Anal. Chem. 1988, BO, 807-811
807
Liquid Chromatographic Artifacts and Peak Distortion: Sample-Solvent Interactions in the Separation of Carotenoids Frederick Khachik,* Gary R. Beecher, Joseph T. Vanderslice, and Greg Furrow’ Nutrient Composition Laboratory, BHNRC, ARS, U.S. Department of Agriculture, Beltsville, Maryland 20705
I n separation of carotenoids by high-performance liquid chromatography (HPLC), the interaction between solute (carotenoids) molecules, Injection &bent, and mobile phase can result in production of HPLC peaks that could be erroneously inferpreted as indicating the presence of Impurities or cis carotenoids. The effect of this sample-solvent interadlon on the resolution of the HPLC peaks for several carotenoids (CY- and &carotene, lycopene, ,f3-apo-8’tarotenal, canthaxanthin, zeaxanthin) injected in various organic solvents and chromatographed under various isocratlc and gradient HPLC conditions has been extensively studied. Depending on the dublllty of the carotenoids in the HPLC mobile phase and the nature of the injecting solvent, single, double, and In some cases multiple HPLC peaks were reproducibly generated. Isolation and structural elucidation of the individual carotenoM components indicated the additional HPLC peaks were artifacts of chromatography. This multiple HPLC peak formation is shown to be dependent on the relative solubility of the carotenoids in the Injection and eluting solvents and on the interaction of the solvents as the sample bolus first interacts with the column.
Carotenoids are among the most abundant naturally occurring group of pigments that are encountered in plants, foods, and animals. The isolation of these pigments from natural produds involves extraction and chromatographywith organic solvent. Since carotenoidsare highly sensitive to light, heat, air, and active surfaces, their isolation from natural sources may be accompanied by degradation, stereoisomerization, rearrangement, and chemical reactions, which in turn may result in carotenoid artifacts. In most cases, the production of carotenoid artifacts is commonly avoided by exercising care in handling these pigments during isolation and extraction procedures. However, in separation and detection of carotenoids by various chromatographic techniques, particularly high-performance liquid chromatography (HPLC), artifacts may be readily produced that are often overlooked or considered to be related to extraction and work-up procedures. There are no known literature reports that have investigated the production of artifacts in HPLC of carotenoids. However, the influence of the injection solvent on production of chromatographic artifacts has been reported in the reversed-phase chromatographyof triglycerides (I). For a review on other separation artifacts, the reader is referred to the publication by Snyder and Kirkland (2). In this report we present a thorough investigation on the HPLC of carotenoids and demonstrate that the C-18 reversed-phase chromatography of these pigments is extremely sensitive to injection conditions and can result in the production of HPLC artifacts. In this report the factors that influence the production of artifacts such as the solubility of carotenoids in the ‘Present address: Eli Lilly and Co., Department KY400,Lilly Corporate Center, Indianapolis, IN 46285.
mobile phase and in the injection solvent, solubility properties of the injection solvent in relation to the mobile phase, and the injection volume are investigated. The injection solvent, in the HPLC of carotenoids extracted from natural sources, must be able to dissolve a wide range of carotenoids and also be compatible with the mobile phase in terms of polarity and solubility. In liquid chromatography, the polarity index (P’) of a solvent as introduced by Rohrschneider (3) is an indicator of its ability to interact with solute molecule and stationary phase. The polarity index P’ is based on actual experimental solubility data (3-5) and provides a rough measure of solvent strength for adsorption liquid chromatography (6). Since changes in P’values of an HPLC solvent mixture result in changes in the value of separation constant, optimum HPLC separation conditions may be established by employing various composition or mixture of solvents with varying polarities. The polarity P’values of a solvent mixture are the arithmetic average of the P’values of pure solvents in the mixture, weighted according to the volume fraction of each solvent (6). For a ternary HPLC solvent mixture of A, B, and C, the polarity for the mixture may be calculated from the following equation:
(1) p’ = @spa + ‘@b + $c?c Where P’is polarity of solvent mixture, 4a,&, and +c are the volume fractions of solvents A, B, and C in the mixture, and pa,p b , and p, refer to polarities (p’)of the pure solvent A, B, and C. Polarities of various solvents have been tabulated by Snyder and Kirkland (6). Large differences between the P’value of the injection solvent and the P’value of the HPLC solvent mixture (mobile phase) are undesirable, often resulting in precipitation of carotenoids on the HPLC column. To ensure compatibility of the injection solvent with the mobile phase, carotenoids preferably should be injected in the HPLC mobile phase. However, owing to the wide range of solubility of carotenoids extracted from natural sources, carotenoids may be more conveniently solubilized in injection solvents ather than the mobile phase. It will be demonstrated that in such cases, the injection solvent must be carefully selected so that the chromatography is not accompanied by false HPLC peaks and peak distortion of carotenoids due to the injection solvent. EXPERIMENTAL SECTION Apparatus. The HPLC separations were monitored on a Beckman Model 114 M ternary solvent delivery system equipped with a Beckman Model 421 controller interfaced into a Hewlett-Packard 1040 A rapid-scanning UV-vis photodiode array detector. The absorption spectra of carotenoids were monitored at 450 and 470 nm. The data were stored and processed by means of a Hewlett-Packard Model-9121disk drive and 7470 A plotter. The HP-85-B computing system with a built-in integration program was used to evaluate the peak area and peak height. The absorption spectra of the carotenoids were recorded between 200 and 600 nm as frequent as 1 scan/5 s (maximum scanning capability 1 scan/100 ms). Column. Analytical separations were carried out on a stainless steel (25cm X 4.6 mm i.d.) Microsorb CI8 (5-pmspherical particles) column ( W i n Instrument) which was protected with a Brownlee
This article not subject to U.S. Copyright. Published 1988 by the American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 60, NO. 8 , APRIL 15, 1988
Table I. Chromatographic Conditions Employed for the Separation of Carotenoids eluents
solvents
70 composition
A
methanol acetonitrile methylene chloride methanol acetonitrile methylene chloride hexane acetonitrile tetrahydrofuran methanol acetonitrile methylene chloride hexane
25 55 20 25 55 10 10 78 22 10," lo* 8 5 , O 50b 2.5," 20b 2.5,"20b
B
C D
"Time 0-15. *Final composition at time 30; gradient begins at time 15. guard cartridge (3 cm X 4.6 mm i.d.) packed with Spheri-5 C18 (5-gm particle size). For semipreparative separation the column was replaced with a stainless steel (25 cm X 10 mm i.d.) Microsorb C,, (5-pm spherical particles) column (Rainin Instrument). Reagents and Materials. The reference samples of alltrans-lycopeneand all-trans-a- a d @-carotene(Sigma,St. Louis, MO) were purified by semipreparative HPLC (procedure described in the text). all-trans-Canthaxanthin and @-apo-8'-carotenal (Fluka Chemical Corp., NY) and nonapreno-@-carotene [synthesized in our laboratory according to the published procedure (7)] were shown to be more than 96% pure as determined by HPLC and molar absorptivity measurements in hexane and ethanol. The reference sample of all-trans-zeaxanthin was provided by Hoffmann-La Roche (Switzerland). Chromatographic Procedures. Various isocratic and gradient HPLC conditions employing eluents A, B, C, and D are listed in Table I. Chromatograms were monitored at 450 nm for zeaxanthin and @-apo-8'-carotenaland at 470 nm for canthaxanthin, lycopene,a-and @-carotene,and
[email protected] flow rate with the analytical column was 0.70 mL/min, while the flow rate with the semipreparative column employing eluents A and B was 3.5 mL/min. Isolation and Purification of Carotenoids. Reference samples of all-trans-lycopene and all-trans-a- and -@-carotene were purified by semipreparativeHPLC employing eluents A and B. The structure of these carotenoids were confirmed by comparison of their 400-MHz proton NMR (8, 9) and UV-vis absorption spectra in various solvents (8,lO) with those published in the literature. Determination of Solubility of j3-Carotene in Various Solvents. Each of the solvents and the mobile phases were individually added dropwise to a weighed amount of crystalline @-caroteneuntil a complete solution resulted. The minimum volume of each solvent necessary to solubilize @-carotenewas recorded and the values were extrapolated in terms of the weight of @-carotenesoluble in 100 mL of the solvents as shown in Table 111.
RESULTS AND DISCUSSION Sample Solvent a n d Mobile Phase Interaction. The chromatogram of a sample of all-trans-@-carotene(purified by semipreparative HPLC) injected in the HPLC solvents (eluent A) contains a single symmetrical peak A (Figure la), while the same sample injected in methylene chloride produces an additional HPLC peak, A' (Figure Ib). Initially the presence of peak A' in this chromatogram was attributed to stereoisomerization of all-trans-@-carotene in chlorinated organic solvents such as methylene chloride and chloroform. These solvents are often contaminated with trace amounts of hydrochloric acid, which can catalyze stereochemical transformations of carotenoids. However, isolation of the two components A and A' by semipreparative HPLC and evaluation of the absorption and NMR spectra of each fraction revealed that both compounds were all-trans-@-carotene, and
8
b
b
0
d
8
4
Time ( m i n )
8 d
Figure 1. Chromatograms of all-trans-&carotene, HPLC conditions (eluent A) described in Table I: (a)upper trace, sample (20 pL) injected in the HPLC solvents (eluent A); (b) lower trace, sample (20 pL) injected in methylene chloride. Peaks A and A' are all-trans-@-carotene. no cis/trans isomerization had occurred. Furthermore, subsequent injection of each of the isolated p-carotene fractions A and A' in methylene chloride resulted in further HPLC peak splitting and produced chromatograms similar to that shown in Figure 1b. It was also demonstrated that when the same solution of p-carotene in methylene chloride is evaporated and the solvent is replaced with hexane, methanol, or the HPLC solvents (eluent A), the chromatograms of p-carotene injected in these solvents can result in the regeneration of a single symmetrical peak for this compound (Figure la). In these experiments it was shown that at a constant mass of @-carotene,the HPLC peak area for this compound injected in solvents such as acetone, methanol, and hexane remains the same as the total peak area of the distorted HPLC peaks (area of peaks A + A'), when p-carotene is injected in solvents such as methylene chloride, THF, and benzene. Tsukida et al. (11)have demonstrated that photochemicallyand thermally induced stereoisomerizations of all-trans-@-caroteneresult in an equilibrium between the all-trans and cis isomers of @carotene; these stereoisomers were found to be stable enough to allow isolation by preparative HPLC and structural elucidation by spectroscopy. Therefore, this single and double HPLC peak formation that is influenced by the injection solvent is unlikely to be associated with a rapid cisltrans isomerization of @-carotenein organic solvents. In addition to eluent A the HPLC peak distortion for @carotene was also experienced with a number of other eluents under isocratic (eluents B and C) and gradient (eluent D)
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Table 11. Polarities of Pure Solvents and Various HPLC Eluents"
4 2
.r(
I
I
II II
II
I1
8
1 5'&6
b
solvents
polarity (P?
acetonitrile mobile phase (eluent D) mobile phase (eluent A) mobile phase (eluent C) acetone methanol mobile phase (eluent B) methylene chloride chloroform tetrahydrofuran benzene to1u en e hexane
5.8 5.6-4.4 5.4 5.4 5.1 5.1 5.0 4.1 4.1 4.0 2.1 2.4 0.1
"Polarity values of pure solvents were obtained from ref 4,and the polarity values for HPLC mobile phases were calculated from eq 1.
Table 111. Solubility of @-Carotenein Various Solvents"
solvents
solubility (mg/100 mL)
solvents
solubility (mg/100 mL)
tetrahydrofuran methylene chloride hexane
930 325 39
acetone eluent A eluent B
21 22 22
Values were generated according to the procedure described in the text.
86
8 Time (min)8
ri
i
8
Q
Flgure 2. Chromatographic profiles of a constant concentration of a mixture of carotenoid reference samples (20111) injected in acetone [(a) upper trace] and methylene chloride [(b) lower trace]: 1, l', all-trans-zeaxanthin;2, 2', all-trans-canthaxanthin; 3, 3', all-trans&apo-8'-carotenal; 4, 4', a//-trans-lycopene;5, 5', a//-trans-a-carotene; 6, 6', all-trans-@-carotene;7, 7', all-trans-C-45-@-carotene HPLC conditions (eluent D) are described in (nonapreno-@-carotene). Table I.
HPLC conditions. The chromatographic profile of a sample of all-trans-@-caroteneinjected in the HPLC solvents (eluent C) results in a single symmetrical peak, while the chromatogram of this compound injected in methylene chloride results in HPLC peak splitting of @-carotenesimilar to that in Figure lb. The production of apparent HPLC artifacts is not unique to @-carotene,as demonstrated by the chromatographic profiles of a mixture of seven carotenoids injected in acetone (Figure 2a) and methylene chloride (Figure 2b) under gradient HPLC conditions (eluent D). This experiment demonstrates that chromatographic peak distortion is associated with not only hydrocarbon carotenoids but other classes of these compounds such as hydroxy, keto, and apo carotenoids with various functional groups and solubility behavior may accordingly result in similar chromatographic artifacts. The HPLC injection solvents employed in the present study can be divided into two groups. The first group contains solvents such as acetonitrile, acetone, methanol, hexane, and the various HPLC eluents (A, B, C, D) that produce a single peak for @-caroteneand other carotenoids under various HPLC conditions. The second group contains solvents such as methylene chloride, chloroform, tetrahydrofuran (THF), benzene, and toluene, which result in the production of HPLC
artifacts. The polarities of these solvents and various HPLC eluents are shown in Table 11. It appears that the injection solvents that do not result in production of HPLC artifacts (Le. acetonitrile, acetone, methanol) have polarity (P? values more compatible with those of the various HPLC mobile phases (eluents A, B, C, D). On the other hand, polarities of the injection solvents that produce artifacts (with the exception of hexane) are less compatible with those of the various HPLC mobile phases. Hexane, despite its much lower polarity value (0.1) than those of the various HPLC mobile phases (eluent A = 5.4, eluent B = 5.0, eluent C = 5.4,eluent D = 5.6-4.4), when employed as an injection solvent, does not generate chromatographic artifacts. This suggests that the production of artifacts may not be solely dependent on compatibility of the polarities of the injection solvent with that of the mobile phase but perhaps more generally on the interaction between solute, injection solvent, and mobile phase. This interaction may more appropriatelybe evaluated in terms of the relative solubility of the solute (carotenoids) in the injection solvent and the mobile phase, which is most likely one of the major factors that govern the production of chromatographic artifacts. The relative solubility of @-carotene in various injection solvents and two of the isocratic HPLC mobile phases (eluents A and B) as determined by solubility measurements are shown in Table 111. From these solubility data and chromatographic profiles it appears that in cases where there is a dramatic difference between the relative solubility of @-carotenein the injection solvents (THF and methylene chloride) and the mobile phases (eluents A and B), chromatographic artifacts result. On the other hand, the solubility of @-carotenein injection solvents such as hexane and acetone is much more compatible with those of the HPLC mobile phases (eluents A and B) and as a result no chromatogrpahic abnormalities are observed. The HPLC peak splitting of all-trans-p-cmoteneinto peaks A and A' (Figure l b ) serves as a good example of HPLC artifacts that may mislead the chromatographer and result in misidentification of carotenoids. For example, if carotenoid
ANALYTICAL CHEMISTRY, VOL. 60, NO. 8, APRIL 15, 1988
810
:I
a
3 ,z5
a
A
!
8
d
8
8
d
4
8
Time (min)
:
b
b
A A'
A
-8I 0
Figure 3. Chromatograms [eluent A, Table I] of a constant concentration of all-trans -@-caroteneat two different injection volumes in hexane: (a) upper trace, sample injectbn in 50 pL of hexane; (b) lower trace, sample injected in 100 pL of hexane. Peaks A and A' are a//-trans-@-carotene.
extracts from biological sources that are suspected to contain a-and 8-carotene are injected in methylene chloride employing isocratic (eluent C) HPLC conditions, the resulting chromatograms may produce artifacts such as peak A', which on the basis of its HPLC retention time and vicinity to peak A may be misidentified as a-carotene. With the implementation of rapid scanning photodiode array detector, under chromatographic conditions employed (eluents A, B, C, D), a- and @-caroteneare readily distinguished from their absorption spectra (a-carotene, & = 446 nm; 8-carotene, A- = 454 nm) monitored in the HPLC solvents. However, in most cases, additional spectroscopicanalyses (NMR and mass spectrometry) of the isolated fractions are necessary to establish the identity and the presence of stereoisomeric carotenoids and avoid misidentification of these compounds as a result of HPLC artifacts. Effect of Sample Injection Volume on Production of HPLC Artifacts. In addition to the nature of the injection solvent in HPLC of carotenoids, the injection volume of the sample is also another factor that could dramatically affect peak shape and resolution and result in chromatographic artifacts. A series of experiments were performed in which the solute mass was kept constant while the injection volume was varied. The chromatographic profiles of a constant mass of all-trans-@-carotenemonitored at two injection volumes in hexane are shown in Figure 3, parts a (injection volume 50 pL) and b (injection volume 100 pL). In this case, an injection
u3 0 l
.
~
'
'
.
'
-
.
~
l
8 Time (min) 8 0 4 R Figure 4. Chromatograms [eluent A, Table I] of a constant concentration of all-trans-&carotene at two different injection volumes in THF: (a) upper trace, sample Injected in 50 pL of THF; (b) lower trace, sample injected In 100 pL of THF. The unresdved broad peaks appearing at the beginning of the lower trace chromatogram (b) correspond to A, A', A", and A"', which indicate ell-trans-p-carotene. 8
solvent such as hexane, under the chromatographicconditions employed, at lower sample injection volume (20 pL) does not produce multiple peaks and at higher sample injection volume results in the HPLC peak distortion of @-carotene. This HPLC peak distortion becomes even more dramatic when a constant concentration of all-trans-8-carotene is injected in 50 and 100 p1 of THF, as shown in the HPLC profiles in Figure 4. We have demonstrated that HPLC artifacts with injection solvents such as methylene chloride, chloroform, THF, benzene, and toluene that usually result in HPEC peak distortion of carotenoids can be eliminated if the injection volume of samples in these solvents is reduced to 5 or 10 pL. This fiiding suggests that at larger injection volumes, immiscibility of the injection solvent containing solute (carotenoids) with the mobile phase plays an important role in generation of the observed HPLC artifacts. To demonstrate the effect of miscibility of the injection solvent with the mobile phase, the HPLC system was redesigned according to the diagrams shown in Figure 5. In the redesigned HPLC system the injection valve was placed in line with methylene chloride and the outlet was then added to a well-mixed solution of acetonitrile and methanol. This redesigned HPLC system enabled the mixing of the injection solvent and solute with two of the HPLC eluents (acetonitrile and methanol) prior to the column. When a solution of pcarotene in methylene chloride was injected in the redesigned
~
~
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ANALYTICAL CHEMISTRY, VOL. 60, NO. 8 , APRIL 15, 1988 HF'LC System
Injection Valve
v
"I
Detector
Recorder
Recorder
Figure 5. Diagrams of the HPLC system before and after redesigning.
811
flow in small bore tubes (12). Thus those factors that inhibit dispersion, Le., disimilar solvent polarity and analyte solubility, may retain the sample as a bolus and as a result cause HPLC peak distortion and artifacts. Unfortunately, the constraint of the real systems are such that it is very difficult to devise a mathematical model that is capable of even a numerical solution. Thus, any insights into the mechanisms that are responsible for the observed artifacts are lacking. However, the experiments reported here indicate that the polarity and the solubility properties of the injection solvent and the mobile phase must be compatible if the artifacts are to be eliminated. Alternatively, intentional mixing of the injection solvent and mobile phase (thus dispersing the original bolus) prior to the column could be used to eliminate these artifacts. Finally, if a single compatible solvent can not be found, binary or even ternary solvent mixtures may be employed. Alternatively, if carotenoids must be injected in a specific solvent, highly concentrated samples may be injected in small volumes (i.e. 10 or 5 pL) to avoid HPLC peak distortion and production of artifacts.
ACKNOWLEDGMENT We thank the F. Hoffmann-La Roche & Co. for the carotenoid samples (including zeaxanthin) and Yiu-Fai Lam (University of Maryland) for running the NMR samples of carotenoids. We also thank Steven J. Schwartz (North Carolina State University) and David Kemper (Shimadzu Scientific Instruments, Inc.) for helpful comments on the paper. Registry No. all-trans-Zeaxanthin, 144-68-3;all-trans-canthaxanthin, 514-78-3; all-trans-@-apo-8'-carotenal,2756-51-2; all-trans-lycopene,502-65-8;all-trans-a-carotene, 432-70-2;alltrans-C-45-@-carotene, 26034-44-6;all-trans-@carotene,7235-40-7. LITERATURE CITED (1) Tsimldou, M.; Macrae, R. J . Chromafogr. Sci. 1985, 23, 155. (2) Snyder, L. R.; Kirkland, J. J. In Introduction to Modern L i q M Chroma tography, 2nd Ed.; Wlley: New York, 1979; Chapter 19, p 791. (3) Rohrschnelder, L. Anal. Chem. 1973, 4 5 , 1241. (4) Snyder, L. R. J . Chromatogr. 1974, 92,223. (5) Snyder, L. R. J . Chromafogr. Sci. 1978, 76, 223. (6) Snyder, L. R.; Kirkland, J. J. In Introduction to Modern Liquid Chroma tography, 2nd ed.; Wlley: New York, 1979; Chapter 6, p 246. (7) Khachik, F.; Beecher, 0. R. Ind. Eng. Chern. Prod. Res. D e v . 1986, 25,671. (8) Tsukida, K.; Saiki, K. J . Nufr. Sci. Vitaminoi. 1983, 29, 11 1. (9) Englert, G. I n Carotenoids Chemistry and Biochemistry; Britton, G., Goodwin, T. W., Eds.; Pergamon: Oxford, 1982; pp 107-153. (10) Ritter, E. D.; Purcell, A. E. I n Carotenoids as Colorants and Vitamin A Precursors: Bauernfeind. J. C.. Ed.: Academic: New York. 1981: Chapter 10, p 883. (11) Tsukida, K.; Saiki, K.; Takii, T.; Koyama, Y. J . Chromafogr. 1982, -245 .- , 359 --- . (12) Vandersllce, J. T.; Rosenfeld, A. G.; Beecher, G. R. Anal. Chim. Acta 1988, 179, 119.
-
0
0
0
d
Time %in1
Flgure 8. Chromatogram [eluent A, Table I] of a/l-trens-@-carotene injected in methylene chloride (20 pL) onto the redesigned HPLC system: A, a//-trans-&carotene.
HPLC system and the resulting solution was then allowed to mix with methanol and acetonitrile prior to loading on the column, a single HPLC peak for this compound was obtained (Figure 6). The HPLC peak broadening of @-carotenein this case results from excessive void volume which is inevitably created by redesigning the HPLC system. Chromatographic profiles of @-caroteneinjected in various solvents under conditions in which premixing of sample with mobile phase was allowed in all cases contained a single HPLC peak for this compound.
CONCLUSION The flow of sample and mobile phase between HPLC injection valve and column, and perhaps flow through the channels of HPLC columns, follows the principals of laminar
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RECEIVED for review September 16,1987. Accepted December 21,1987. Part of this paper was presented at the 8th International Symposia on Carotenoids, Boston, MA, July 27-31, 1987. Partial support was by the National Cancer Institute through reimbursable Agreement Y01-CN-30609. Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture, and does not imply its approval to the exclusion of other products that may also be suitable.
