a c t i c a l detector i n HPLC, but i t has been treated, or so i t would seem, l i k e an orphan of the manufacturer. Hopefully, t h i s unfortunate state-ofa f f a i r s w i l l soon change, or else the PCD w i l l fade from the market place and disappear altogether. A New

Photoelectrochemical Detector for HPLC and FIA (HPLC-PED)

The e a r l i e r work with the photoelectroanalyzer system, HPLC-hv-EC, led



156


us quite n a t u r a l l y to consider placing the l i g h t d i r e c t l y within the EC c e l l , and onto the working electrode surface (144, 145)· The idea of using electrochemistry to measure or monitor photochemical products, intermediates, or processes, has been suggested and attempts described for several years (146-156). Most modern texts on e l e c t r o a n a l y t i c a l chemistry have at least a small section dealing with photoelectrochemistry, though there are many v a r i a t i o n s on t h i s term. What we mean here i s the use of photochemistry or l i g h t to generate new species having d i f f e r e n t electrochemical properties for detection than the o r i g i n a l , unphotolyzed compound/analyte. Though others have used t h i s approach i n s o l u t i o n , using rotating ring-disk electrodes and other approaches, i t i s now f e l t that they were looking at ground-state intermediates or photoproducts of the photolysis reaction. It seems unlikely i n view of their experimental systems that they were ever studying the electrochemical properties of e l e c t r o n i c a l l y promoted excited states, such as singlets or t r i p l e t s . Though that e a r l i e r work may s t i l l stand on i t s own, i t seems to bear l i t t l e r e l a t i o n or resem blance to that which we are now doing i n HPLC-PED or FIA-PED areas. We should point out, i n a l l fairness and s c i e n t i f i c o b j e c t i v i t y , that numerous publications attest to the impossibility of electrochemically detecting, by conventional oxidative or reductive techniques, any short-lived, e l e c t r o n i c a l l y promoted excited states generated photochemically, such as singlets or t r i p l e t s . Our- approach, Figure 8, u t i l i z e s a high i n t e n s i t y , broad spectrum, mercury or xenon/mercury discharge lamp that produces a l i g h t beam of high intensity that i s then focused and directed through the top-half of a two part, t h i n layer, flow-through amperometric transducer c e l l sold commercially for more conventional LCEC applications and studies (Bioanalytical Systems, Inc., West Lafayette, Indiana). The l i g h t beam then impinges through a quartz window cemented into the top h a l f (auxiliary electrode) of this two part c e l l * d i r e c t l y onto the working electrode surface operated at any desired oxidative or reductive p o t e n t i a l . Single or dual, series or p a r a l l e l arrangements of the c e l l can be used, with various working p o t e n t i a l s and materials, such as glassy carbon, gold/mercury, p l a t i n u , or s i l v e r . In the dual electrode configuration, these could be of the same or d i f f e r e n t materials, one or both could be i r r a d i a t e d , they could be operated at the same or d i f f e r e n t working p o t e n t i a l s . A l l of these conditions, as w e l l as changing the wavelength of l i g h t used, increase the o v e r a l l possible s e l e c t i v i t i e s available f o r any given analyte amenable to PED detec t i o n (145). Mobile phase requirements are very similar to those needed for successful LCEC, s a l t s and solvents that have no EC properties, and no photolytic products derived from these constituents that would then have t h e i r own unique EC properties e i t h e r . Any conventional, commercial HPLC system i s compatible with the PED, again requiring r e l a t i v e l y pulseless solvent delivery, and the mobile phase must be thoroughly degassed of dissolved oxygen. This i s now possible v i a many techniques, but the simplest i s surely the use of a zinc (granular) oxygen scrubber placed on-line, just p r i o r to the sample i n j e c t i o n valve i n the HPLC system. A l l parts of the HPLC-PED system must then be stainless s t e e l , so that no oxygen from the a i r can redissolve i n the mobile phase after i s has passed the i n j e c t i o n valve stage (157). The l i g h t source must be contained i n a black box to protect laboratory workers, and the EC c e l l should be contained i n a small Faraday cage to reduce background noise levels and spikes.


9. KRULL


157

.HIGH PRESSURE PUMP .SOLVENT CONDITIONING COLUMN jNJECTOR LC. COLUMN

[REACTION

SYSTEM DELAY COIL

Ï INLET , SPLITTER

REACTOR BLOCK Ç ICOIL

LAMP


_ _ J U _ _ _ T DIFFERENTIAL CONDUCTIVITY CELL

t _ - _ -

Ί

- — ~-J4 •WASTE !

"TWASTE OR RESERVOIR

J CONDUCTIVITY SIGNAL PROCESSOR

OUTPUT DEVICES

I

I

DETECTION SYSTEMj

Figure 7. Block diagram showing the relationship of the photo conductivity detector (PCD) to the other components of the HPLC system. Reproduced with permission from Ref. 142. Copyright 1979, Preston Publications, Inc.

— [

m OPTICAL

LAMP

BENCH

Figure 8. The o v e r a l l HPLC-photoelectrochemical or FIA-PED detec t i o n system using the modified BAS flow-through detection c e l l . Reproduced with permission from Ref. 144. Copyright 1984, BAS Press, Inc.



158

CHROMATOGRAPHY

A N D SEPARATION

CHEMISTRY

This use of a thin-layer, flow-through, amperometrie EC c e l l to generate, i n s i t u , new electrochemically active species appears very d i f f e r e n t from the work of Weber and colleagues (148, 149). That work used a l i g h t beam that passed right across the surface of the working electrode i n a w a l l - j e t configuration, but never impinged d i r e c t l y on to the surface at 90 angles. Our own approach puts the l i g h t d i r e c t l y onto the working electrode surface at 90°, there i s no question that the surface i s undergoing i r r a d i a t i o n at a l l times. As the i n d i v i d u a l HPLC analytes elute from the HPLC column into the PED c e l l , they are i r r a d i a t e d , presumably at the working electrode surface or j u s t above t h i s , and something i s produced that then has i t s own unique EC properties. None of the compounds i n Table I I I , which i s but a p a r t i a l l i s t of those compounds already shown responsive under PED conditions, have t h e i r own EC responses with the lamp o f f , except for 2-cyclohexen1-one, a somewhat s p e c i a l case. None of the known solution photoproducts of the other compounds i n Table III having PED responses with the lamp on, have been found to have any EC responses under these p a r t i c u l a r operating conditions and p o t e n t i a l s . Thus, i t would appear that something formed between the ground state s t a r t i n g material and ground state product(s) i s responsible for the PED responses being seen. That could be a singlet excited state, a t r i p l e t excited state, a ground-state free r a d i c a l derived from the s i n g l e t or t r i p l e t a f t e r a hydrogen abstraction reaction with the hydrogen donating alcohol solvent, or a r a d i c a l anion ground-state intermediate leading to the observed f i n a l photoreduced products of these ketones and aldehydes. Numerous experiments are s t i l l needed to conclusively prove the actual mechanism operative i n PED operation. We are acutely aware, indeed p a i n f u l l y aware after c e r t a i n recent s c i e n t i f i c meetings and presentations, that on both experimental and t h e o r e t i c a l grounds, i t should be impossible to electrochemically detect a s h o r t - l i v e d , e l e c t r o n i c a l l y excited (promoted) singlet or t r i p l e t species. V i r t u a l l y a l l carbonyl ketones and aldehydes exhibit varying responses on the PED, and MDLs for many of these compounds are equal to or better than the best HPLC-UV conditions. Many compounds that are very poor UV responders appear to have quite usable and p r a c t i c a l MDLs v i a PED. The PED therefore appears to be a quite s e n s i t i v e (low ppb) and selective detector that could prove quite useful for many carbonyl derivatives, e s p e c i a l l y ketones and aldehydes. Together with dual electrode approaches, as suggested above, analyte s e l e c t i v i t y appears unique for the compounds amenable to PED. A number of standard chromatograms have already been obtained for mixtures of carbonyls, and actual applications with p r a c t i c a l samples are now being developed (158). I t i s expected that a number of publications i n the coming years w i l l more f u l l y describe and depict the PED, as well as what i t can be applied for and what unique approaches i t may o f f e r the trace analyst. A l l of this i s i n addition, of course, to the rather exciting idea of perhaps using electrochemistry to characterize and describe and better understand photοchemically promoted, e l e c t r o n i c a l l y excited states derived from promotion of a ground-state electron into a nonbonding o r b i t a l (η-Pi*). Even i f i t turns out that electrochemistry cannot be used to detect or characterize e l e c t r o n i c a l l y excited, short-lived species, the PED w i l l s t i l l have i t s own interest as far as what i t can be used for i n HPLC/FIA applications i n trace a n a l y t i cal chemistry.



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Table I I I . Some Organic Compound Responses i n FIA-PED* EC Response ( - O 0 6 V ) Lamp On Lamp Off

Organic Compound HPLC mobile phase Acetone 2-Cyc1ohexen-1-one Acetophenone 2,3-Butanedione Cinnamaldehyde Mesityl oxide Benzaldehyde Indanone o-Aminobenzaldehyde 1,4-Cyclohexanedione Fructose L-Alanyl-L-phenylalanine

no no yes no no no no no no no no no no

no yes yes yes yes yes no yes yes yes yes no no

a. FIA-PED conditions used a mobile phase of 20%MeOH/80% HOH + OdM NaCl, flow rate of 0.8 ml/min, i n j e c t i o n volumes 20ul, -0.6V (reductive) vs Ag/AgCl reference electrode, GC working electrode, photochemical lamp 500W Hg arc, lOmV f u l l scale EC attenuation. Source: Reproduced BAS P r e s s .

w i t h p e r m i s s i o n from R e f . 144. C o p y r i g h t 1984,


160



Conclusions and Future Advances i n Selective and Sensitive Detectors for HPLC and FIA We have t r i e d to depict some of the more recent advances i n the devel opment of new and p o t e n t i a l l y novel s e n s i t i v e and s e l e c t i v e detectors of use i n HPLC and/or FIA. Our e f f o r t s and descriptions have been, i n the main, limited to those areas of most f a m i l i a r i t y and experience i n our own research e f f o r t s . The development of new approaches to detection of organics and inorganics i n chromatography or flow i n j e c t i o n must be guided by c e r t a i n inherent, basic p r i n c i p l e s of selec t i o n . These could be outlined as follows: 1. The detector should meet an e x i s t i n g need i n the a n a l y t i c a l community, and provide detection l i m i t s , methods, and r e s u l t s having serious advantages over e x i s t i n g approaches. 2. The detection approach should be widely applicable and usable by a very large number of analysts, and i t should find ready acceptance and u t i l i t y by that community. 3. The detector should be easy to construct or inexpensive to purchase, easy to operate, easy to maintain, easy to learn, have a short warm-up time, and be modular i n design so that i t w i l l f i t into any laboratory already having HPLC instruments 4. The detection method should be both s e n s i t i v e and s e l e c t i v e , i n order to find the widest application to r e a l samples, and to provide a high degree of analyte i d e n t i f i c a t i o n and/or confirmation. 5. The detection method should be quite d i f f e r e n t from a l l other existing approaches, that i s , i t should be t r u l y novel and provide us with further understanding and knowledge i n a n a l y t i c a l chemistry. 6. The detector should be compatible with both normal and rever sed phase chromatography, nonaqueous and aqueous based solvents. To some extent, we have chosen to explore new and p o t e n t i a l l y novel detection areas, because they seemed to offer promise of unusual success, and at times, because they seemed to o f f e r solutions to existing problems of pressing need and concern. Hopefully, these and other c r i t e r i a w i l l p r e v a i l i n the future as w e l l . What about the future? We have t r i e d to indicate already some of the areas where we and others are continuing to develop detection approaches for HPLC and FIA, and how those developments might proceed i n s p e c i f i c regards. What else i s there to be done i n the future, aside from the use of lasers i n o p t i c a l detectors, which some of our colleagues are already developing or have successfully developed? Along the l i n e s of the photoelectrochemical detector, we are toying with the possible uses of electrogenerated luminescence or HPLC/FIA, as a possible a l t e r n a t i v e to the now well-established chemiluminescence post-column detection methods described i n recent years (158-164). Electrogenerated chemiluminescence i s very similar to photoelectro chemical detection, i n that we are using l i g h t and electrochemistry, but i n reversed modes (146, 147). Whereas the PED uses l i g h t to gen erate a new species that then has novel electrochemical properties for f i n a l detection, the ELD approach w i l l use electrochemistry to gene rate new species that react with one another to generate luminescence, thereby decaying to neutral species. That luminescence could then be monitored by any suitable photomultiplier or monochromator. This approach would c l e a r l y be limited to those combinations of compounds


9.

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that are able to undergo electrochemical promotion or reduction and oxidation to produce two oppositely charge species i n nearby proximity at the l i g h t output region of the flow-through c e l l . Whatever l i g h t were produded would then have to be c o l l e c t e d , focused, collimated, and directed into the f i n a l l i g h t detector. It i s possible that i n direct or quenched electrogenerated luminescence, a^ lji room tempera ture phosphorescence i n HPLC, could also be devised i n the future. Though c e r t a i n l y a new detection approach i n HPLC and FIA, having inherent novelty, i t i s questionable at t h i s time i f i t w i l l have widespread a p p l i c a b i l i t y and acceptance for p r a c t i c a l applications. However, i n view of the current decided popularity and application of chemiluminescence detection i n HPLC/FIA, the ELD method j u s t might r i v a l this now established approach, at least for c e r t a i n analytes.


Acknowledgments We wish to thank the editor, S. Ahuja, for the o r i g i n a l i n v i t a t i o n to contribute to t h i s ACS Symposium Series p u b l i c a t i o n , and to be able to enjoy describing some of our recent e f f o r t s i n the areas of selec t i v e and sensitive detection for HPLC/FIA. Much of our own work de scribed here was made possible by various grants and/or contracts to Northeastern University over the past several years. We wish to acknowledge firms who have made such commitments, including: Instru mentation Laboratory ( A l l i e d A n a l y t i c a l Systems, Inc.), HNU Systems, Inc., B i o a n a l y t i c a l Systems, Inc., P f i z e r , Inc., EM Science, Inc., The Barnett Fund for Innovative Research at Northeastern University, and others, for their continued support, instrumentation donations, advice, collaboration, i n t e r e s t , encouragement, respect, and most of a l l , confidence i n what we have been together undertaking. This work would not have been possible, as w e l l , without the input along the way of many graduate and undergraduate students, as well as several V i s i t i n g Chinese S c i e n t i s t s from the People's Republic of China. We especially acknowledge the input and contributions of the following: D.S. Bushee, B. Karcher, W.R. LaCourse, CM. Selavka, R.J. Nelson, J . Burton, Wm. Costa, K-H. Xie, X-D. Ding, L-R. Chen, M. Swartz, S.W. Jordan, and others. We are e s p e c i a l l y g r a t e f u l to our colleagues within The Barnett I n s t i t u t e and Department of Chemistry for encourag ing us along the way, making many valued and valuable suggestions, and for constantly taking an i n t e r e s t i n the progress of the R&D programs described above. Special gratitude and acknowledgment goes to B.L. Karger, W.C. Giessen, P.L. Vouros, and R.W. Giese, a l l at NU. This i s contribution number 244 from The Barnett I n s t i t u t e at NU.

Literature Cited 1. 2.

3.

Scott, R.P.W. "Liquid Chromatography Detectors"; Elsevier Scientific Publishing Co.: Amsterdam, Holland, 1977. Kissinger, P.T.; Felice, L.J.; Miner, D.J.; Preddy, C.R.; Shoup, R.E. In "Contemporary Topics in Analytical and Clinical Chemistry, Volume 2"; Hercules, D.M.;Hieftje, G.M.; Snyder, L.R.; Evenson, M.A., Eds.; Plenum Press, Inc.: New York, 1978; Chap. 3. Snyder, L.R.; Kirkland, J.J. "Introduction to Modern Liquid Chromatography" Second Edition; J. Wiley & Sons, Inc.: New York, 1979; Chap. 4.


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Vickrey, T.M., Ed. "Liquid Chromatography Detectors"; Marcel Dekker: New York, 1983. Yeung, E.S. In "Advances in Chromatography, Volume 23"; Giddings, J.C.; Grushka, E.; Cazes, J.; Brown, P.R., Eds.; Marcel Dekker: New York, 1984; Chap. 1. Hancock, W.S.; Sparrow, J.T. "HPLC Analysis of Biological Com pounds, A Laboratory Guide"; Marcel Dekker: New York, 1984; Chap. 4. Kissinger, P.T., Ed. "An Introduction to Detectors for Liquid Chromatography" First Edition; BAS Press: West Lafayette, Indi ana, 1981. Lawrence, J.F. "Organic Trace Analysis by Liquid Chromatography"; Academic Press: New York, 1981; Chap. 5. DiCesare, J.L.; Ettre, L.S. J. Chromatogr., Chromatogr. Revs. 1982, 251, 1. Varadi, M.; Balla, J.; Pungor, E. Pure & Appl. Chem. 1979, 51, 1177. Borman, S.A. Anal. Chem. 1982, 54, 327A. Brenner, M.; Sims, C.W. American Laboratory (Fairfield, Ct.) 1981, 13(10), 78. Ettre, L.S. J. Chromatogr. Sci. 1978, 16, 396. Wise, S.A.; May, W.E. Research/Development Magazine 1977, 54 (October). Krull, I.S.; Lankmayr, E.P. American Laboratory (Fairfield, Ct.) 1982, 14(5), 18. Xie, K-H.; Colgan, S.; Krull, I.S. J. Liquid Chrom. 1983, 6(S-2), 125. Colgan, S.; Krull, I.S. In "Post-Column Reaction Detectors in HPLC" Krull, I.S., Ed.; Marcel Dekker: New York, 1985, in press. Brinckman, F.E.; Bernhard, Μ., Eds. "The Importance of Chemical Speciation in Environmental Processes"; Springer-Verlag: West Berlin; Heidelberg, 1985, in press. Krull, I.S. In "Environmental Analysis by Liquid Chromatography"; Lawrence, J.F., Ed.; Humana Press: Clifton, N.J., 1984; Chap. 5. Krull, I.S.; Jordan, S. American Laboratory (Fairfield, Ct.) 1980, 12(10), 21. Krull, I.S. Trends in Anal. Chem. 1984, 3(3), 76. Krull, I.S.; Panaro, K; Gershman, L.L. J. Chromatogr. Sci. 1983, 21, 460. Berman, E. "Trace Metals and Their Analysis"; Heyden: London, 1980. Risby, T.H., Ed. "Ultratrace Metal Analysis in Biological Science and Environment"; Advances in Chemistry Series 171; American Chemical Society: Washington, D.C., 1979. Jewett, K.L.; Brinckman, F.E. In "Detectors in Liquid Chromatog raphy"; Vickrey, T.M., Ed.; Marcel Dekker: New York, 1983; Chap.6. Schwedt, G. "Chromatographic Methods in Inorganic Analysis"; A.H. Verlag: Heidelberg, 1981. Uden, P.C.; Bigley, I.E.; Walters, F.H. Anal. Chim. Acta 1978, 100, 555. Bushee, D.S.; Krull, I.S,; Demko, P.R.; Smith, S.B., Jr. J. Liquid Chrom. 1984, 7, 861. Cassidy, R.M. In "Trace Analysis, Volume 1"; Lawrence, J.F., Ed.; Academic Press: New York, 1981; p. 122. MacCrehan, W.A.; Durst, R.A. Anal. Chem. 1978, 50, 2108. MacCrehan, W.A.; Durst, R.A. Anal. Chem. 1981, 53, 1700. MacCrehan, W.A. Anal. Chem. 1981, 53, 74.


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