Peter T. Kissinger,' Lawrence J. Felice, William P. King, Lawrence A. Pachla, Ralph M. Riggin, and Ronald E. Shoup
Purdue Univers~ty
west Lafayeite, Indiana 47907
High Performance Liquid Chromatography Experiments for Undergraduate Laboratories
In recent vears dramatic imnrovements have been made in the theory and practice of both liquid chromatography and electrochemistrv. Althoueh these develonments have led to revived interest in using such methods for routine analytical nurnoses. thev have vet to make a sianificant imnact on un. . dergradualr eduration. Now that inexpensive insrrumentation is avi~llable,cons~dcrationcan he riven ro utilization of hoth kinds of experiments in undergrad&te laboratories. Although several liquid chromatographs have recently entered the teaching market, they remain prohibitively expensive for many due to costly uv detectors employed. 0&r the past f&rycar.i we have d&'loped simple instrumenratinn incorporating theadvantages of hoth liquid rhromatngraphy and electrochemiitry. Liquid rhromatography with electrochrmical detection II.CKC) has been used with considerable success in our laboratory by undergraduate students on a variety of hioanalytical problems of practical significance. The purpose of the present paper is to briefly review the principles involved and to suggest apparatus and examples which have merit for undergraduate experiments and honors research projects. If we consider one electroactive compound passing by an electrode in a flowing stream, the amount of charge, Q, exchanged with the electrode will he given by Faraday's law
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Q = nFN where n is the number of electrons transferred per molecule (or the number of equivalents per mole), F is the number of coulombs per equivalent, and N is the number of moles of reactant converted into nroduct. The current. it. can he derived by differentiatio; of Faraday's law with respect to time ""
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The observed current is therefore directly proportional to the number of equivalents converted in the flow cell per second (ndNldt) and the Faraday constant is the proportionality factor. The current may also he expressed as it= nF(C, - COu
where C ; and Ct are the initial (enterins) ". and final (leaving) ". concentration of the electroactive analyte species moving throueh the cell (in moles/cm3) and u is the volume flow rate in cmqsec. since the fraction bf Ci converted by the cell is a constant, (dependent on velocity, viscosity, temperature, etc.), the current it will be directly proportional to Cj. I t is thus a simple matter to use an amperometric transducer to measure concentration as a function of time in flowing streams. Furthermore, it is frequently possible to detect picomole amounts of material eluting from a high performance liquid chromatography column. We have advocated the use of very simple thin layer electrochemical cells for this pwpose.(l, 2). To date cells containing carbon paste electrodes have been employed for the,oxidation of phenolic natural products, drugs, and aromattc amiues (14);compounds which can often be easily separated by HPLC in a few minutes. Figure 1 illustrates a typical experimental arrangement. Chromatographic Components . . ('sing amp~rometricdetection permits one to construct R c o n ~ ~ lhirh e t ~performanct: liquid rhromatczra~hfor less than
' To whom correspondence should be addressed 50 / Journal of Chemical Education
Figure 1. (len)Typical experimentel arrangement for LCEC. All components are clamped to a vertical aluminum stand. Figure 2. (right) Low pressure glass pump $500 (including constant pressure pump, injector, columns, detector, and packing materials, but excluding the usual time base recorder). Such an instrument can be expected to have subnanomole sensitivity for a great many electroactive compounds. Besides the ahsolute economic advantage, an LCEC unit can nrovide sensitivitv and selectivitv"s u ~.e r i o to r analytical systems which are far more costly. The educational opportunities for such an instrument are unique in that the two major analytical tools relying on surface phenomena (electrochemistry and chromatography) can be introduced in a unified way. An additional advantage is the applicability to some interesting problems involving complex sample matrices. Pumps
Due to the use of small-diameter column packing materials and high viscosity mobile phases in HPLC, some form of pumping system i ~ n e c e s ~deliver t o the liquid mohile phase to the column in an accurate and reproducible fashion. Such a system should be capable of givingprecise, resettable flows for pressures usually in the range of 5Q500 psi. It is a common misconception that "high performance" implies very "high pressure" in a liquid chromatography system. Often excellent efficiency is achieved a t less than 500 psi. There are two classifications of pumping systems: constant volume and constant pressure. Constant volume pumps are of two types: reciprocating ist ton or screw-driven svrinee DumDs. These divisions refer to the type of linkage betwee; the piston, which directly applies the linear motion to the mohile phase, and the pump motor assembly. Reciprocating piston constant volume Dumw, such as the Milton Rov "miniPum~"line (- $600). i r e characterized by pulsating fluid delivery due to the re-
ciprocal action of the piston. Pulsations are generally quenched by some sort of hydraulic dampener, but new designs utilizing two matching reciprocal pistons 180° out of phase are aimed a t eliminating this problem. The latter aoproach is not entirely successfd and contributes significantiy to the expense (- $4,000). With low internal volumes and external solvent reservoirs, reciprocating pumps are advantageous in facilitating rapid solvent changeovers. Since this type of pump delivers constant volume, flow is constant over the long run regardless of small changes in flow resistance in the column. The screw-type constant volume pump utilizes a syinge mechanically operated by a motor-driven screw. The greatest advantage of this system is its obviouslv oulseless .. operation, so that constant~olumeflow rates are assured. High pressures (10,000 psi) are possible. The obvious disadvantages lie in the amount of time necessary for mobile phase changes and degassing (to prevent bubble formation in the detector). These operations may require disassemhly of the cylinder. In the case of column occlusion all constant volume pumps will cause the pressure to rise until eventually the weakest link in the system, usually the tubing or a tube fitting, bursts. Because the constant flow pumps do not contain gas under compression, they are not mechanically hazardous even a t several thousand psi. Constant pressure pumps utilize a gas-driven piston to apply pressure directly on the mobile phase in the reservoir and sophisticated models usually involve some hydraulic amplification technique so that very high pressures can he achieved. The greatest advantage of this class of pumping system lies in its pulse-free flow; a t high sensitivites, the elimination of pulsations can dramatically enhance the detection limits. These pumps can be inherently simple in desian and are usually inexpensive to maintain. ~ o w e v i rconstant , flow is possible only if the column flow resistance remains constant. In the case of shifts in packing density or the repeated injection of materials which will eventually clog the column (such as complex biological samples which have not undergone deproteinization), flow rates will drop as time goes on since the pressure is held constant. The necessarv internal solvent reservoirs of these pumps require more"effort in changing the mobile phase than do constant volume reciorocating pumps with external reservoirs. A primary consideration in choosing a pump for LCEC applications is the amount of metallic surface that the mohile phase must contact in passing through the pump. Metals appear to either release or catalyze the formation of materials which may give high detector background currents. Another factor which can he important i s t h e tendency to pick up electrical interferences due to the contact of metal with the electrolyte solution used as the mohile phase. Therefore, the best pump materials for LCEC would appear to he either glass, Teflon-lined stainless steel, or a t the very least, one with a minimum amount of exposed metal, such as a reciprocating constant volume pump with small internal volume and external glass reservoir. We have devised a crude but simole constant oressure pump, capable of pressures up to 180 psi. Made from glass, it is easily fabricated from commonlv available materials bv a glassblower and is satisfactory and safe for use a t the required for 25-50 um beads in 15-25 cm X 2-4 mm i.d. columns thnt hare hac;k pressures of not more rhan 100 psi. The hodv (Fia. 2, was fashioned from two lenerhs of Fischer and ~ o r t e i ~ a b k r ePipe s t Combustion Tube fi4in. i.d., 1in. 0.d.). One piece is formed to give a rounded bottom a t the non-flanged end and serves as ;solvent reservoir. To the other length is attached two pieces of 7 mm o.d. X 2 mm i.d. heavv wall plnss ruhina, flan&d to fit standilrd Altex scientific coiumn e n d - f ~ t t ~ n rThe s . tube runninr t h n ~ u c hthe cao extends to within 1cm bf the bottom of thireserv& pieceahen the sections are joined together. A pressure-tight seal is made via a Teflon O-ring gasket and a triangular two-piece clamp bolted across the seal (available from Fischer and Porter). The
entire assembly may be housed in a steel box for added security. There are several inexpensive constant pressure pumping systems available which make use of a stainless steel reservoir (e.g., from Varian Associates and Gow-Mac Instruments). These systems can he used with LCEC; however. it is essential coelrctrically isolate the pump housingfrmn thenitrogen m k hy using hravy wall plastic tuhina as the nitrocen line. For experiments involving sh& analyticalcolumns and column packings with sufficiently low hack pressures, this type of pressure pump may be all that is necessary. It is inherently simple to use and does well within the limits of applicability mentioned above. However, in the case of much rigorous use and in experiments a t higher pressures, with longer columns and/or smaller packing materials, a reciprocating constant volume pump would offer the best balance among the factors of cost. durahilitv. .. and ease of oneration that i l l s t he considered f i r use in an undergraduate instrumentation lahoraturs. The details of mure roohisticated 1.C pumps have been reGiewed in several monographs ( 7 , B ) .
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Injection Ports and Valves
Injection ports and valves provide a convenient means of applying the sample to the column without interrupting the flow. Operationally analogous to those used in gas-liquid chromatography, septum injectors are usually less expensive than valve types, hut manual injections limit reproducibility, and hoth precision and accuracy degrade a t higher pressures. Syringe damage is always a major economic factor in undergraduate experiments and over the long run a modest injection valve will usuallv.. Drove to be a good investment. Injection valves incorporate either a rotor or slider, hoth of which operate on a two-step, load-injection cycle. During the load step, the slider or rotor is positioned so that the sample is drawn into an iniection chamber or looo while the solvent flow is directed to tile column from the pump. When the slider or rotor is moved to the inject ~osition. oncomine solvent from . . the pump flushes the charnheramtenk unw t h i c o ~ u m nhed. The volume of sample ran be varied hv exrhancine - slidersor loops. From the previous discussion, it may be seen that the syringe offers the most convenient way of injecting variable sample volumes while the valve prqvides superior reproducibility in application of sample to the column. Both features can he realized by the combination of syringe and valve to form a third class of injection port. The syringe can be used to place a variable amount of sample in an injection chamber or l w o a t atmosoheric Dressure which is then flushed onto the column as before. since manipulations with the syringe are not done against hiah oressure. loss of orecision a t this ooint in the procedure will be less than with the septum injector alone, and the svringes . . will also have loneer life exoectancies. These mmhinat~oniniection ports, such as the Waters ASSOcintrs' Model 1'6K lniecror, or the Rheodvne .Model 7105, can withstand pressures"p to 5000 psi w h h still maintaining increased versatility in having both variable sample volumes and greater precision. The greatest drawback is their relatively high price (-51000).
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Columns ~-~ -
In tII'I.C, the n h m n and the manner in which it is parked are critical to optimum chromatographic performance. For this reirson. cdumns are designed with uniform bores and low dead volume end fittings to reduce band spreading. They are usualls fahrirated from elass or stainless steel. The elass rol~-~ umns -are generally use2 a t pressures up to 500 psi, while stainless steel columns can he used a t pressures exceeding 5,000 psi. When stainless steel columns are used with electrochrmiral detectnrs, rare must be takrn to avoid electrical ~nterf~,rrnres since, in effect, the metill a,lumn is a Dart of the electrochemical cell and should not be connected the same common as the detector electronics. Columns are available in lengths ranging from 10-100 cm ~
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Volume 54, Number 1. January 1977 / 51
with inner diameters of 2-5 mm (a centimeter or more for work). " both the canacitv . . and .nrenarative . . . the size affecting resolution of the system. For those packing materials with narticle sizes of 25-50 em. the columns can easilv be drv packed (8).In the case of the smaller highly efficiend5-l0&1 particle sizes, balanced densitv slurries are usuallv packed "nder pressuie (8).Stainless steel frits incorporated in the end-fittings serve as the column bed supports. When a given packing material is no longer satisfactory it can he easily pumped from the column by removing the lower end-fitting. The column is then cleaned and dried before repacking with stationary phase. Although there are a variety of column sizes and packing materials from which to choose, one column size can usually solve a variety of separation problems with proper choice of packing and mobile phase. We strongly recommend the 2.1 mm i.d. glass columns availahle from Altex Scientific. Inc. Two sizes. 25 cm and 50 cm, are satisfactory for most &plications, including all of those described here. Besides the glass columns, an array of satisfactory Teflon tubing, end fittings, unions, injection ports, and valves are availahle from Altex. Several other manufacturers also sell compatible components (e.g., Laboratory Data Control, Pierce Chemical Co., and Durrum Instruments). The component approach to LC using glass and plastic parts is far less expensive than stainless steel. In addition, it is a simple matter to replace parts or redesign asystem in a "tinker toy" fashion. Because of oressure limitations the nellicular stationarv phases (>30 pk)are used with glass cdlumns. The packing materials are expensive (typ $5/g) and therefore the 2 mm diameter columns are chosen to minimize the amount of material required, while giving adequate performance. A 25 cm X 2 mm column requires about 0.7 g of a pellicular stationary phase. Detailed discussions of HPLC columns and packing materials are availahle (7-10); however, the glass-Teflon approach has not been considered in these reviews.
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Electronics
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The electronics necessarv for amoerometric detection ~ ~ a t~ modest sensitivity levels is extremely simple. Several designs hased on our earlier work have been described in the literature (3, 4 ) and a third is now commercially available from Bioanalvtical Svstems Inc. (P. 0. Box 2206. West Lafavette. . . Ind. 47906). ~ e i y i n ~ the o n earlier sources f i r details, we need only indicate the general principles here. Figure 3 illustrates three simple approaches, all involving the same F E T input integrated circuit amplifiers such as are now availahle for a few dollars each (i.e., $5-10). In the first case a follower-type potentiostat is used to maintain a constant potential difference between the reference and working electrodes while supplying sufficient current via an auxiliary electrode. In the second case a voltage follower is used simply to minimize loading of the source or perhaps to convert a three-electrode instrument into one suitable for two-electrode operation. The third circuit implies the realistic assumption that the source will not he loaded by the small currents needed for LCEC. In all three cases an operational current follower is used to convert the current (usually in the nanoampere range) to a voltage suitahle for a strip chart recorder. A more detailed explanation of these circuits for those not familiar with operational amplifiers is available on request. There is a distinct advantage to circuit b for undergraduate experiments in that there is no danger that the feedback loop of the follower might be opened by an unknowing or careless student who forgets to connect the reference el&trode. Allowing the potenti&tat amplifier to run into saturation in this manner can alter the working electrode surface to a degree which makes it useless for further experimentation. ~
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Applications Rather than suggesting one application in great detail, we have chosen six examples from our on-going research which 52 / Journal of Chemical Education
we feel to he particularly suitable. All six involve molecules of hioanalytical interest. Prior to the analysis of an organic compound in a homage. nized or fluid biological sample matrix, several clean-up steps are usuallv necessarv. The first of these commonlv involves the remo;al of proteins from the sample. This is readily accomplished hy the addition of an appropriate agent which causes the proteins to precipitate. A few of the common reagents used for this Duruose are nerchloric acid. ammonium &fate, ethanol, a n d tri&loroac&ic acid. After separation of the proteins. a method is chosen to isolate the-class(es) of co&ounds'of interest, while eliminating as many potential interferences as is oossihle. Often this is accompliihed by one or more solvent extractions. By careful p H adjustment and judicious selection of the solvent. it is possible to he quite selective. For example, extraction of urine a t p H 7 with ethyl acetate will remove most of the neutral aromatic comoounds. while leavine the acidic and basic compounds in the aqueous phase. At p H 2, the organic acids will he undissociated and will partition into ethvl acetate while leaving the bases and strong acids (e.g., sulfuric acid esters) behind. In some cases, liquid-liquid extraction is not effective, as for many biogenic amines. lsolation may then he accomplished using a liquid-solid or liauid-eel extraction. . Alumina, XAD resins, and ion-exchange resins have been used. In thesnalys~iof urinnry and tiiauecntrrholamines hy W E C , fur example, ad\.mtage is tnkrn o i the fact thnt cntrc h d compoundi urcsrlectively udsort~edontoalumina at p H 8.5 and then released a t low (5). Sometimes it is necessary to use several different extraction procedures in series. Even then one mav be left with a comnlex mixture containing several hundred cbmpounds. Analysis of a given compound must then he achieved by thin-layer chromatography, electrophoresis, HPLC, or glc. Combinations of high resolution chromatoma~hictechniaues with more or less specific detectors can the deteimination of a given compound with considerable certaintv. In-general, three approaches are often practical for LCEC sample preparation 1) simple dilution and direct iniection. 2) ~ ~ ~ ~ ~ ~ ~ ~ isolation by extraction prior to injection, and 3) extraction A d tlc prior to injection. The complexity of the matrix relative to detector selectivity for the analyte of interest determines which approach will be satisfactory. When the first option is taken there is usuallv. a nroblem of nermanentlv chaneine . " - the characteristics of the column by contamination with protein and other material in the sample. It is therefore advisable to use a short replaceable precolumn after the injection port (14). The precolumn can then he changed periodically to lengthen the useful life of the expensive analytical column.
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Figure 3. Operational amplifier circuits for electrochemical detection. A. three-electrode configuration; 8, two-electrode configuration; and C. twoelectrode configuration.
Ascorbic Acid Although ascorbic acid is best known as a vitamin and possible therapeutic or prophylactic drug it is also widely used as an antioxidant in food products. While vitamin C can be monitored by lc with uv detection ( I I ) , there are a number of substances which may interfere. A chemically irreversible electrochemical oxidation converts ascorbic acid into dehydroaacorhic acid at low potentials (12,13) making this vitamin well suited to analysis by LCEC. Besides its low cost LCEC offers several other advantages. There is a minimum amount of sample preparation required, and the detector is hoth more sensitive and selective. Satisfactory samples for use in undergraduate experiments include multivitamin preparations, urine, and artificial fruit drinks (e.g., Tang, Hawaiian Punch, etc.). In these cases the experimental procedure consists of a simple dilution with 0.05 M HC104 and direct injection onto the chromatographic column. An important consideration for any undergraduateexperimenr is thr stability of the rearenti and stmdards which an. to be used. We have found that solid samples and standards in most cases can be weighed into drv volumetric flasks and stored for long periods. just prior to-the analytical determination asimple dilution with cold 0.05M HC104 followed by refrigeration or storage in an ice bath affords excellent stability over a 6-hr period. A short duPont Zipax column (2.1 X 250 mm) employing acetate buffer (pH = 4.75) as the mobile phase with an applied potential of 700-800 mV provides adequate selectivity and sensitivity for the above samples. Detailed procedures have been presented elsewhere (2,15,16). Due to the ease of sample preparation, and the chromatographic conditions which are employed it is possible to analyze many samples in three hours. Uric Acid Uric acid is a major constituent of body fluids which is often analyzed calorimetrically in hospital laboratories (17). LCEC has recently been introduced as a possihle reference method for the monitoring of this important metabolite (2,18,I9). An analytical method for uric acid is well suited to laboratory courses designed with the medical technologist in mind. Although high performance liquid chromatography is not commonlv used in routine clinical situations, it is imbortant at the time to introduce state of the &t techniques to students who will he confronted with these tools in the future. Lyophilized control serum or urine standards are available and make convenient samples. Since only 100 fil is needed to perform an LCEC assay the expense involved is minimal. The basic assay for a serum sample is unusually simple: 100 fi1of the reconstituted serum is diluted with 5 ml of water and 4 p1 of the final solution is injected directly onto an anion exchange column. The method is linear up to 1g/l and as little as 1 pg can he detected depending on the chromatographic conditions employed. Since uric acid is extremely stable, it is possible to prepare standard solutions and unknowns in advance and freeze them. The average undergraduate student can prepare a series of standard control samples and analyze an unknown serum sample in an afternoon. Recommended procedural details have been published (19). Acetaminophen Acetamiuoohen (N-acetvl-D-aminonhenol or APAP) is a - . commonly used analgesic available oveithe counter in various liauid Temora) and solid formulatious . (e.e.. NvQuil. " . . Tvlenol. . (e.g., Excedrin, Vanquish, Tylenoi, ~ i n u t a hetc.). , While the analysis of APAP in dosage forms can he accomplished by lc with uv detection, LCEC offers several advantages. Theselectivitv of amoerometric detection oermits one to detect AI'AI' jn the pr&ence of other speries more diffirult tooxidue (re.. snlicvlamidc and r~hcnacrtin~. In addition the extreme sensitiviti affords the'detection of trace amounts of hoth APAP and its toxic hydrolysis product p-aminophenol (PAP). It is important to be able to detect trace amounts of PAP in order to evaluate the quality of a dosage form. The quantita-
tive determination of PAP at the 100 pg level is straightforward. At the normal operating potential for APAP detection (1.0 V versus AgIAgCl) the APAP chromatographic peak will he huge compared to the peak representing trace amounts of PAP. Because the oxidation of APAP is more difficult than for PAP hv about 200 mV. one can discriminate against .. the .ZPAI' wnile n~aintniningexcellent sensitivity for PAP. In this mas tracr amounts of PAP as low as 0.005%in do;iare rorms can he determined. A recent publication describes the procedure for analysis of APAP in formulations, urine, and serum (4). The dosage form assay simply involves a dilution prior to injection on a strong cation exchange resin. The urine and serum assays involve a preliminary ethyl acetate extraction in the workup followed by injections onto a polyamide column. A different stationary phase was used in this case because uric acid interferrd ahen the ration column was used. The polyamide n h n n is suitahlt. for Imth hodv fluid and dosage fiwm assajr. whereas the cation exchange resin is better suited for determining traces of PAP. The samule and chromatoeranhic conditions . .nrenaration . are such that an experiment based on one or two dosage forms or body fluid samples can he completed in one afternoon.
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SalicyluricAcid Everyday we are exposed to amvriad of foreim substances. Many of these chemicals are injested in the foods we eat and in the many pharmaceuticals we use. Our bodies act upon these compounds with a variety of enzymes, converting them to chemicals which are more or less toxic. These reactions can aenerallv he termed detoxification nrocesses. For examnle. ~, the common analgesic aspirin is hydrolyzed to the phenolic acid, salicylic acid (2-hydroxy-henzoic acid). The salicylic acid is in turn hydroxylated to.gentisic acid (2,5-dihydroxybenzoic acid) or coniuaated with a molecule of elvcine to form salicyluric acid-('-hydroxyhippuric acid). ~ m phenolic t acids will undergo . these reactions to varvina . -degrees. - . as well as several other common reactions. In the case of aspirin, salicyluric acid is the maior metabolite. This can he dramaticallv demonstrated by analyzing the aciafraction of urine after thk ingestion of aspirin, in which case the concentration of salicyluric acid will have increased several orders of magnitude. The analysis of salicyluric acid in urine is very straightforward. A strong anion exchange resin is used with acetate buffer in the aqueous mobile phase. Typical results are illustrated in Figure 4. Chromatogram A was obtained for a morning urine sample from an individual who had taken two aspirin tablets early the previous evening. Salicyluric acid (SUA) is responsible for the major peak present ( t =~ 14.2 min). Chromatogram B was obtained for a urine pool from healthy
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Figure 4. Analysis of salicyluric acid in urine. A, emact of urine following aspirin dose (220 ng SUA injected): and B, extract of urine pool from healthy individuals.
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Volume 54, Number 1, January 1977 / 53
individuals. A small peak is consistent with the presence of SUA in normal urine as established by tlc and other techniques. In each case the urine was acidified to pH 2 with 6M HC1 and a 5-ml aliquot was extracted three times with 5-ml portions of ethyl acetate. The ethyl acetate was evaporated to dryness at 40-50°C under a stream of nitrogen. The residue was dissolved in 0.4 M acetate buffer, transferred to a 50-ml volumetric flask and diluted to volume with acetate buffer. Four microliters were injected onto the column. Chlorogenic Acid
During the processing and storage of many food products, undesirable color formation ("browning") often results. The causes of "hrowninp" are the oxidation of phenolic components and the cumpiexation of these compounds with transition metal ions (20). "Browning" is a serious problem which has prevented the acceptance of many potentially important food sources. For example, the sunflower is one of the world's largest sources of vegetable oil and is also a source of high protein flour. Since use of this flower in cooked and processed foods results in dark green and hrown coloration, this high protein source has generally been used only in animal feed and not for human nutrition. Analvsis indicates that chloro~enic acid is the major phenolic component of the flour and may he responsible for "browning" (21,22). Chlorogenic acid is formed from the combination of a molecule of caffeic acid with quinic acid. H
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PH
Sunflower meal can be purchased from most health food stores. The meal is Soxhlet extracted with aqueous ethanol. The extract is concentrated on a rotary evaporator. The residue is brought up in an aqueous buffer and injected onto a polyamide HPLC column (Reeve Angel, Pellonex). One major peak is observed which can easily be quantitated by comparison with a standard solution. An extension of the present example to other common plant phenolics could well provide a good topic for undergraduate honors research. We wish to acknowledge Profs. Ralph Adams, Royce Murray, and Charles N. Reilley for stimulating discussions leading to the preparation of this manuscript. The authors are also indebted to the many undergraduate students who collaborated on developing various assays a t the University of Kansas. Michigan State Universitv. " .. and Purdue Universitv. Developments described were supported by funds from the National Science Foundation. the National Institute of General Medical Sciences, and t h e Showalter Trust Fund. Literature Cited 11) Kinminer, P.T.. Refshaugc.
C. J.. Dreiling.R..and Adam8.R. N.,Anal.Lett.. 6.465 (21 . . Kissineer. P.T..Felice.L. . . J..Rinzii.R.M..Pachls. L. A..md Wenke.D.C..Clin.Chem.. 20. k2'11974). 13) Reirhsuge,C. J.,Kissinger,P.T.,Dreiling,R.,Blsnk.L..Freeman,R..andAdams,R. ~.,~i/esci., an 1 1 9 ~ (41 Riggin,R. M..Schmidt,A. L.,andKiasinger,P. T., J Phorm. Sci. 64.68011975). Ihl Kirringer. P. T.. Rimin. R. M.. Alcorn. R. L.. and Rsu. L.~D.. Rioehem. Med.. 13.299 II9iii. (61 R i g i n , R. M., Rau. L-D., Alcorn. R. L., and Kissinger, P. T., A n d Lett., 7. 791 txe""! \.-.-,. \.",",. ,307Qt
(71 Kirklsnd, J. J. IEdiLnli."ModornPraetieeofLiquidChromatography." Wiley-Intorscience, New York. 1971. 18) Snyder. L. R.. and Kirkland. J. J.,''lntroduetIon to Modern Liquid Chramatagraphy," ~ i l e y l ~ t eNew ~ ~York. ~ i 1974. ~ ~ ~ ~ . (9) Leivh. R. E.. and DP Stelano. J. J.. J. Chmmotog. Sci. 11.105 119731 i,l n ~ l Lwh. R. J..RPs. D~uPI.. . 4. ~ , . . JuIY. .. 1 9 7 4... ~ 2 i l l ] Majors. R. E.. Americon Lnborofory. 13, IOctaber 19751. (121 Perone,S. P.,and Kretlaw, W . J.. A n d Cham.. 38,1760 119701. i1:lI Adams, R. N.. "El~trochemistryat Solid Electrodes.). Marcel Oekker. New York.
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1909 .....
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The presence of the acrylic acid group conjugated with the aromatic ring facilitates oxidation and makes this compound well suited to analysis by LCEC. Chlorogenic acid is found in most fruits and vegetables and in large quantities in coffee and tobacco. The high level of chlorogenic acid and the absence of interferences make sunflower seeds a good choice for an undergraduate experiment (23).
54 1 Journal of ChemicalEducation
114) Pachla. L. A.,snd Kissinper. P. T..Annl. Chem., 48. 237 11976!. 1161 Thrivikraman, K. V.,Refshauge,C.,snd Adams, R. N.,Life Sci, 15,1335 11974). 1161 Pachls. L.A.,andKiaiin~er,.PT.,Anoi. Chern. 48,364 119761. 1171 Faulkner. W. R..snd King,J. W..in"FundamontalsofClinicalChemistry."(Editor: Tie*. N. W.).W. B. Saundors Co.. Philadelphis. 1970, pp 726729. 1181 Pachla, L. A.,and Kissinger,P. T,Ciin. Chim.Acto, 59.30911975). 1191 Slaunwhite.0. W., Pach1a.L.A..Wenke,D.C.,andKissinger,P.T..Clin. Chpm., 21. 1427 11975). im M ~ P AW C.. and Parnia. H.A. B.. "Fmd Bmunine as a Polvohcnol Reaction" in
