Douglas W. Bastian, Rochelle L. Miller, Allan G. Halline, Fred C. Senftleber', and Hans ~ e e n i n g Bucknell University Lewisburg, Pennsylvania 17837
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High Performance Liquid Chromatography Of Urinary Compounds An undergraduate experiment
High performance liquid chromatography (LC) is a relatively new analytical technique which has experienced an enormous increase in reseurch activity during the last 10 years. The subject has heen re\.iewed in several recent hooks ( i , 2 J and naurrs I3 . 5).'l'he drvelonment olsoohisticated LC instrumentation during recent years has made LC a powerful complement t o gas chromatography (GC). The advantage of LC is that thermally unstable, nonvolatile compounds which cannot he eluted hv GC can freouentlv be seoarated hv LC. since columns are operated a t ambient temperature. Hieh oressure ion exchanee LC has been used extensively for the separation and determination of organic acids, carbohydrates, amino acids, indoles, and many other components found in physiological fluids such as urine and blood plasma (4. . . 6.. 7). . These comuounds are of ereat interest because of their association with the molecul~rbasis of disease. Separations of biochemically active compounds have been obtained by gradient elution on a basic anion exchange resin a t a column inlet pressure of 200011000 psi. Nearly 150 components have been clearly resolved during one single run (6,7). One of the most important breakthroughs in LC during the last few years h ~been i the development ul packing materials in which the orranic stationary phase is permanently honded to a high surface area porous siiica support (3).Such chemically bonded stationary phases do not easily strip from the support particles, they are thermally and hydrolytically stable, and they can be used in either isocratic or gradient elution. A group of materials, which is especially suitable for the elution of aqueous samples (i.e. physiological fluids) is the so-called "reversed phases" (8).This type of packing contains a nonpolar, chemically honded stationary phase such as the saturated octadecyl hydrocarbon group (CIS). Nonpolar components which have a high affinity for the Clg environment elute with relativelv lone retention times. whereas the more oolar materials (iniluding ionic compounds) elute early. ~ h use k of madient elution. emolovine a D o h nonwlar solvent Dair nt (e.g. water methan&,kifo&en result i n k x c e ~ ~ eresb~ution of c o m ~ l e xmixtures. It has been shown in many recent reports tha; "reversed-phase" LC is rapidly lwcon&g the method of chuice for the separatiun and detectiun of hiochemicals in physiological fluids. LC is no douht being utilized more and more in undergraduate analytical chemistry courses; although only two reports have thus far been pul)lished (9, 10, 131. We report an undergraduate instrumental analysis experiment in which a mixture of five clinically important urinary compounds can he seoarated and determined bv reversed-ohase LC. The compounds include uracil, uric acid, xanthine, allopurinol, and nicotinamide. T h e senaration is achieved hv use of isocratic elution and ultraviolet (uv) detection. T h e experiment is designed to demonstrate how LC can he used as a rapid and efficient analytical method, and how various chromatographic parameters such as theoretical plates and height equivalent
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'Present address: Department of Chemistry, Providence College, Providence Rhode Island 02918. ?4tocksolutions were made up two decimal Dlaces in order to provide at least three significant figures in the results. 3The amount of base should be kept to a minimum in order to prevent the hydrolysis of nicotinamide to nicotinic acid. 766 1 Journal of Chemical Education
per theoretical plate can be determined. The experiment d s o illustrates the technique of peak identification hy use of uv spectrophotometry. Instrumentation The liauid chromatoeraoh utilized for this exmriment consisted'of a solvent r&ekoir equipped with a magnetic stirrer. a high Dressure solvent deliverv svstem (Model 711. L a h o r k o r y ~ ~Control, ta Riviera Beaih, FL 334'04), a 100 pi samule l o o ~iniection svstem (Model 70-10. Rheodvne. Berkeley, Cbr 9 k 1 0 ) , a 36 cm X 4 mm (i.d.), t'hermostked (25°C) analytical column packed with 10 pm silica particles honded with octadecylsilane (p-Bondapak-Cia; Waters Associates, Inc., Milford, MA 01757), a Laboratory Data Control uvMonitor (254 nm), and a Perkin-Elmer Model 56 linear recorder. The entire flow system was made of stainless steel. and connections (beyond the injector) were made using 1.6 mm X 0.23 mm (id.) stainless steel tubing, and low volume Swagelok fittings. Acetic acid (0.0500 M),buffered to a p H of 4.50, was used as the eluent. The moving phase was stirred continuously in order to remove dissolved gases. The flow rate was 2.1 mllmin, and the column inlet pressure was 2000 psi. A Cary 14 uv Recording Spectrophotometer (Varian, Palo Alto, CA 94303) was used for LC peak identification.
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Procedure Solutions Stock solutions20f uracil (2.43 mgll00 ml),uricacid (11.74 mgI100 ml), xanthine (4.67 mg1100 ml), allopurinol (5.43 mg1100 ml), and nicotinamide (18.09 mg1100 ml) were prepared by dissolving the respective compounds (Sigma Chem. Co., St. Louis, MO 63178) in doubly distilled water and adding several drops of 40%NaOH to enhance solubility.3 Samples were refrigerated (7-8%) until ready to use. Uric acid standards were prepared on the same day they were used because of their known instability in aqueous solution ( 1 1 ) . Uracil was chosen as the internal standard heeause of its observed stabilityin solution,and heeause of its short retention time. An aliquot (5.00 ml) of uracil stock solution was pipetted into each of five 100 ml volumetric flasks. Varvine , ..aliauots . (1-5 ml) of each of the four other srwk s~lutiunswere then a h pipetted inro the volumetric flasksand each wlutiun was dilursd to the mark with doullly distilled water. Compment concrnrmtiona ranged from 0.05-0.9 my. 1110ml. Unknown solutions containing an identical amount of the internal standard were prepared similarly. Results Analysis of Unknown Solutions A typical chromatogram showing the five component separation is shown in Figure 1. It can be seen that the total analysis e m he completed in 10 min. Peak area ratios for each component in the five oeak areas standard solution trial runs were calculated hv. dividing,.the . of the individud components by the peak area of the internal rtandard. The ratios were plutted against cmnponrnr nmrentratinns t o give linear working curves as shown in Figure 2. Correlation coefficients for each plot were 1.00. Peak area ratios were measured for each component in the unknown sample, and the concentrations of the individual compounds were read from the calibration plats. Average relative standard deviations for six determinationsof each of the four components (0.1 to 0.7 mgll00 ml) in synthetic mixtures werere: uric acid, 2.2%; xanthine, 4.7%; allopurinol, 2.5%; and nicotinamide, 1.9%. ~~~
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Uracil
Nicotinamide
Figure 2. Workina- curves tor allopurinol,xanthine, nicotinamide, and uric acid. lntemai standard: uracil. I
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10 TIME ( M I N ) Figwe 1. Liquidchromatogram twtheseparationof 0.12 pguracil. 0.47 ~ w i c acid, 0.19 pg xanlhine. 0.22 pg ailopurinol. and 0.74 pg nicotinamide. 4
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Calculation of N and HETP The efficiency of a liquid chromatographic column is conveniently described by its number of theoretical plates ( N ) ,a quantity which can beestimated from theretention time (t.) and theoeakwidth (w). N = Ifi (t./w)2. eauivalent oer theoretiEd nlate tff) is . .. . The heieht u . anutherway I,y whir.hcolumnrfficlenry;an hedeioib&. Ti = /..Ar. (I. is thecolumn length in cm.J 'IIYPICRI r m u l t ~10r the rercnled-phase column varied from 150Q2000 plates a t a flow rate of 2.0 mllmin depending upon which component was chosen. ~
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Peak Identification by UV Spectrophotometry When seoamtine ..cnm~lexmixtures hv. LC.. it is freauentlvnecessarv . .
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tu idrnr~fythe rhromntographic peaks. This can be accompl~ahedby collecting thnr purtnm ofthe moving phase which conrnms t h desired ~ component. The collerted samplescan rlwn be conwnientl!. ~denrified by uv spectrophotometry. Appropriate allowance must he made for the dead volume hetween the detector flow cell and the fraction collector. This technique can be illustrated by collecting any one of the components in the mixture. A uv spectrum is obtained for the eluted fraction by using the moving phase (0.0500 M acetic acid) as a spectrophotometric reference. The result is compared against the spectrum of the authentic compound recorded under identical conditions; i.e. the compound was dissolved in the moving phase, and the pH was readjusted to 4.50. The resulting spectra matched those of the atandards and showed characteristic ahsorotion maxima a t 258 nm (unc:l), 292 nm (uric mid!, 26: nm txanrhine,, ?f,l nm ralh~purindJ. and 262 nrn (n~rormnmidr).
Analysis of Urine
The final portion of this experiment involved the analysis of urine. A urine sample (5 ml) was diluted to 15 ml with doubly distilled water and the urinary proteins were precipitated by treating the sample with 1.5 ml of ZnSOa solution (100 gfl) and 0.8 ml of 0.1 M NaOH (22).This solution was centrifuged for 10 min a t 5000 rpm. The supernatant liquid was filtered through a 0.2-pm Nalgene membrane filter (Syhron Corp., Rochester, NY 14602) to remove any remaining particulate
Figure 3. Liquid chromatogram tor urine.
matter. One milliliter of the filtrate was diluted to 10 ml. The sample was then injected directly into the column without further pretreatment. Twenty minutes after injection of the sample, the run was stopped, and the column was purged by pumping with 1:l methanol-water for 15 min, followed by pure methanol. A typical chromatogram for a pooled urine sample is shown in Figure 3. Individual compounds were identified on the resulting chromatogram by uv spedrophotometry and comparison to retention data published previously (8). Because several of the peaks in the urine chramatogram are known to contain more than one component (8).quantitative Volume 54, Number 12, December 1977 1 767
determinations were not attrrnpwd. Hmvcvrr, several o i t h r
