Analysis of carotenoids by high performance liquid chromatography and diode-array detection

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<ul><li><p>RE VIEW PA PER Analysis of Carotenoids by High Performance Liquid Chromatography and Diode-array Detection </p><p>Peter M. Bramley Department of Biochemistry. Royal Hollowey and Bcdford New Collcgc (Univcrsity of London), Egham Hill, Egham. Surrey TW20 OEX. U K </p><p>The use of diode-array detectors with high performance liquid chromatography enables the separation and analysis of carotenoids from biological samples to be achieved in a significantly shorter period than when conventional ultraviolet/visible detectors are used. This relates to the ability of diode-array detectors to provide full spectral analysis of the compounds during chromatography. The features of diode-array detectors, including their advantages and drawbacks, are discussed, particularly with respect to the identification and quantitation of carotenoids. </p><p>KcJywords: Carotenoids; high performance liquid chromatography: diode-array detection. </p><p>INTRODUCTION </p><p>The carotenoids represent one of the most important and widespread groups of pigments in Nature. The structures of well over S O 0 are known, and they are responsible for many of the yellow and red colours of flowers, fruits, birds. insects and other animals (Weedon, 1971; Goodwin, 1980, 1983, 1986; Schiedt, 1990). Their universal presence in photosynthetic tissue is on ly noticeable at the onset of leaf senescence when the chlorophylls disappear. The dramatic changes in the colour of ripening fruits also reflect the degradation of chlorophylls and a concomitant and massive increase in carotenoids. </p><p>Carotenoids play vital roles in photosynthetic tissues, acting as accessory light harvesting pigments (Jeffrey ei ul . , 1974; Larkum and Barrett, 1983; Moore et ul . , 1990), protectors against photoxidation (Knox and Dodge, 1985) and precursors of abscisic acid (Milbor- row, 1983). Their importance as dietary precursors of vitamin A (Goodwin, 198t)) as well as their increasing use as protectors against photosensitive skin disorders (Krinsky, 1989, 1990) and as anticancer agents (Mathews-Roth, 1989) has led to an increase in their commercial use, especially when derived from biologi- cal sources rather than produced synthetically (Taylor, </p><p>This upsurge of commercial interest, the on-going research into the enzymology and regulation of carote- noid biosynthesis and of their function, requires accur- ate techniques for the separation, identification and quantitation of these pigments from a variety of sources. Traditional chromatographic techniques of column and thin layer chromatography (TLC) are being replaced by high performance liquid chroma- tography (HPLC), and most recently the use of HPLC coupled to a diode-array detector. The purpose of this review is to explain the underlying principles of diode </p><p>0958-0344/92/030097-OX $09.00 0 1992 by John Wiley &amp; Sons. Ltd. </p><p>1990). </p><p>array detection and to illustrate its advantages over conventional ultravioletfvisible (UViVIS) detectors, especially in the analysis of carotenoids. </p><p>STRUCTURE, NOMENCLATURE AND PROPERTIES OF CAROTENOIDS </p><p>Carotenoids are isoprenoids which generally contain eight isoprene units joined together so that the linking of the units is reversed at the centre of the molecule. The main feature of the symmetrical tetraterpenoid is a polyene chain which may extend from 3 to 1.5 conju- gated double bonds. Cyclization of the carbon skele- ton, at one o r both ends of the chromophore, occurs for example in (3-carotene (6,P-carotene; Fig. l ) , while xanthophylls are formed from the hydrocarbon caro- tenes by the introduction of oxygen functions, e.g. lutein (3,3-dihydroxy-f!,~-carotene) and violaxanthin (5,6,5 ,6 -diepoxy-S,6,5 ,6 - tetrahydro- B$-carotene - 3,3-diol; Fig. 1). </p><p>Common carotenoids have well established trivial names, and also IUPAC-recommended systematic nomenclature (Commission on Biochemical Nomenclature, 1971, 1975). Trivial names are used throughout this review; the corresponding semi- systematic names are quoted at the first mention of each carotenoid. The structures of the carotenoids can be found in Straub (1987), whilst regular reviews on advances in carotenoid chemistry are published by the Chemical Society of Great Britain (e.g. Britton, 1989). The structures of predominant plant carotenoids are shown in Fig. 1. Progress in the structural elucidation of plant carotenoids has been reviewed recently (Szabolcs, 1990). </p><p>The length of the carotenoid chromophore deter- mines the absorption spectrum of the molecule. Thus </p><p>Received 20 October 1991 Accepted freoised) 2 December f Y Y l </p></li><li><p>08 PETER M. BRAMLEY </p><p>I </p><p>OH </p><p>11 HO </p><p>OH </p><p>111 HO </p><p>OH </p><p>HO </p><p>OH </p><p>HO </p><p>Figure 1. Structures of carotenoids found in green leaves: ( I ) Ikarotene, (11) antheraxanthin, (Ill) lutein, (IV) neoxanthin, (V) violaxanthin and (VI) zeaxanthin. </p><p>phytoene (7,8,11,12,7,8, 11,12-octahydro-q~,~-caro- tene) has three conjugated double bonds, and absorbs in the UV at 285 nm, whilst b-carotene (11 conjugated double bonds) absorbs at 451 nm. Both the absorption wavelengths and spectral persistence are characteristics of carotenoids (e.g. Fig. 2 ) , and are frequently used to identify and quantify these compounds (Davies, 1976). The spectral properties are fully exploited in diode array detection. It should be noted, however, that carotenoids are unstable to heat, light and oxygen, and so care must be taken in the extraction and handling of samples throughout analysis. Detailed experimental procedures can be found elsewhere (Britton and Goodwin, 1971; Davies, 1976; Britton, 1985; Goodwin and Britton, 1988). </p><p>PRINCIPLES OF DIODE ARRAY DETECTORS </p><p>The diode- or photodiode-array detector was intro- duced i n the early 1980s by Hewlett-Packard. It is now available from several manufacurers (Table 1). </p><p>Although it shares many elements with a conventional UV/VIS detector, the essential difference is that it can record the entire spectral range (190-800 nm) during analysis, thus monitoring the chromatogram at selected wavelengths whilst recording the spectra of eluants simultaneously (Fig. 3 ) . Although conventional detec- tors are able to monitor at several wavelengths. they cannot record spectra simultaneously since they separ- ate the incident light with a diffraction grating before it reaches the sample. </p><p>In a diode-array detector, polychromatic light from the lamp source (usually deuterium) is transmitted through an aperture (typically 5 nm), lens and shutter directly onto the sample in a flow cell. This arrange- ment of optical components, with the flow cell posi- tioned before the grating, is often called reverse optics. The shutter closes when the measurement of dark current is required. Transmitted light is then focussed via a slit onto a diffraction grating which separates the light into a spectrum, which travels t o a n array of up t o 512 photodiodes (Fig. 4). The spacing of the diodes and the slit width determine the overall wavelength resolu- tion of the detector. The slit width also affects the intensity o f light reaching the array and the noise of the response. Decreasing the slit width increases optical resolution, but also increases noise. </p><p>The photodiode array is a series of light-sensitive elements on a silicon chip, and is normally fully charged. As each diode is struck by light of a given wavelength, it discharges. The amount o f current required to recharge the photodiode is a function of the quantity of light striking it. Both the dark current, i.e. the electrical current discharged from the array when no light is striking the photodiodes, and the reference current, the result of light transmitted by the solvent. are also measured. The reference energy spectrum is </p><p>350 400 450 500 550 Wavelength (nm) </p><p>Figure 2. Absorption spectra of (+-carotene (- - -) and lycopene (---I in hexane. </p></li><li><p>ANALYSIS OF C'AKOTENOIDS BY HPLC 99 </p><p>Y </p><p>t </p><p>Table 1 . Features of selected diode array detectors Wavelength range Accuracy Number Minimum band </p><p>Manufacturerlmodel (nm) (nm) Lampa of diodes width inm) </p><p>Applied Biosystems 1000s 190-370; 370-555 1 .O D 76 5 Beckman 168 190-600 1 .o D 51 2 1 Hewlett-Packard 1040 190-600 1 .o D 21 1 4 Hitachi L-3000 200-520 5.0 D 70 10 LCD Analytical 5000 190-360 1 .o D 70 5 Merck-Hitachi L3000 200-360; 360-520 5.0 D 36 5 Phillips PU4121 190-590 1.5 D, T - Pharmacia-LKB 2140 190-370 0.9 D 200 4 Waters 990 190-800 1 .o D 512 1.4 a D=deuterium: T=tungsten. </p><p>I n m Absorbance </p><p>Chromatogram \ Wavelength \ 2 </p><p>Y </p><p>t Absorbance I </p><p>Figure 3. Chromatographic data obtainable with a diode-array detector. (Reproduced with permission of Millipore Corporation.) </p><p>used as a n indicator o f lamp intensity and instrument performance. The quantity of light at varying wavc- lengths is thus accurately and instantly measured after the diodes are recharged. </p><p>Holographic </p><p>grating piit t </p><p>\ S ' A L I ] </p><p>,Second order filter </p><p>Spectrum\ </p><p>Figure 4. The optical assembly of a Waters 990 diode-array detector. A = aperture; L = lens; S = shutter. The bold, arrowed line indicates the light psth. (Reproduced with permission of Millipore Corporation.) </p><p>DATA COLLECTION AND ANALYSIS ~ ~~ ~ ~ </p><p>Because of the enormous quantity of data generated during a typical chromatographic separation, diode- array detectors are coupled to computers with compre- hensive software for data analysis. Typically, the com- puter is programmed to scan the diodes (the sampling period) repeatedly during periods as short as 10ms. The entire array is recharged and read at the start of each sampling period as an analogue signal. Once the array is scanned, the diodes are allowed to discharge until the beginning of the next sampling period, when the scan is repeated. The process of recharging takes the same time, regardless of how much each diode was discharged, so that the total sampling time is the sum of the scan of the array and the waiting period whilst the diodes are recharging. </p><p>Scanning all wavelengths for the duration of one chromatogram and saving each data point requires a great deal of space on the hard disk of the computer. Therefore, all the data for each scan are not stored in memory. Several parameters can he optimized in order to minimize the size of files: </p><p>(a) Wurieleiigrh runge. Only specific wavelengths appropriate to the absorption spectra of the compounds need to be stored: for carotenoids a range of 250- 650 nm is sufficient. </p><p>(b) Meusirrrrnerif time urzd iiirerrd. Data need only he iiccumulated during the elution times of compound of interest. The interval defines how many data points are stored by setting the frequency at which data are saved: H smaller interval collects larger amounts of data. During periods of no interest in the separation, e.g. between peaks. the computer can be programmed not t o collect data. </p><p>(c) Acc~irrnir/ririoii. The data points from several scans can be averaged t o a single point. The accumu- lation parameter determines this number: increasing the value decreases the noise of the dispay. </p><p>(d) Hesolrrrion. For the identification of carotenoids by spectral analysis, the resolution of the diode array should be at ;I maximum. However. if absolute resolu- tion is not required, a larger value can be used to reduce disk space required for storage. </p><p>Post-run analyses. using new algorithms, provide extremely sophisticated methods of visualizing data, usually o n a colour monitor which can be connected to a colour printer. Isograms (contour plots. Fig. 5 ) and three-dimensional topographical plots (Fig. 6) are readily obtaincd. as well as composites of elution pro- </p></li><li><p>I00 PETER M. BRAMLEY </p><p>files and spectra of each component (Fig. 7). The data printout can be graphical or numerical. A spectral library feature enables comparison of experimental spectra with standards to be made, whilst integration, and hence quantitation, of elution peaks can be achieved once peak areas have been calibrated with standard amounts of solute. </p><p>ADVANTAGES AND DISADVANTAGES </p><p>Probably the greatest advantage of using a diode-array detector is that spectral and chromatographic profiles are recorded simultaneously (Fig. 3), thus eliminating the need to generate multiple chromatograms at differ- ent wavelengths and/or collecting fractions to record spectra later. This facility significantly reduces the time taken for analysis. </p><p>The data handling features enable peak priority to be determined quickly-a very useful feature in methods development and quality control. The high wavelength resolution of the detector enables identification of peaks to be achieved accurately, whilst contaminants can aIso be recognized. Spectra can be recorded on the up-slope, apex and down-slope of a chromatogram peak, normalized and overlaid, pinpointing differences in spectral shape and hence purity. Ratios of peak areas can also be calculated to determine purity. These features are often very useful in highlighting carotenoid </p><p>isomers which may co-elute, but have slightly different absorption spectra. Spectral libraries, stored on hard disk, make comparisons with standards easy, while baseline substructions enable comparisons to be made once a gradient blank has been stored. </p><p>A limitation of diode-array detectors is their sensitiv- ity in comparison to conventional UV/VIS detectors. This is most noticeable in the visible regions of the spectrum, as the deuterium lamp has relatively low spectral energy between 400 and 600nm. For this reason, some machines have been developed with both deuterium and tungsten lamps. However, the lamps cannot be used simultaneously, and require switching when needed. This is difficult during the chromatog- raphy of compounds whose spectra are unknown. In the UV region, instruments are sensitive as the best single or variable wavelength detectors and can be set at 0.001 AUFS. Generation of spectra at such levels of sensitivity is more problematical, however, due to signal-to-noise ratios, and may require a scale which is an order of magnitude less sensitive. </p><p>Other disadvantages relate to the complexity of the instruments themselves and of the software. The instru- ments do take longer to customize than conventional detectors, due to the large number of parameters to be set. Machines invariably require changes in parameter settings when required by another user. The time taken for alterations can be minimized by a mouse-driven system. The hard disk capacity is soon saturated, and so files must be deleted, or transferred to floppy disks or mainframe computers for storage purposes. </p><p>i. </p><p>3 </p><p>1 5 2 'I 51 </p><p>Figure 5. Contour plot of an HPLC profile, showing a three-dimensional plot of absorbance (AU) vs. wavelength (nm) vs. elution time (min). </p></li><li><p>ANALYSIS OF ('AROTENOIDS BY HPLC </p><p>we.., 31111 SlOl </p><p>1 AT( 1 4 7 1 A Q </p><p>Figure 6. Alternative representation of the three-dimensional plot of Fig. 5 showing absorbance (AU) vs. wavelength (nm) vs. elution time (min). </p><p>................................... Waters 990 Spectrum index plot ton17.fiT3 Y-scalr S l o p e Sanipl ing t...</p></li></ul>


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