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Anal. Chem. 1980, 52, 1228-1233

Table 11. Aromatic Retention Data compound benzene naphthalene fluorene anthracene chrysene benz[u]anthracene benz[a]pyrene retention time
3:OO

the fused silica capillary column.

LITERATURE CITED
Snyder. L. R.; Bueli, B. E. Anal. Chem. 1968, 4 0 , 1295-1302. Snvder. L. R. Anal. Chem. 1989. 4 1 . 314-323. Scfiiikr', J. E.: Mathiason, D. R. Ana/: Chem. 1977, 49, 122551228, McKay, J . F.; Weber, J. H.; Latham, D. P. Anal. Chem. 1978, 48, 891-898. Jones, A. R.; Guerin, M. R . : Clark, 8. R . Anal. Chem. 1977, 49, 1766-1771. Jewell, D. M.; Roberto, R. G.; Davis, B. E. Anal. Chem. 1972, 4 4 , 2318-2321. DiSanzo, F. P.; Siggia, S.; M e n , P. C. Anal. Chem. 1980, 52, 906-909. Suatoni, J. C.; Swab, R. E. J . Chromatogr. Sci. 1975, 13, 361-366. Suatoni, J. C.; Garber, H. R. J . Chromatogr. Sci. 1976, 1 4 . 546-548. Suatoni, J. C.; Swab, R. E. J . Chromatogr. Sci. 1978, 74, 535-537. Dark, W. A.; McFadden, W. H.; Bradford, D. L. J . Chromatogr. Sci. 1977, 15, 454-460. Schomburg, G.; Hussman, H. Chromatographia, 1975, 8 , 519-530. Robilhrd, M. V. Ph.D. Dissertation, University of Massachusetts, Amherst, Mass., 1979. Robiihrd, M. V.; Siggia, S.; Uden, P. C. Anal. Chem. 1979, 57, 435-439. Mourey, T. H.; Siggia, S.; Uden, P.C.; Crowiey, R. J. Anal. Chem. 1980, 52, 885-891.

3:40 4:51 5:19


8:24

8:26

13:55

the shale aromatic fractions were composed mainly of 1-4 ring systems. The finding corroborated that previously noted by Mourey e t al. (15).

ACKNOWLEDGMENT We are grateful to Mark T. Atwood of the Oil Shale Corporation (TOSCO) and to the Laramie Energy Technology Center for the provision of shale oil samples. We also thank S.B. Dave of Johns Manville Corporation for provision of HPLC substrates and helpful advice, and D. Gere and B. D. Quimby of Hewlett-Packard Corporation for making available

RECEIVED review February 19, 1980. Accepted April 21, for


1980. This work was supported by the National Science Foundation Grant CHE74-15244.

Determination of Limonin and Related Limonoids in Citrus Juices by High Performance Liquid Chromatography
Russell L. Rouseff" and James F. Fisher
Florida Department of Citrus, AREC,

P.O.Box

1088, Lake Alfred, Florida 33850

Four maior citrus limonoids are separated in less than 15 min on a nitrile (CN) column in the normal phase mode using a ternary solvent system. This is the first high speed liquid chromatographic technique to separate limonin from related juice limonoids. The detection limits at 207 nm are 3.5 ng of deoxyllmonln or obacunone and 10 ng for llmonin or nomllln. Maximum column efficiency is observed at 40 ' C . Optimum flow in terms of maximum column efficiency and maximum detector response per unit time occur at 1.0 mL/min. Limonin, in a 5.0 ppm grapefruit juice, can be determined with a relative standard deviation, RSD, of 2.2% ( n = 5). I n a low limonin ( X = 0.75 ppm) orange juice, the limonin RSD is 3.2% ( n = 5). Nomilin can be determined in the same juices with RSDs of 8.9 and 9.4%, respectively.

Limonin, an intensely bitter triterpenoid dilactone, is the major bitter component in several varieties of oranges ( I , 2 ) and one of the important bitter components in grapefruit ( 3 ) . T h e richest source of citrus limonoids is found in the seeds, and limonin is the limonoid found in highest concentration. A tasteless precursor to limonin exists in the juice sacs. This precursor has been identified as limonate A ring lactone ( 4 ) . After the juice is expressed from the fruit, the tasteless precursor comes in contact with the acidic juice and is slowly lactonized into the bitter limonin. The delayed bitterness is most noticeable in the juice of the navel orange. While some bitterness is desirable in grapefruit juice, excessive bitterness is considered objectionable. Any bitterness in orange juice is undesirable. Therefore, limonin can be detrimental to juice
0003-2700/80/0352-1228$01 .OO/O

quality, and a rapid objective method for measuring limonoid bitterness in citrus juices would be extremely important to the citrus industry. Other studies have reported the presence of several limonoids besides limonin in citrus (5-7). Nomilin is reported t o be bitter while deoxylimonin and obacunone are tasteless (7, 8). Therefore, any method designed to quantitate limonoid bitterness should be able to distinguish between bitter and nonbitter limonoids as well as between limonoids of various degrees of bitterness. Several thin-layer chromatographic (TLC) procedures to estimate limonin values in citrus have been reported ( S I I ) . They are characterized by involved sample preparation and/or the inability to distinguish limonin from other limonoids. Quantitation is achieved by visual comparison with standards and is subject to individual interpretation. A gas-liquid chromatographic (GLC) (12) as well as a fluorometric (13) method has been reported but they also require lengthy sample preparation and are specific for limonin only. Early high-performance liquid chromatographic (HPLC) methods were designed t o measure limonin only, as it was not known if other limonoids existed in juice. The first method (14) used a 10-pm porous silica packing with a refractive index detector. The solvent system was chloroform-acetonitrile (95:5 v/v). However, because of the low sensitivity and the poor thermal stability of the refractive index detector, the method was abandoned in favor of a reverse phase system employing a water-methanol solvent system with chemically bonded nitrile (CN) column packing material and a variable wavelength UV detector set a t 210 nm (15). There were several disadvantages with this procedure that made it unsuitable for
0 1980 American
Chemical Society

ANALYTICAL CHEMISTRY, VOL. 52, NO. 8, JULY 1980 routine analysis. After repeated injection of juice extracts, the base line became very irregular owing to late eluting peaks from previous injections. T h e retention time of limonin decreased with each subsequent injection. All solvents or solvent combinations employed were unsuccessful in restoring t h e column t o its original condition. After approximately 200 injections, t h e column was essentially useless, apparently poisoned by some component in t h e juice extracts that was irreversibly retained by t h e column. T h e objective of this work was t o develop a stable chromatographic method capable of separating and quantitating limonin a s well as other limonoids in citrus juice with good sensitivity a n d precision. EXPERIMENTAL Reagents a n d Standards. All chromatographic solvents as well as the chloroform used in the extractions were of high purity TTV grade purchased from Burdick and Jackson Lab., Inc. Limonin was extracted from ground, defatted grapefruit seeds using a method similar to that of Emerson ( 5 ) . Crude limonin was purified by repeatedly dissolving it in methylene chloride, then recrystallizing it with methanol, mp 295-299 "C dec. -124.2 "C. The other limonoids were supplied by Shin Hasegawa of the Western Regional USDA Laboratory in Pasadena, Calif. Chromatographic analysis of these standards indicated that they could be used without further purification. Standard solutions were prepared in acetonitrile. Working solutions were prepared by making dilutions with the mobile phase solvent. All standard solutions were refrigerated when not in use. Apparatus. A DuPont (Wilmington, Del.) model 848 pump equipped with a Valco Valve (Houston, Texas) 20-pL loop injector and a Tracor (Austin, Texas) model 970A variable wavelength detector set a t 207 nm were used as the chromatographic system. The detector had a 6-nm band pass and an 8-pL sapphire window flow cell. A 1-V full scale absorbance output was used with a 3-s time constant. Peak areas were determined by integration using a Spectra-Physics (Santa Clara, Calif.) minigrator or SP-4000 chromatographic data system. A Perkin-Elmer (Norwalk, Conn.) model 56 strip chart recorder was used to record the chromatograms. Chromatographic Conditions. Citrus limonoids were separated isocratically using DuPont's Zorbax CN 5-pm column, 25 cm X 4.6 mm i.d. The mobile phase consisted of hexane, isopropanol (IPA), and methanol in varying proportions. A ratio of 11:12:2 (v/v) was used with a flow rate of 1.0 mL/min when optimum resolution and sensitivity were required. For routine quality control purposes, a mixture of 6:3:1 (v/v) with a flow rate of 1.5 mL/min was found to be satisfactory. (Heptane was occasionally substituted for hexane when irregular base lines were observed with only a slight decrease in column efficiency.) Each solvent was filtered, then degassed separately by applying a mild vacuum while it was placed in an ultrasonic bath for approximately 3 min. Recycle Chromatography. A Waters Associates (Milford, Mass.) M-6000 pump with a U-6K injector and a Tracor 970A variable wavelength UV detector set a t 207 nm was used for the recycle experiment. The mobile phase consisted of isopropanol-hexane (7:3, v/v) at a flow rate of 1.0 mL/min. A Waters p-Bondapak CN column, 30 cm X 4.6 mm, was used to separate limonin from the other juice components. Temperature a n d Flow Rate Studies. These studies were carried out with a DuPont Zorbax CN column, 25 cm X 4.6 mm and the same pump, injector, and detector system used in the recycle experiments. The mobile phase consisted of isopropanol, hexane, and methanol (60:3010 v/v). Column temperatures were maintained using an insulated water jacket connected to a GCA/Precision Scientific model 253 circulating constant temperature bath. Column temperatures were maintained at fO.l O C of the desired temperature. Values reported are the average values of triplicate analyses. Sample Preparation. Approximately 40 mL of single strength grapefruit juice was centrifuged for approximately 15 min to remove the pulp and most of the suspended material. Exactly 25 mL of the supernatant grapefruit juice or 35 mL of the supernatant orange juice were placed in a 125-mL separatory funnel

1229

, I -

12

16

21)

24

28

TIME imin)

Figure 1. Chromatogram of the four limonoid standards (100 ppm each) obtained under reverse phase conditions. Chromatographic conditions: p-Bondapak C column at 25 OC,mobile phase; watermethanol (4555, N v/v); flow, 1.5 mL/min; pressure 1500 psi: injection volume, 15 I.LL

and extracted with three 10-mL aliquots of chloroform on a shaker (Kraft apparatus) set for 4 min at minimum agitation. (Smaller amounts of juice may be used with a slight loss of precision.) The layers were allowed to separate and the chloroform was drawn off the bottom. The combined chloroform layers were evaporated t o dryness in a Bdchi rotary evaporator a t 40 "C under vacuum. Finally, the residue was dissolved in 2.0 mL of mobile phase and filtered through a 1.2-pm Millipore filter before injection. Identification a n d Quantitation of Peaks. The peaks of interest were identified by five methods. First, retention times of the peaks of interest were compared with retention times of the standard limonoids. Standard limonoids were then added to the sample and rechromatographed t see if the peak of interest o increased in height. The resulting peak was also analyzed for symmetry. Chromatographic peaks were repeatedly collected, concentrated, and spotted on TLC plates. The plates were developed using ethyl ether-acetic acid-water (15:3:1) and R, values were compared to those of authentic compounds and published literature values ( 4 ) . Each peak was tested with Ehrlich's reagent (p-dimethylaminobenzaldehyde) which if positive is indicative of limonoids. Finally, UV spectra were obtained and compared to absorption maxima in the literature. Precision Studies. An 8-02. can of single strength orange juice was thoroughly mixed before five aliquots of juice were removed and analyzed in the usual manner. The juice was packed in Florida on December 31, 1979. The grapefruit juice used was a composite of four commercial, single strength juices from Florida. They were packed between January 16--18,1980. Equal volumes from the 48-02. can were blended for 1 h with a magnetic stirrer before five aliquots of juice were removed and analyzed in the usual manner. Averages of triplicate analyses were used to determine the concentrations of limonoids in each aliquot. A DuPont Zorbax CN column was used with a mobile phase of heptane, isopropanol, and methanol (11:12:2 v/v). Flow rate was 1.0 mL/min and the column was heated to 40 OC. A Waters Associates WISP automatic sample injector was used to introduce 50 and 25 pL of the orange and grapefruit juice extracts, respectively, to the head of the column.

RESULTS A N D DISCUSSION N o r m a l vs. R e v e r s e d - P h a s e C h r o m a t o g r a p h y . T h e decision t o approach t h e separation of citrus limonoids via normal phase chromatography was based upon the type of problems encountered when t h e separation was carried out in t h e reverse phase mode. Figure 1 illustrates the low efficiency separation of four limonoid standards on a CN column in t h e reverse phase mode. It should be noted that while deoxylimonin and obacunone are well resolved, the resolution between limonin and nomilin is poor. Alkyl bonded phase

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ANALYTICAL CHEMISTRY, VOL. 52, NO. 8, JULY 1980

I
W

Table I. Effect of Solvent Composition o n Limonin Retention Timea mobile phase composition %P A % hexane 50
55

I1

limonin t R , min 26.7 24.0 21.8 19.5 17.1 16.0 15.2

60 65 70 75 80
a

50 45 30 35 30 25
20

Conditions: Sample: 30 ppm limonin standard, inj. volume: 20 pL, flow rate: 1.0 mL/min, column: Zorbax CN, pressure: 1000-1400 psi, temperature: 40 'C, detector: 210 nm/0.05 AUFS. Table 11. Temperature Effects on Retention Time and Column Efficiencya teyperature,
C
tR:,

20
25
0

IO TIME ( m i n )

15

30 35 40
45 50

Flgure 2. Chromatogram o the four limonoid standards (100 ppm each) f obtained under normal phase conditions. Chromatographic conditions: Zorbax CN column at 40 OC; mobile phase, isopropanokheptane: methanol (12:11:2, v/v); flow, 1.0 mL/min; pressure, 500 psi; injection volume, 15 KL

min 15.93 13.67 11.97 10.51 9.30 8.27 7.44

N , plates/ 25 cm 827 1950 3560 4910 5250 4440 2030

NltR

51.9 139 297 467 565 537 273

a Mobile phase: isopropanol, hexane, methanol (6:3:1, v/v); flow rate: 1.0 mL/min.

columns ((28, CIS) used in the reverse phase were more efficient, but all limonoids could not be adequately resolved from other components in juice samples with these columns. However, low efficiency and poor resolution were not the primary reasons for seeking an alternate approach to the separation of these compounds. The major problem with the reverse phase separation was its chromatographic instability and the ease and speed with which the column was poisoned. T o determine if the component(s) responsible for the column poisoning was nonpolar, it was proposed that the limonoids be separated using normal phase chromatography. If the compounds responsible for the poisoning were nonpolar, they should have a greater affinity for the nonpolar solvent than for the weakly polar column. Thus, they should elute a t the solvent front and not be retained. There should be very few polar compounds in a chloroform extract of juice to elute a t long retention times under these conditions. Many of the solvents normally used in polar partition separations could not be utilized because their UV solvent cut-off was higher than the 207-nm absorbance maximum of limonin. Fortunately, a reasonable separation could be achieved with isopropanol and hexane, both of which are reasonably transparent a t 207 nm. Figure 2 illustrates the normal phase separation of four limonoid standards. In comparing the two separations, it should be noted that the normal phase separation gave much greater resolution and required less time than the reverse phase separation. As expected, the elution order of the four limonoid standards is reversed. All four limonoids are well separated and nomilin is cor ,pletely resolved from limonin. R e t e n t i o n T i m e vs. Solvent Composition. Isopropanol is usually considered the polar modifier when used in normal phase systems and is used in small amounts (typically 2-15%). As seen in Table I unusually high proportions of isopropanol were required to elute limonin. Even with 50% isopropanol, it took over 28 min for the limonin peak to elute. However,

typical of normal phase separations, limonin retention time continued to decrease as the proportion of polar modifier in the mobile phase was increased. As the mobile phase isopropanol proportion was increased above 70%, isopropanol became increasingly less effective in reducing the retention time of limonin. In looking for a supplementary solvent to reduce the retention time of limonin to more reasonable values, it was found that small amounts of methanol (5-lo%), when added to IPA and hexane, greatly decreased the retention time of limonin and substantially improved the resolution between the limonoid standards. Thus, a combination of IPA and methanol was used to adjust the retention time of limonin to its desired value. T e m p e r a t u r e Effects. Temperature effects have often been overlooked in modern liquid chromatography. Like solvent composition, temperature is an experimental condition to be optimized. Snyder (16) and Scott and Lawrence (17) have shown that temperature programming can be used to effect a separation in the same manner as solvent programming. Other investigators (18,19) have shown that changing column and mobile phase temperature can improve resolution and alter elution order. Elevating column and mobile phase temperature should reduce the mobile phase viscosity and increase the rate of mass transfer between the mobile and stationary phases. As shown in Table 11, the retention time of limonin decreased with increasing temperature. As expected, column head pressure decreased from 2400 t o 1500 psi over the temperature range studied. Column efficiency as measured by N, the number of theoretical plates generated by the 25-cm column using limonin standards, increased with increasing temperature up to 40 "C where it reached a maximum of 5200 plates. As seen in Table 11, it fell off rapidly a t temperatures above 40 "C. Since temperature affects both retention time and column efficiency, the ratio of N / t R was calculated to determine the temperature a t which column efficiency was maximized and retention time minimized. These values are shown in Table 11. A t 40 "C, both absolute column efficiency, N , as well as

ANALYTICAL CHEMISTRY, VOL. 52, NO. 8 , JULY 1980

1231

Table 111. Limonin Flow Rate Characteristics at 4 0 " C


flow rate,

mL/min
0.5 1.0 1.5 2.0 2.5 3.0
a

retention time, t R , min


23.06 11.67 7.67 5.13 4.67 3.77

pressure, psi
350 1000 1400 1600 2000 2500

peak area,
A , pV.s
4 60 375 245 188 151 122

peak height, mm
11 5 163 141 130

peak area/
t R ~

Pv

plates/min
362 504 2 24 144 137 158

NltR

pm/plate
29.9 42.5 146 303 388 418

118
116

3.33 5.36 5.33 5.47 5.42 5.43

HETP = height equivalent t o a theoretical plate.

N/tR were a t their maximum values. Thus, all subsequent chromatograms were run at 40 "C to reduce the column head pressures (thus reducing equipment wear and frequency of repair), reduce analysis time, and maximize column efficiency. Flow R a t e Effects. Column efficiency as measured by N or H E T P , the height equivalent of a theoretical plate, is a direct function of the rate of solvent flow relative to the rate of mass transfer between the mobile and stationary phases (20). Since the rate of mass transfer is fixed a t a given temperature, the column efficiency decreases as the mobile phase flow rate increases. As illustrated in Table 111, other factors such as pressure, retention time, peak area, and peak height are also affected by flow rate. While it would be desirable to reduce analysis time by increasing flow, the price paid in decreased column efficiency, decreased sensitivity, and increased pressure might not justify the savings in time. Bakalyar and Henry (21) reported peak area and retention time to be inversely proportional to flow rate and peak height relatively independent of flow rate. However, their study was done over a narrow range of flow rates (1.76 to 2.00 mL/min). As shown in Table 111,similar relationships were observed for pressure, retention time, and peak area. However, peak height was found to be inversely proportional to flow rate between 1.0 and 3.0 mL/min. Below 1.0 mL/min, peak height decreased with decreasing flow rate. Therefore, maximum sensitivity in terms of peak height can be obtained a t 1.0 mL/min. T o determine optimum flow conditions where peak area is maximized and retention time, tR, is minimized, the ratio of peak area/tR is plotted as a function of flow rate. As illustrated in Table 111, peak area/tR is almost constant between 1.0 and 3.0 mL/min but falls off sharply a t 0.5 mL/min. Thus, in terms of peak area per unit time, optimum flow rate would be anywhere between 1.0 and 3.0 mL/min. The relationship between column efficiency (as measured by H E T P , where H E T P = 250000 pm/N) and flow rate is shown in Table 111. As expected, maximum column efficiency is achieved a t minimum flow rates (i.e., H E T P is a minimum and N is a maximum a t 0.5 mL/min). Thus, the optimum flow rate for the Zorbax CN column operating a t 40 "C in terms of sensitivity and efficiency is observed a t 0.5 mL/min. However, if time is considered and a time dependent efficiency defined by N/tR is used to determine optimum operating conditions, then optimum flow no longer occurs at 0.5 mL/ min. As shown in Table 111, the maximum column efficiency per unit time occurs a t 1.0 mL/min. Slower flow rates will increase column efficiency slightly but only with greatly increased analysis time. Higher flow rates could be used with some sacrifice in peak area (sensitivity) and a considerable loss in column efficiency. For routine juice analyses, flow rates u p to 2.0 mL/min can be used if analysis time becomes more important than resolution and sensitivity. Peak height would be more suitable for quantitation purposes under these circumstances as it is less affected by flow rate (see Table 111). R o u t i n e Analysis. In juice samples, the major limonoid found is always limonin. Smaller amounts of nomilin are observed. Obacunone and deoxylimonin are not usually ob-

E
t IC

cv

LL

v)

d 0 0

II
0

. .
5

TiME (min)

io

Flgure 3. Chromatogram of commercial grapefruit juice containing 5 ppm limonin and 0.4 ppm nomilin run under quality control conditions: Zorbax CN column at 40 "C; mobile phase, isopropano1:heptane: 1, methanol (6:3: v/v); flow, 1.5 mL/min; pressure, 900 psi; injection volume, 25 p L

served. Therefore, to decrease analysis time for routine applications a t the expense of the resolution between limonin and deoxylimonin, as well as between nomilin and obacunone, the composition of the mobile phase was altered. The relative portions of the polar modifiers, isopropanol and methanol, were increased to increase the affinity of the limonoids for the mobile phase. Resolution between the two pairs of limonoids was sacrificed in the interest of speed because abacunone and deoxylimonin were normally not observed and the additional resolution was unnecessary. T h e resulting chromatogram for a typical grapefruit juice extract is shown in Figure 3. There is still excellent resolution between nomilin and limonin and the chromatographic analysis is complete within 10 min. P e a k Identification a n d Purity. To determine if another compound in addition to limonin might be found under the limonin peak, recycle experiments were conducted. After seven passes through the chromatographic column, the limonin peak became shorter and broader because of dilution effects but remained perfectly symmetrical. There was no evidence of a second peak. If another compound from the juice extract had coeluted with limonin i t should begin to separate from the limonin peak with each successive pass through the column.

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ANALYTICAL CHEMISTRY, VOL. 52, NO. 8, JULY 1980

Table IV. Summary of Juice Precision Study Orange Juice Results average limonin std. dev., sample OJ-1 OJ-2 OJ-3 05-4
05-5
S

% RED, s IX

0.76 0.72 0.74 0.73 0.78 4.9 4.9 4.9 5.1 5.1

0.015
0.015

2.0
2.1

0.006 0.030 0.020


0.115

0.8 4.1 2.6

average nomilin c o n c n 2 ppm, ( X) 0.032 0.026 0.033 0.031 0.030 0.28 0.24 0.31 0.29 0.27

std. dev.,
S

% RBD,

six
14.7
15.8 12.1

0.0047
0.0041

0.0040 0.0041 0.0061 0.026 0.031


0.010

13.2 20.3 9.2 12.9 3.2


7.2

Grapefruit Juice Results GFJ-1 GFJ-2 GFJ-3 GFJ-4 GFJ-5 3. T h e UV spectra from the collected limonin and nomilin peaks compared favorably with literature descriptions (22,23). Unfortunately, the UV spectra of these compounds are not very informative, consisting primarily of end absorption in the region of 200 nm. The material from the collected peaks along with authentic compounds was spotted on TLC plates and developed according to the procedure of Maier and Beverly (4). Ehrlichs reagent was used to visually determine the location of the spotted material. This reagent has been used as a specific test for limonoids (4,9),but it can react with other compounds (primarily those with a furan ring). However, Dreyer (7) has reported that limonoids will give a characteristic color. In each case, only a single spot was observed and the collected nomilin and limonin spots were the same color as their standards. In addition, R, values of the collected limonin and nomilin peaks were the same as those in the literature (4). Thus, the combination of evidence from the two chromatographic (HPLC and TLC) systems, the UV spectra, and the positive Ehrlichs test, all indicate that the collected peaks are nomilin and limonin. There was no evidence to suggest that additional compounds coelute with either limonin or nomilin. Thus, limonin or nomilin content can be quantitated from extract chromatograms with confidence. Chromatographic Stability and Column Lifetime. In the normal phase mode, no irreversible absorption, shifting retention times, or subsequent column poisoning was observed. Retention times were extremely stable and reproducible. Column lifetime appears to be limited by the stability of the silica support. This is a considerable improvement over the lifetime of the same column used in the reverse phase mode. Thus, the supposition that the material responsible for the reverse phase column poisoning is nonpolar appears valid. Extraction Efficiencies. Limonoids are separated from other components in citrus juices with a chloroform extraction. This extraction acts both as a sample cleanup and a concentration step, as the extract is evaporated to dryness and redissolved in a smaller amount of mobile phase. Using a triple extraction, essentially all added limonin was recovered (range 94-103%). Other workers have also reported similar recoveries (12, 14). Limits of Detection, Concentration Linearity Range, and Precision. Under conditions of maximum sensitivity, approximately 10 ng of limonin and an equal amount of nomilin wl give a signal to noise ratio of 2. The absorbtivities i l of obacunone and deoxylimonin are considerably greater than those of limonin and nomilin (see Figure 2); thus, 3.5 ng of these compounds will give the same signal to noise ratio. Since it has been shown (24) that the volume in which a sample is 0.173 0.321
0.115

2.3 3.5 6.6 2.3


1.1

0.021
0.015

0.058

5.6

5 IO TIME ( m i n )

15

Figure 4. Chromatogram of orange juice extract from low limonoid precision study. Chromatographic conditions: Zorbax CN column at 40 OC; mobile phase, isopropanol:heptane:methanol(l2: 11:2, v/v); flow, 1.0 mL/min; pressure, 500 psi; injection volume, 50 pL

injected will alter detection limits, detection limits given are those obtained from 2 0 - ~ L injection. The results of five replicate limonoid determinations from a typical orange and grapefruit juice are shown in Table IV. A low limonin orange juice was chosen to demonstrate the sensitivity and precision of the method, while the grapefruit juice was chosen to determine precision levels under more typical conditions. The orange juice separation achieved under conditions of maximum sensitivity and resolution is illustrated in Figure 4. While the limonin peak is reasonably large and well shaped, the nomilin peak is small, representing approximately 26 ng of material, which is close to its detection limit. This chromatogram and corresponding results are noteworthy in that they demonstrate for the first time that limonoid concentrations can be determined with good precision in juices where the limonin concentration is well below its taste threshold. From an examination of the five replicate deter-

Anal. Chem. 1980, 5 2 ,

1233-1239

1233

minations, it can be seen that the overall precision of the method is excellent. Limonin and nomilin concentrations in t h e orange juice were 0.75 f 0.03 and 0.030 f 0.003 ppm, respectively. T h e corresponding values for grapefruit juice were 5.0 f 0.14 and 0.25 f 0.03 ppm. (Confidence limits were determined a t the 95% level, n = 5 . ) As a result of concentrating t h e limonoids from the orange juice by extracting a larger volume of juice and injecting twice as much of the extract, the relative standard deviation, RSD, for orange juice limonin values is very similar to that of the grapefruit juice (3.2 vs. 2.2%, n = 5) and the RSD for nomilin results is slightly lower (8.9 vs. 9.4%). I t is interesting to note that the RSDs of individual samples were approximately the same magnitude as the overall RSD suggesting that most of the random error is associated with injection and integration variations and little is due to sample preparation. The relatively large nomilin RSDs (12.1 to 20.3%) represent reasonable precision when measuring amounts of a material very close to its detection limits. Approximately twice as much nomilin is present in the grapefruit injections (70 ng) and, as seen in Table IV, the individual RSDs are considerably lower (3.2 to 12.9%). Using standard limonin and nomilin solutions of various concentrations a n d constant injection volumes, a linear detector response was established from 0.02 to 4 gg ( r = 0.999).

ACKNOWLEDGMENT
The authors express their gratitude to Gail Allen, Ellie Case, and Faye Martin for their invaluable technical ilssistance. The authors would also like to express their appreciation to E. I. du Pont de Nemours & Co. for the generous use of the 848 liquid chromatograph.

LITERATURE CITED
(1) Chandler, B. V.; Kefford. J. F. J. Sci. FoodAgric. 1966, 5 0 , 193-97. (2) Scott, W. C. Proc. Fla. State Hort. SOC.1970, 83, 270-77. (3) Maier, V. P.; Dreyer, D. L. J. Food Sci. 1965, 30, 874-75. (4) Maier, V. P.; Beverly, G. D. J. Food Sci. 1968, 30, 488-92. (5) Emerson, Oliver H. J. Am. Chem. SOC.1946, 70, 545-49. (6) Emerson, Oliver H. J. Am. Chem. SOC.1951, 73,2621-23. (7) Dreyer, David L. J. Org. Chem. 1965, 30, 749-57. (8) Dreyer, David L. Abstr. Citrus Res. Conf. 1963, Pasadena, Calif., Dec.

CONCLUSIONS A nonsubjective, highly sensitive technique for determining limonin and, for the first time, other citrus limonoids in citrus
juices has been developed. Quantitation is achieved without pre- or post-chromatographic reactions. The chromatographic system is exceptionally stable and not subject to column poisoning as earlier HPLC techniques were. Analyses can be carried out rapidly and with good precision.

9. (9) Maier, V. P.: Grant, Edward R. J. Agric. FoodChem. 1970, 78, 250-52. (IO) Chandler, B. V. J. Sci. Food Agric. 1971, 22, 473-78. (11) Tatum, James H.; Berry, Robert E. J. Food Sci. 1973, 38, 1244-46. (12) Kruger, A. J.; Colter, C. E. Proc. Fla. State Hort. SOC. 1972, 85, 206- 10. (13) Fisher, James F. J. Agric. Food Chem. 1973, 27, 1109-10. (14) Fisher, James F. J . Agric. Food Chem. 1973, 23, 1199-1201. (15) Fisher, James F. J . A@. Food Chem. 1978, 2 6 , 497-99. (16) Snyder, L. R. J. Chromatogr. Sci. 1970, 692-706. (17) Scott, R. P. W.; Lawrence, J. B. J . Chromatogr. Sci. 1970, 8.619-24. (18) Saner, W. A.; Jadamec, J. R.; Sazer, R . W. Anal. Chem. 1976, 50, 749-53. (19) Chmielowiec, J.; Sawatzky, H. J. Chromatogr. Sci. 1979, 17,245-52. (20) Karger. Barry T. "Modern Practice of Liquid Chromatography"; WileyInterscience: New York, 1971; Chapter 1. (21) Bakalyer, Stephen R.; Henry, Richard A. J. Chromatogr. 1976, 726, 327-33. (22) Dean, F. M.; Geissman, T. A. J. Org. Chem. 1956, 23, 596-603. (23) Barton, D. H. R.; Pradham, S. K.; Sternhell, S.; Templeton, J. F. J. Chem. SOC.1961, 255-75. (24) Karger, B. L.; Martin, M.; Guiochon, G. Anal. Chem. 1974, 1640-47.

RECEIVED review November 16, 1979. Accepted April 1, for 1980.

Gas Chromatography with Detection by Laser Excited Resonance Enhanced 2-Photon Photoionization
Charles M. Klimcak and John E. Wessel"
Ivan A. Getting Laboratories, The Aerospace Corporation,

P.0. Box

92957, Los Angeles, California 90009

Resonance enhanced 2-photon photolonization has been applied to detection of gas chromatograph effluents using a small volume proportional counter cell and a low power laser. Detection llmlts on the order of 10 picograms were obtained for the aromatic hydrocarbons anthracene, phenanthrene, benzanthracene, acenaphthene, naphthalene, and assorted halonaphthalenes. Spectral differentiationbetween coeluting anthracene and phenanthrene was also demonstrated. The influence of excitation pulse duratlon was Investigated.

There is a n increasing need for selective gas chromatographic detectors with high sensitivity. In this report, we describe application of resonance enhanced 2-photon photoionization (R2PI) to detection of chromatographic effluents. Spectrally selective detection of several aromatic hydrocarbons is demonstrated. At ambient pressure the process, which is shown in Figure 1, involves excitation from ground state So to excited state S2,followed by collisionally induced relaxation to SI and subsequent ionization. This new method which has been described in recent reports (2-3) is applicable to most
0003-2700/80/0352- 1233$01.OO/O

molecular species, and is expected to attain sub-picogram detection limits. Related multiphoton photoionization methods have been applied extensively for molecular spectroscopic investigations (4-25) and RBPI has been used for single atom detection (26, 27). Recently a commercial photoionization detector based on one-photon photoionization induced by a n incoherent UV source has achieved widespread use for gas chromatographic applications (28, 29). This device, whose development was based on extensive prior photoionization work, provides limited although highly valuable spectral selectivity in that the photon energy can be chosen in order to selectively ionize species with low ionization potentials while leaving species with higher ionization potentials unexcited (28). Thus, aromatics can be detected in the presence of higher concentrations of alkanes. By utilizing short wavelength UV sources, all molecules of interest can be ionized; therefore the method is widely applicable. Picogram detection limits have been obtained in favorable cases. The multiphoton detection method studied in this report offers the following potential advantages with respect to conventional one-photon photoionization detection: (1)greatly
1980 American Chemical Society