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Simultaneous measurement of chlorophyll and astaxanthin in Haematococcus pluvialis cells by first-order derivative ultraviolet-visible spectrophotometry
JOURNAL OF BIOSCIENCE AND BIOENGINEERING Vol. 101, No. 2, 104–110. 2006 DOI: 10.1263/jbb.101.104
© 2006, The Society for Biotechnology, Japan
Simultaneous Measurement of Chlorophyll and Astaxanthin in Haematococcus pluvialis Cells by First-Order Derivative Ultraviolet-Visible Spectrophotometry Abdolmajid Lababpour1 and Choul-Gyun Lee1* Department of Biotechnology, Institute of Industrial Biotechnology, Inha University, 253 Younghyun-Dong, Nam-Gu, Incheon 402-751, Republic of Korea1 Received 5 September 2005/Accepted 27 October 2005
A first-order derivative spectrophotometric method has been developed for the simultaneous measurement of chlorophyll and astaxanthin concentrations in Haematococcus pluvialis cells. Acetone was selected for the extraction of pigments because of its good sensitivity and low toxicity compared with other organic solvents tested; the tested solvents included acetone, methanol, hexane, chloroform, n-propanol, and acetonitrile. A first-order derivative spectrophotometric method was used to eliminate the effects of the overlaping of the chlorophyll and astaxanthin peaks. The linear ranges in 1D evaluation were from 0.50 to 20.0 µg⋅ ml–1 for chlorophyll and from 1.00 to 12.0 µg⋅ ml–1 for astaxanthin. The limits of detection of the analytical procedure were found to be 0.35 µg⋅ ml–1 for chlorophyll and 0.25 µg ⋅ml–1 for astaxanthin. The relative standard deviations for the determination of 7.0 µg⋅ml–1 chlorophyll and 5.0 µg⋅ml–1 astaxanthin were 1.2% and 1.1%, respectively. The procedure was found to be simple, rapid, and reliable. This method was successfully applied to the determination of chlorophyll and astaxanthin concentrations in H. pluvialis cells. A good agreement was achieved between the results obtained by the proposed method and HPLC method. [Key words: chlorophyll, astaxanthin, first-order derivative, UV-visible spectrophotometry]
thin in H. pluvialis cells has already been reported (8–10). In these reports, the analysis of chlorophyll and astaxanthin requires a separation step that necessitates costly instrumentation of HPLC. The separation by HPLC is not as convenient or as rapid as that by spectrophotometry. The spectrophotometric determination of chlorophyll has already been reported (11); however, to the best of our knowledge, no analytical techniques have been reported for the simultaneous measurement of chlorophyll and astaxanthin in H. pluvialis cells by UV-visible spectrophotometry. The simultaneous measurement of chlorophyll and astaxanthin by common UV-visible spectrophotometry from their absorbance (zero-order derivative) is difficult, as these two pigments mutually interfere caused by a considerable overlaping of the peaks of their carbon chains that normally requires a separation step. Derivative spectrophotometry is a useful technique that can be used to resolve the overlapping of spectral peaks. This technique has been utilized for the simultaneous determination of a system of two or more components in analytical chemistry (12). The first (and higher) derivative absorption spectrum is the first (and higher) derivative of the absorbance as a function of wavelength. The higher derivative leads to a decrease in sensitivity and a loss of spectral information. Derivative spectrophotometry for the simultaneous measurement of chlorophyll and astaxanthin has not yet been reported; therefore, it is one of the main features of this study. Additionally, the re-
Astaxanthin, a natural red ketocarotenoid (3,3′-dihydroxyβ,β-carotene-4,4′-dione), has been used in clinical studies for its role in health conditions. It is also used as a pigment source in aquaculture and poultry, and as a food additive to supply nutrients or color, or for other biological activities (1, 2). The photosynthetic microalga, Haematococcus pluvialis, is a rich, natural source of astaxanthin. The cultivation of H. pluvialis has already been reported for a largescale production of natural astaxanthin (3). Motile green H. pluvialis cells mainly contain chlorophyll and start astaxanthin biosynthesis under some stress conditions, such as nutrient deficiency and high light intensity (4, 5). The monitoring of chlorophyll and astaxanthin concentrations is necessary to control H. pluvialis cultivation. The control of this cultivation also necessitates an accurate, precise, and easy handled method for the rapid and inexpensive determination of pigments both on the laboratory and industrial scales. The methods reported in the literature for the determination of chlorophyll and astaxanthin concentrations can be used to quantify pigments extracted by mechanical or solvent methods (6, 7). The extracted pigments are then subjected further separation and identification by high-performance liquid chromatography (HPLC). The simultaneous measurement of pigments, such as chlorophyll and astaxan* Corresponding author. e-mail: [email protected] phone: +82-32-860-7518 fax: +82-32-872-4046 104
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sults of this study can be used for the easier, precise, and accurate measurement of chlorophyll and astaxanthin concentrations in H. pluvialis cells. Accordingly, in this paper, we describe the effects of organic solvents for pigment extraction and the application of first-derivative UV-visible spectrophotometry to the simultaneous measurement of chlorophyll and astaxanthin in H. pluvialis cells, thereby eliminating the need for a separation step during pigment measurement.
MATERIALS AND METHODS Microorganism and cultivation conditions The photosynthetic microalga, H. pluvialis UTEX 16 (Culture Collection of Algae, University of Texas, Austin, TX, USA), was precultivated in a 200-ml Erlenmeyer flask under continuous light intensity of 40 µE⋅ m–2 ⋅s–1 using a fluorescent lamp in the photoautotrophic mode of operation. Modified Bold’s basal medium (MBBM) containing 246.5 mg ⋅ l–1 NaNO3, 24.99 mg⋅ l–1 CaCl2 ⋅ 2H2O, 73.95 mg ⋅l–1 MgSO4 ⋅7H2O, 74.9 mg ⋅l1 K2HPO4, 175 mg ⋅ l–1 KH2PO4, 25.13 mg ⋅l–1 NaCl, 49.68 mg ⋅l–1 C10H16N20Na, 30.86 mg ⋅l–1 KOH, 4.98 mg ⋅ l–1 FeSO4 ⋅ 7H2O, 1.0 mg ⋅l–1 H2SO4, 11.13 mg ⋅l–1 H3BO3, 8.83 mg ⋅ l–1 ZnSO4 ⋅7H2O, 1.0 mg ⋅l–1 MnCl2 ⋅ 4H2O, 6.06 mg ⋅l–1 MoO3, 2.0 mg ⋅ l–1 CuSO4⋅5H2O, 0.49 mg ⋅l–1 Co(NO3)2 ⋅6H2O, and 1.19 mg ⋅ l–1 Na2MoO4 ⋅2H2O (pH =6.3 ±0.5) was used for cultivation. The preculture was then used for the preparation of seed culture in a 500-ml column photobioreactor, which is 45 cm high and 2.8 cm in diameter. Aeration (0.2 vvm) and mixing were carried out by bubbling of 95% air and 5% CO2. The light was supplied by two white fluorescent lamps (FL 20 SSEX_D/18; Osram) from both sides at 40 µE⋅ m–2 ⋅ s–1 at the inner surface of the column. The exponentially growing cells of the seed organism were inoculated into 500-ml column photobioreactors at a working volume of 400 cm3. The pH was kept constant at 6.3 ±0.5 by introducing CO2. Monitoring of H. pluvialis cells during cultivation A Coulter Counter Multisizer II equipped with a Channelyzer Z2 256 (Coulter Electronics, Hialeah, FL, USA) with a 200-µm aperture was used to determine the mean cell number, cell size, and fresh weight of H. pluvialis cells during cultivation. The data were analyzed using AccuComp Software, ver. 2.01. A microscope (CSBHP3; Samwon Scientific Ind., Seoul, Korea) was used to determine the color, viability, and morphology of the cells. Conditions for determination of chlorophyll and astaxanthin concentrations All spectrophotometric measurements were carried out using an HP8453B, Hewlett Packard UV-visible spectrophotometer equipped with 1-cm path length quartz cells. All spectra were recorded from 420 to 520 nm with a 1.0 nm bandwidth. This range includes the absorbance peak of astaxanthin and the main peaks of chlorophyll. First-order derivative spectra were recorded by digital differentiation (convolution method). All solvents were of analytical grade and were used without further purification (Merck, Darmstadt, Germany). Authentic astaxanthin and chlorophyll (Sigma Chemical, St. Louis, MO, USA) were used to determine the calibration curves. Standard stock solutions of astaxanthin and chlorophyll were prepared by dissolving 5.0 µg each of astaxanthin and chlorophyll in 50.0 ml of acetone, respectively. The stock solutions were serially diluted with acetone for the preparation of standard solutions for the purpose of calibration. Preparation and analysis of samples H. pluvialis cells showed morphological changes from green, motile, flagellated cells to red haematocysts without flagella during cultivation. During cultivation, samples of H. pluvialis were taken, which were motile green cells of a relatively small size (18 µm) and red haematocyst
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cells of a relatively large size (about 50 µm). The samples were divided into four types on the basis of their color: green, greenbrown, brown-red, or red. Culture broths (1.0 ml) of green, greenbrown, brown-red, and red H. pluvialis cells were centrifuged at 3000×g for 10 min. After discarding the supernatant, 1.0 ml of acetone was added to the cell pellet. The pigments were extracted in acetone and disrupted using a specially-designed tissue homogenizer (Bodine Electric Company, Chicago, IL, USA) for 2 min with 12 strokes at 4000 rpm. Pigment extraction was stopped when cell debris became colorless and the extracted supernatant showed no absorbance. Samples were then kept at 4°C for 20 min, and UVvisible spectra were taken after 2–20 times dilution depending on the cell concentration (14). In the investigation of a suitable solvent for the extraction of chlorophyll and astaxanthin from cells, 1.0 ml of culture broths of green, green-brown, brown-red, and red H. pluvialis cells was centrifuged at 3000×g for 10 min. After discarding the supernatant, the cells were resuspended in 1.0 ml of solvent. The pigments were extracted with six different water-miscible organic solvents of acetone, methanol, hexane, chloroform (CHCl3), n-propanol, and acetonitrile (CH3CN) (volume ratio, 5% acetone + 95% of the six different solvents (v/v). The absorbance of the green and red cells was measured after 2 min of cell disruption using a tissue homogenizer (Bodine Electric Company) at 4000 rpm. The cells were disrupted under high shear stress in a narrow space between the vessel wall and the rotor. The optimal duration homogenization was tested at three levels of 1, 2, and 3 min with 12 strokes. In a separate experiment, acetone was added to cell pellets (without mechanical extraction by a tissue homogenizer), and visual observation was carried out to check the efficiency of pigment extraction by acetone for the different types of cell. For the preparation of the astaxanthin calibration curve, stock astaxanthin solutions of various volumes (20.0–150.0 µl) were diluted with acetone in 1.0-ml falcon tubes to obtain astaxanthin working solutions (2.0, 3.0, 4.0, 6.0, 9.0, and 12.0 µg ⋅ml–1). The same procedure was performed for the preparation of chlorophyll calibration solutions. Stock chlorophyll solutions of various volumes (40.0–200.0 µl) were diluted with acetone in 1.0-ml falcon tubes to obtain chlorophyll working solutions (4.0, 8.0, 12.0, 16.0, and 20.0 µg ⋅ml–1). Spectra were recorded using acetone as the blank, and the concentrations of chlorophyll and astaxanthin were determined by first-derivative spectrophotometry for all samples. To calculate the concentrations of chlorophyll and astaxanthin by first-derivative spectrophotometry, the concentration of astaxanthin was kept constant at 3.0 µg⋅ml–1 and that of chlorophyll was varied from 0.0 to 20.0. The concentration of chlorophyll was then kept constant at 4.0 µg⋅ml–1 and that of astaxanthin was varied from 0.0 to 12. The absorbance of all samples was determined and their first-derivative altitudes were calculated. The calibration curves were then obtained for known standard solutions, and the curves for unknown samples were compared with the calibration curves. The same samples were also analyzed by HPLC (Younglin Instrument, Anyang, Korea) using two M930D pumps and M730D photodiode array (PDA) detector equipped with a reverse-phase column (C18 column, 300 ×3.9 mm; 5 µm; Waters, Milford, MA, USA). The mobile phase (acetone at a flow rate of 1.0 ml ⋅min–1) and the absorbance of the effluent solution were measured at 477 nm and 680 nm, respectively. The concentration was determined by comparing the peaks of samples with those of the known standards.
RESULTS AND DISCUSSION Effects of solvent
Figure 1 shows the absorption
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spectra of the chlorophyll and astaxanthin extracted from H. pluvialis cells with different solvents. Chlorophyll showed maximum absorbance in acetone (the subtraction of the chlorophyll peak and the lowest points of the spectrum was highest) and showed minimum absorbance in chloroform. Astaxanthin showed maximum absorbance in acetonitrile and showed minimum absorbance in n-propanol. The absorbance of astaxanthin in acetone remained stable for a long time, but the absorbance of chlorophyll in acetone drifted after 30 min (data not shown). Acetone has good sensitivity and has frequently been used for pigment extraction; moreover acetone is a considerably less toxic than other solvents. In this study, acetone was chosen as the working solvent. Figure 2a shows chlorophyll and astaxanthin extracted with acetone. No mechanical cell disruption was utilized in this experiment. A larger amount of chlorophyll was extracted from green cells with thin cell walls than red cells
FIG. 1. (a) Absorption spectra of chlorophyll in different solvents. (b) Absorption spectra of astaxanthin in different solvents. Symbols: open squares, chloroform; closed rhombuses, acetone; open triangles, methanol; open circles, hexane; closed circles, acetonitrile; and open rhombuses, n-propanol.
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with thick cell walls. On the other hand, almost no astaxanthin was extracted from cells with thick cell walls under the same conditions. Figures 2b and 2c show the absorption spectra of chlorophyll and astaxanthin after 1, 2, and 3 min of extraction in acetone after homogenization. Compared with astaxanthin, higher amounts of chlorophyll were extracted from the cells after exposure to a homogenization for 1 min, which suggests that the extraction of the pigments from green cells with thin walls is easier than that from red heterocyst cells with thick walls. Graph calibration and precision Figure 3a shows the spectra of the zero-order derivatives of chlorophyll (curve 1), astaxanthin (curve 2), and a mixture of chlorophyll and astaxanthin (curve 3) in the range of 420–520 nm. As shown in Fig. 3a, in this range, astaxanthin shows the maximum absorption at 477 nm, whereas chlorophyll shows two maxima, at 431 nm and 455 nm. Figure 3b shows the spectra of zero-crossing wavelengths of chlorophyll and astaxanthin at 431 nm, 455 nm, and 477 nm. The peak maxima for chlorophyll and astaxanthin were obtained from first-derivative spectra at 455 nm and 477 nm (dA/dλ ≅ 0 at peak maxima). In preliminary experiments, it was found that the sensitivity of the determination of chlorophyll and astaxanthin concentrations is higher at 455 nm than at 431 nm, and 455 nm was selected for the determination of chlorophyll and astaxanthin concentrations (data not shown). That is, the spectral band of astaxanthin generally overlaps with that of chlorophyll within the range of 440–520 nm, rendering the simultaneous determination of these pigments impossible by UVvisible absorbance measurement. The greatest overlaping of chlorophyll and astaxanthin peaks is at 455 and 477 nm;
FIG. 2. (a) Extraction of chlorophyll and astaxanthin from green, green-brown, brown-red, and red cells without mechanical disruption with homogenizer. (b) Chlorophyll extraction and (c) astaxanthin extraction for 1 (curve 1), 2 (curve 2), and 3 (curve 3) min.
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FIG. 3. (a) Zero- and (b) first-derivative spectra of chlorophyll (curve 1), astaxanthin (curve 2), and a mixture of chlorophyll and astaxantion (curve 3). CChl = 4.0 µg⋅ ml–1, CAs = 3.0 µg⋅ ml–1, CChl = 4.0 µg⋅ ml–1 + CAs = 3.0 µg⋅ml–1.
FIG. 4. (a) Zero- and (b) first-derivative spectra of astaxanthin and chlorophyll mixtures. Cas = 3.0 µg⋅ ml–1 and Cchl = 0.0, 4.0, 8.0, 12.0, 16.0, and 20.0 µg⋅ml–1. These concentrations correspond to lines 1, 2, 3, 4, 5, and 6, respectively.
therefore, these two values were selected for the measurement of chlorophyll and astaxanthin concentrations in the mixture (Fig. 3). The molar absorptivity of astaxanthin was calculated from the slopes of the regression lines of absorption versus different concentrations of pure astaxanthin at 477 nm. The molar absorptivity was determined to be 6.96 ×104 l ⋅ mol–1 ⋅cm–1 (R2 = 0.9982). The same procedure was applied to chlorophyll at 455 nm, and the molar absorptivity was determined to be 8.87 ×103 l ⋅mol–1 ⋅ cm–1 (R2 = 0.9974). Figures 4a and 4b show a set of zero- and first-derivative spectra of mixtures containing 3.0 µg ⋅ ml–1 astaxanthin and increasing amounts of chlorophyll (0.0, 4.0, 8.0, 12.0, 16.0, and 20.0 µg⋅ml–1). Figures 5a and 5b show a set of the zero- and first-derivative spectra of mixtures containing 4.0 µg ⋅ml–1 chlorophyll and increasing amounts of astaxanthin (0.0, 2.0, 4.0, 6.0, 9.0, and 12.0 µg ⋅ ml–1). The results shown in Figs. 4b and 5b indicate that when the concentration of astaxanthin was kept constant and the concentration of chlorophyll was varied, the peak amplitudes at 455 nm were unaltered. When the concentration of chlorophyll was kept constant and the concentration of astaxanthin was varied, the peak amplitudes at 477 nm were unaltered. The amplitudes at 477 nm (h1) and 455 nm (h2) were proportional
to chlorophyll and astaxanthin concentrations, respectively. That is, the signals of the first-derivative at 477 nm (zerocrossing wavelength of astaxanthin) were only proportional to chlorophyll concentration, whereas the first-derivative signals at 455 nm (zero-crossing wavelength of chlorophyll) were proportional to astaxanthin concentration regardless of chlorophyll concentration. A peak maximum of 477 nm was selected for chlorophyll, and a peak maximum of 455 nm was selected for the measurement of chlorophyll and astaxanthin concentrations. The concentration of chlorophyll was determined from the height (h1) of the first-derivative signal (zero-crossing point for astaxanthin) at 477 nm, and the astaxanthin concentration was determined from the height (h2) of the first-derivative signal (zero-crossing point for chlorophyll) at 455 nm (Figs. 4 and 5). To construct calibration graphs, the first-derivative absorbances (1Ds) of the chlorophyll series containing 3.0 µg⋅ ml–1 astaxanthin and those of the astaxanthin series containing 4.0 µg⋅ml–1 chlorophyll were measured against acetone as the blank for the calibration mixtures. The concentrations of chlorophyll and astaxanthin were determined by measuring 1 Ds at 477 and 455 nm, respectively. Calibration graphs for chlorophyll and astaxanthin were constructed by plotting the amplitudes of peaks at 477 nm (h1) for chlorophyll and
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FIG. 6. Calibration diagram for chlorophyll determination by firstorder derivative spectrophotometry in the presence of astaxanthin. Cas = 3.0 µg⋅ ml–1 and curve: 4.0–20.0 µg⋅ ml–1 chlorophyll.
FIG. 5. (a) Zero- and (b) first-derivative spectra of chlorophyll and astaxanthin mixtures. Cchl = 4.0 µg⋅ml–1 and Cas = 0.0, 2.0, 4.0, 6.0, 9.0, and 12.0 µg⋅ml–1. These concentrations correspond to lines 1, 2, 3, 4, 5, and 6, respectively.
455 nm (h2) for astaxanthin of the first-derivative signal (dA/dλ) against the different concentrations of chlorophyll and astaxanthin. The concentrations of astaxanthin and chlorophyll were estimated from the linear calibration curves of 1 Ds versus concentration (Figs. 6 and 7). The calibration curve for chlorophyll using 1Ds can be represented by the following equation for concentrations between 0 and 20.0 µg ⋅ml–1 chlorophyll: D477 = 1.9875Cchl − 0.0991
1
(R2 = 0.9958)
(1) 1
The calibration curve for astaxanthin using Ds can be represented by the following equation for concentrations between 0 and 12.0 µg ⋅ml–1 astaxanthin: D455 = 0.5789Cas − 0.5872
1
(R2 = 0.9975)
(2)
The precision and reproducibility of the method were determined using 10 identical samples in a mixture containing 7.0 µg ⋅ cm–3 chlorophyll and 5.0 µg ⋅ cm–3 astaxanthin. The detection limits for the analytical procedure were 0.25 and 0.35 µg⋅ ml–1 for astaxanthin and chlorophyll (signal-to-noise ratio = 2), respectively. The relative standard deviations (n = 10) for the determination of 7.0 µg ⋅ml–1 chlorophyll and 5.0 µg ⋅ ml–1 astaxanthin were 1.2 and 1.1%, respectively.
FIG. 7. Calibration diagram for astaxanthin determination by firstorder derivative spectrophotometry in the presence of chlorophyll. Cchl =4.0 µg⋅ ml–1 and curve: 2.0–12.0 µg⋅ ml–1 astaxanthin.
Simultaneous determination of astaxanthin and chlorophyll in H. pluvialis cells Tables 1 and 2 show a comparison of the results of analyses using the developed method and conventional HPLC for the green, green-brown, brownred, and red H. pluvialis cells. After the calculation of the pooled estimate of standard deviation, the null hypothesis was tested using the equation: X1 − X2 = ±Spooled[(N1+N2)/N1N2]1/2
(3)
obtaining t = 1.75, which was less than the critical value of (t0.95). This finding showed that the null hypothesis could not be rejected. Thus, the two population means are of comparable precision, meaning that the two methods gave similar results with no significant differences in means at a 95% confidence level. Table 3 shows relative changes in chlorophyll and astaxanthin concentrations during the morphological changes of H. pluvialis cells. Conclusion A simple, rapid derivative spectrophotometric method has been developed for the simultaneous analysis of chlorophyll and astaxanthin mixtures and samples of H. pluvialis cells using acetone as the solvent. The
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TABLE 1. Comparison of the precisions of the HPLC and developed method in the measurement of chlorophyll in 1.0 ml of H. pluvialis culture broth Parameters
HPLC G 46.6 2.00 43.6 49.4 1.60 10
GB 30.6 1.02 28.8 32.0 0.817 10
BR 18.1 1.76 16.2 21.3 1.25 10
Mean SD Minimum value Maximum value Variance Number of measurements (N) t-test (95% confidence level) G, Green; GB, green-brown; BR, brown-red; R, red.
R 1.70 1.21 0.467 4.15 0.776 10
G 46.7 1.48 44.7 48.7 1.01 10 0.137
Developed method GB BR 30.8 18.2 1.37 1.68 28.4 16.7 32.7 21.3 0.966 1.14 10 10 0.317 0.100
R 1.68 1.19 0.459 4.09 0.759 10 −0.0336
TABLE 2. Comparison of the precisions of the HPLC and developed method in the measurement of astaxanthin in 1.0 ml of H. pluvialis culture broth Parameters
HPLC G 2.92 1.18 1.57 4.52 0.980 10
Mean SD Minimum value Maximum value Variance Number of measurements (N) t-test (95% confidence level) G, Green; GB, green-brown; BR, brown-red; R, red.
GB 5.95 1.50 3.98 8.43 1.13 10
BR 29.1 1.72 27.1 32.6 1.32 10
TABLE 3. The percentage of chlorophyll and astaxanthin in different morphological conditions of H. pluvialis cells Cells Green Green-brown Brown-red Red
HPLC Chlorophyll Astaxanthin 15.96 0.06 5.14 0.19 0.62 16.1 0.03 36.56
Developed method Chlorophyll Astaxanthin 16.06 0.09 5.18 0.33 0.63 17.24 0.03 36.66
method does not require a separation step conventionally used in HPLC. Beer’s law was satisfied in terms of the concentration ranges, 0–12.0 µg ⋅ml–1 for astaxanthin and 0–20.0 µg ⋅ ml–1 for chlorophyll. The molar absorptivities for astaxanthin and chlorophyll at λmax = 477 nm and λmax = 455 nm were 69513.65 l ⋅ mol–1 ⋅cm–1 and 8873.55 l ⋅mol–1 ⋅ cm–1, respectively. The analytical characteristics of the proposed method, such as sensitivity, detection limit, and coefficient of variation were determined. The method was applied to the determination of astaxanthin and chlorophyll in H. pluvialis cells. The proposed method is suitable for routine analysis. The procedure is simple, rapid, and reliable. A good agreement was achieved between the results obtained by the proposed method and the conventional HPLC. ACKNOWLEDGMENTS This study was supported by the Korea Research Foundation Grant (KRF-2004-005-D00002), for which the authors are grateful.
R 62.2 1.95 59.5 64.9 1.45 10
G 2.91 1.17 1.58 4.48 0.964 10 −0.0219
Developed method GB BR 5.94 29.0 1.48 1.76 4.03 27.5 8.32 33.1 1.13 1.29 10 10 −0.0191 −0.0759
R 61.7 1.56 60.6 64.3 1.09 10 −0.623
REFERENCES 1. Guerin, M., Huntley, M. E., and Olaizola, M.: Haematococcus astaxanthin: applications for human health and nutrition. Trends Biotechnol., 21, 210–216 (2003). 2. Gross, G. J. and Lockwood, S. F.: Cardioprotection and myocardial salvage by a disodium disuccinate astaxanthin derivative (Cardax™). Life Sci., 75, 215–224 (2004). 3. Olaizola, M.: Commercial production of astaxanthin from Haematococcus pluvialis using 25,000-liter outdoor photobioreactors. J. Appl. Phycol., 12, 499–506 (2000). 4. Choi, S.-L., Suh, I.-S., and Lee, C.-G.: Lumostatic operation of bubble column photobioreactors for Haematococcus pluvialis cultures using a specific light uptake rate as a control parameter. Enzyme Microb. Technol., 33, 403–409 (2003). 5. Lababpour, A., Hada, K., Shimahara, K., Katsuda, T., and Katoh, S.: Effects of nutrient supply methods and illumination with blue light emitting diodes (LEDs) on astaxanthin production by Haematococcus pluvialis. J. Biosci. Bioeng., 98, 452–456 (2004). 6. Yuan, J.-P. and Chen, F.: Chromatographic separation and purification of trans-astaxanthin from the extracts of Haematococcus pluvialis. J. Agric. Food Chem., 46, 3371–3375 (1998). 7. Yuan, J.-P. and Chen, F.: Isomerization of trans-astaxanthin to cis-isomers in organic solvents. J. Agric. Food Chem., 47, 3656–3660 (1999). 8. Lacker, T., Strohschein, S., and Albert, K.: Separation and identification of various carotenoids by C30 reversed-phase high-performance liquid chromatography coupled to UV and atmospheric pressure chemical ionization mass spectrometric detection. J. Chromatogr., 854, 37–44 (1999). 9. Yuan, J.-P., Chen, F., Liu, X., and Li, X. Z.: Carotenoid composition in the green microalga Chlorococcum. Food Chem., 76, 319–325 (2002). 10. Macías-Sánchez, M. D., Mantell, C., Rodríguez, M., Martínez de la Ossa, E., Lubián, L. M., and Montero, O.: Supercritical fluid extraction of carotenoids and chlorophyll a
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© 2006, The Society for Biotechnology, Japan
Simultaneous Measurement of Chlorophyll and Astaxanthin in Haematococcus pluvialis Cells by First-Order Derivative Ultraviolet-Visible Spectrophotometry Abdolmajid Lababpour1 and Choul-Gyun Lee1* Department of Biotechnology, Institute of Industrial Biotechnology, Inha University, 253 Younghyun-Dong, Nam-Gu, Incheon 402-751, Republic of Korea1 Received 5 September 2005/Accepted 27 October 2005
A first-order derivative spectrophotometric method has been developed for the simultaneous measurement of chlorophyll and astaxanthin concentrations in Haematococcus pluvialis cells. Acetone was selected for the extraction of pigments because of its good sensitivity and low toxicity compared with other organic solvents tested; the tested solvents included acetone, methanol, hexane, chloroform, n-propanol, and acetonitrile. A first-order derivative spectrophotometric method was used to eliminate the effects of the overlaping of the chlorophyll and astaxanthin peaks. The linear ranges in 1D evaluation were from 0.50 to 20.0 µg⋅ ml–1 for chlorophyll and from 1.00 to 12.0 µg⋅ ml–1 for astaxanthin. The limits of detection of the analytical procedure were found to be 0.35 µg⋅ ml–1 for chlorophyll and 0.25 µg ⋅ml–1 for astaxanthin. The relative standard deviations for the determination of 7.0 µg⋅ml–1 chlorophyll and 5.0 µg⋅ml–1 astaxanthin were 1.2% and 1.1%, respectively. The procedure was found to be simple, rapid, and reliable. This method was successfully applied to the determination of chlorophyll and astaxanthin concentrations in H. pluvialis cells. A good agreement was achieved between the results obtained by the proposed method and HPLC method. [Key words: chlorophyll, astaxanthin, first-order derivative, UV-visible spectrophotometry]
thin in H. pluvialis cells has already been reported (8–10). In these reports, the analysis of chlorophyll and astaxanthin requires a separation step that necessitates costly instrumentation of HPLC. The separation by HPLC is not as convenient or as rapid as that by spectrophotometry. The spectrophotometric determination of chlorophyll has already been reported (11); however, to the best of our knowledge, no analytical techniques have been reported for the simultaneous measurement of chlorophyll and astaxanthin in H. pluvialis cells by UV-visible spectrophotometry. The simultaneous measurement of chlorophyll and astaxanthin by common UV-visible spectrophotometry from their absorbance (zero-order derivative) is difficult, as these two pigments mutually interfere caused by a considerable overlaping of the peaks of their carbon chains that normally requires a separation step. Derivative spectrophotometry is a useful technique that can be used to resolve the overlapping of spectral peaks. This technique has been utilized for the simultaneous determination of a system of two or more components in analytical chemistry (12). The first (and higher) derivative absorption spectrum is the first (and higher) derivative of the absorbance as a function of wavelength. The higher derivative leads to a decrease in sensitivity and a loss of spectral information. Derivative spectrophotometry for the simultaneous measurement of chlorophyll and astaxanthin has not yet been reported; therefore, it is one of the main features of this study. Additionally, the re-
Astaxanthin, a natural red ketocarotenoid (3,3′-dihydroxyβ,β-carotene-4,4′-dione), has been used in clinical studies for its role in health conditions. It is also used as a pigment source in aquaculture and poultry, and as a food additive to supply nutrients or color, or for other biological activities (1, 2). The photosynthetic microalga, Haematococcus pluvialis, is a rich, natural source of astaxanthin. The cultivation of H. pluvialis has already been reported for a largescale production of natural astaxanthin (3). Motile green H. pluvialis cells mainly contain chlorophyll and start astaxanthin biosynthesis under some stress conditions, such as nutrient deficiency and high light intensity (4, 5). The monitoring of chlorophyll and astaxanthin concentrations is necessary to control H. pluvialis cultivation. The control of this cultivation also necessitates an accurate, precise, and easy handled method for the rapid and inexpensive determination of pigments both on the laboratory and industrial scales. The methods reported in the literature for the determination of chlorophyll and astaxanthin concentrations can be used to quantify pigments extracted by mechanical or solvent methods (6, 7). The extracted pigments are then subjected further separation and identification by high-performance liquid chromatography (HPLC). The simultaneous measurement of pigments, such as chlorophyll and astaxan* Corresponding author. e-mail: [email protected] phone: +82-32-860-7518 fax: +82-32-872-4046 104
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sults of this study can be used for the easier, precise, and accurate measurement of chlorophyll and astaxanthin concentrations in H. pluvialis cells. Accordingly, in this paper, we describe the effects of organic solvents for pigment extraction and the application of first-derivative UV-visible spectrophotometry to the simultaneous measurement of chlorophyll and astaxanthin in H. pluvialis cells, thereby eliminating the need for a separation step during pigment measurement.
MATERIALS AND METHODS Microorganism and cultivation conditions The photosynthetic microalga, H. pluvialis UTEX 16 (Culture Collection of Algae, University of Texas, Austin, TX, USA), was precultivated in a 200-ml Erlenmeyer flask under continuous light intensity of 40 µE⋅ m–2 ⋅s–1 using a fluorescent lamp in the photoautotrophic mode of operation. Modified Bold’s basal medium (MBBM) containing 246.5 mg ⋅ l–1 NaNO3, 24.99 mg⋅ l–1 CaCl2 ⋅ 2H2O, 73.95 mg ⋅l–1 MgSO4 ⋅7H2O, 74.9 mg ⋅l1 K2HPO4, 175 mg ⋅ l–1 KH2PO4, 25.13 mg ⋅l–1 NaCl, 49.68 mg ⋅l–1 C10H16N20Na, 30.86 mg ⋅l–1 KOH, 4.98 mg ⋅ l–1 FeSO4 ⋅ 7H2O, 1.0 mg ⋅l–1 H2SO4, 11.13 mg ⋅l–1 H3BO3, 8.83 mg ⋅ l–1 ZnSO4 ⋅7H2O, 1.0 mg ⋅l–1 MnCl2 ⋅ 4H2O, 6.06 mg ⋅l–1 MoO3, 2.0 mg ⋅ l–1 CuSO4⋅5H2O, 0.49 mg ⋅l–1 Co(NO3)2 ⋅6H2O, and 1.19 mg ⋅ l–1 Na2MoO4 ⋅2H2O (pH =6.3 ±0.5) was used for cultivation. The preculture was then used for the preparation of seed culture in a 500-ml column photobioreactor, which is 45 cm high and 2.8 cm in diameter. Aeration (0.2 vvm) and mixing were carried out by bubbling of 95% air and 5% CO2. The light was supplied by two white fluorescent lamps (FL 20 SSEX_D/18; Osram) from both sides at 40 µE⋅ m–2 ⋅ s–1 at the inner surface of the column. The exponentially growing cells of the seed organism were inoculated into 500-ml column photobioreactors at a working volume of 400 cm3. The pH was kept constant at 6.3 ±0.5 by introducing CO2. Monitoring of H. pluvialis cells during cultivation A Coulter Counter Multisizer II equipped with a Channelyzer Z2 256 (Coulter Electronics, Hialeah, FL, USA) with a 200-µm aperture was used to determine the mean cell number, cell size, and fresh weight of H. pluvialis cells during cultivation. The data were analyzed using AccuComp Software, ver. 2.01. A microscope (CSBHP3; Samwon Scientific Ind., Seoul, Korea) was used to determine the color, viability, and morphology of the cells. Conditions for determination of chlorophyll and astaxanthin concentrations All spectrophotometric measurements were carried out using an HP8453B, Hewlett Packard UV-visible spectrophotometer equipped with 1-cm path length quartz cells. All spectra were recorded from 420 to 520 nm with a 1.0 nm bandwidth. This range includes the absorbance peak of astaxanthin and the main peaks of chlorophyll. First-order derivative spectra were recorded by digital differentiation (convolution method). All solvents were of analytical grade and were used without further purification (Merck, Darmstadt, Germany). Authentic astaxanthin and chlorophyll (Sigma Chemical, St. Louis, MO, USA) were used to determine the calibration curves. Standard stock solutions of astaxanthin and chlorophyll were prepared by dissolving 5.0 µg each of astaxanthin and chlorophyll in 50.0 ml of acetone, respectively. The stock solutions were serially diluted with acetone for the preparation of standard solutions for the purpose of calibration. Preparation and analysis of samples H. pluvialis cells showed morphological changes from green, motile, flagellated cells to red haematocysts without flagella during cultivation. During cultivation, samples of H. pluvialis were taken, which were motile green cells of a relatively small size (18 µm) and red haematocyst
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cells of a relatively large size (about 50 µm). The samples were divided into four types on the basis of their color: green, greenbrown, brown-red, or red. Culture broths (1.0 ml) of green, greenbrown, brown-red, and red H. pluvialis cells were centrifuged at 3000×g for 10 min. After discarding the supernatant, 1.0 ml of acetone was added to the cell pellet. The pigments were extracted in acetone and disrupted using a specially-designed tissue homogenizer (Bodine Electric Company, Chicago, IL, USA) for 2 min with 12 strokes at 4000 rpm. Pigment extraction was stopped when cell debris became colorless and the extracted supernatant showed no absorbance. Samples were then kept at 4°C for 20 min, and UVvisible spectra were taken after 2–20 times dilution depending on the cell concentration (14). In the investigation of a suitable solvent for the extraction of chlorophyll and astaxanthin from cells, 1.0 ml of culture broths of green, green-brown, brown-red, and red H. pluvialis cells was centrifuged at 3000×g for 10 min. After discarding the supernatant, the cells were resuspended in 1.0 ml of solvent. The pigments were extracted with six different water-miscible organic solvents of acetone, methanol, hexane, chloroform (CHCl3), n-propanol, and acetonitrile (CH3CN) (volume ratio, 5% acetone + 95% of the six different solvents (v/v). The absorbance of the green and red cells was measured after 2 min of cell disruption using a tissue homogenizer (Bodine Electric Company) at 4000 rpm. The cells were disrupted under high shear stress in a narrow space between the vessel wall and the rotor. The optimal duration homogenization was tested at three levels of 1, 2, and 3 min with 12 strokes. In a separate experiment, acetone was added to cell pellets (without mechanical extraction by a tissue homogenizer), and visual observation was carried out to check the efficiency of pigment extraction by acetone for the different types of cell. For the preparation of the astaxanthin calibration curve, stock astaxanthin solutions of various volumes (20.0–150.0 µl) were diluted with acetone in 1.0-ml falcon tubes to obtain astaxanthin working solutions (2.0, 3.0, 4.0, 6.0, 9.0, and 12.0 µg ⋅ml–1). The same procedure was performed for the preparation of chlorophyll calibration solutions. Stock chlorophyll solutions of various volumes (40.0–200.0 µl) were diluted with acetone in 1.0-ml falcon tubes to obtain chlorophyll working solutions (4.0, 8.0, 12.0, 16.0, and 20.0 µg ⋅ml–1). Spectra were recorded using acetone as the blank, and the concentrations of chlorophyll and astaxanthin were determined by first-derivative spectrophotometry for all samples. To calculate the concentrations of chlorophyll and astaxanthin by first-derivative spectrophotometry, the concentration of astaxanthin was kept constant at 3.0 µg⋅ml–1 and that of chlorophyll was varied from 0.0 to 20.0. The concentration of chlorophyll was then kept constant at 4.0 µg⋅ml–1 and that of astaxanthin was varied from 0.0 to 12. The absorbance of all samples was determined and their first-derivative altitudes were calculated. The calibration curves were then obtained for known standard solutions, and the curves for unknown samples were compared with the calibration curves. The same samples were also analyzed by HPLC (Younglin Instrument, Anyang, Korea) using two M930D pumps and M730D photodiode array (PDA) detector equipped with a reverse-phase column (C18 column, 300 ×3.9 mm; 5 µm; Waters, Milford, MA, USA). The mobile phase (acetone at a flow rate of 1.0 ml ⋅min–1) and the absorbance of the effluent solution were measured at 477 nm and 680 nm, respectively. The concentration was determined by comparing the peaks of samples with those of the known standards.
RESULTS AND DISCUSSION Effects of solvent
Figure 1 shows the absorption
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spectra of the chlorophyll and astaxanthin extracted from H. pluvialis cells with different solvents. Chlorophyll showed maximum absorbance in acetone (the subtraction of the chlorophyll peak and the lowest points of the spectrum was highest) and showed minimum absorbance in chloroform. Astaxanthin showed maximum absorbance in acetonitrile and showed minimum absorbance in n-propanol. The absorbance of astaxanthin in acetone remained stable for a long time, but the absorbance of chlorophyll in acetone drifted after 30 min (data not shown). Acetone has good sensitivity and has frequently been used for pigment extraction; moreover acetone is a considerably less toxic than other solvents. In this study, acetone was chosen as the working solvent. Figure 2a shows chlorophyll and astaxanthin extracted with acetone. No mechanical cell disruption was utilized in this experiment. A larger amount of chlorophyll was extracted from green cells with thin cell walls than red cells
FIG. 1. (a) Absorption spectra of chlorophyll in different solvents. (b) Absorption spectra of astaxanthin in different solvents. Symbols: open squares, chloroform; closed rhombuses, acetone; open triangles, methanol; open circles, hexane; closed circles, acetonitrile; and open rhombuses, n-propanol.
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with thick cell walls. On the other hand, almost no astaxanthin was extracted from cells with thick cell walls under the same conditions. Figures 2b and 2c show the absorption spectra of chlorophyll and astaxanthin after 1, 2, and 3 min of extraction in acetone after homogenization. Compared with astaxanthin, higher amounts of chlorophyll were extracted from the cells after exposure to a homogenization for 1 min, which suggests that the extraction of the pigments from green cells with thin walls is easier than that from red heterocyst cells with thick walls. Graph calibration and precision Figure 3a shows the spectra of the zero-order derivatives of chlorophyll (curve 1), astaxanthin (curve 2), and a mixture of chlorophyll and astaxanthin (curve 3) in the range of 420–520 nm. As shown in Fig. 3a, in this range, astaxanthin shows the maximum absorption at 477 nm, whereas chlorophyll shows two maxima, at 431 nm and 455 nm. Figure 3b shows the spectra of zero-crossing wavelengths of chlorophyll and astaxanthin at 431 nm, 455 nm, and 477 nm. The peak maxima for chlorophyll and astaxanthin were obtained from first-derivative spectra at 455 nm and 477 nm (dA/dλ ≅ 0 at peak maxima). In preliminary experiments, it was found that the sensitivity of the determination of chlorophyll and astaxanthin concentrations is higher at 455 nm than at 431 nm, and 455 nm was selected for the determination of chlorophyll and astaxanthin concentrations (data not shown). That is, the spectral band of astaxanthin generally overlaps with that of chlorophyll within the range of 440–520 nm, rendering the simultaneous determination of these pigments impossible by UVvisible absorbance measurement. The greatest overlaping of chlorophyll and astaxanthin peaks is at 455 and 477 nm;
FIG. 2. (a) Extraction of chlorophyll and astaxanthin from green, green-brown, brown-red, and red cells without mechanical disruption with homogenizer. (b) Chlorophyll extraction and (c) astaxanthin extraction for 1 (curve 1), 2 (curve 2), and 3 (curve 3) min.
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FIG. 3. (a) Zero- and (b) first-derivative spectra of chlorophyll (curve 1), astaxanthin (curve 2), and a mixture of chlorophyll and astaxantion (curve 3). CChl = 4.0 µg⋅ ml–1, CAs = 3.0 µg⋅ ml–1, CChl = 4.0 µg⋅ ml–1 + CAs = 3.0 µg⋅ml–1.
FIG. 4. (a) Zero- and (b) first-derivative spectra of astaxanthin and chlorophyll mixtures. Cas = 3.0 µg⋅ ml–1 and Cchl = 0.0, 4.0, 8.0, 12.0, 16.0, and 20.0 µg⋅ml–1. These concentrations correspond to lines 1, 2, 3, 4, 5, and 6, respectively.
therefore, these two values were selected for the measurement of chlorophyll and astaxanthin concentrations in the mixture (Fig. 3). The molar absorptivity of astaxanthin was calculated from the slopes of the regression lines of absorption versus different concentrations of pure astaxanthin at 477 nm. The molar absorptivity was determined to be 6.96 ×104 l ⋅ mol–1 ⋅cm–1 (R2 = 0.9982). The same procedure was applied to chlorophyll at 455 nm, and the molar absorptivity was determined to be 8.87 ×103 l ⋅mol–1 ⋅ cm–1 (R2 = 0.9974). Figures 4a and 4b show a set of zero- and first-derivative spectra of mixtures containing 3.0 µg ⋅ ml–1 astaxanthin and increasing amounts of chlorophyll (0.0, 4.0, 8.0, 12.0, 16.0, and 20.0 µg⋅ml–1). Figures 5a and 5b show a set of the zero- and first-derivative spectra of mixtures containing 4.0 µg ⋅ml–1 chlorophyll and increasing amounts of astaxanthin (0.0, 2.0, 4.0, 6.0, 9.0, and 12.0 µg ⋅ ml–1). The results shown in Figs. 4b and 5b indicate that when the concentration of astaxanthin was kept constant and the concentration of chlorophyll was varied, the peak amplitudes at 455 nm were unaltered. When the concentration of chlorophyll was kept constant and the concentration of astaxanthin was varied, the peak amplitudes at 477 nm were unaltered. The amplitudes at 477 nm (h1) and 455 nm (h2) were proportional
to chlorophyll and astaxanthin concentrations, respectively. That is, the signals of the first-derivative at 477 nm (zerocrossing wavelength of astaxanthin) were only proportional to chlorophyll concentration, whereas the first-derivative signals at 455 nm (zero-crossing wavelength of chlorophyll) were proportional to astaxanthin concentration regardless of chlorophyll concentration. A peak maximum of 477 nm was selected for chlorophyll, and a peak maximum of 455 nm was selected for the measurement of chlorophyll and astaxanthin concentrations. The concentration of chlorophyll was determined from the height (h1) of the first-derivative signal (zero-crossing point for astaxanthin) at 477 nm, and the astaxanthin concentration was determined from the height (h2) of the first-derivative signal (zero-crossing point for chlorophyll) at 455 nm (Figs. 4 and 5). To construct calibration graphs, the first-derivative absorbances (1Ds) of the chlorophyll series containing 3.0 µg⋅ ml–1 astaxanthin and those of the astaxanthin series containing 4.0 µg⋅ml–1 chlorophyll were measured against acetone as the blank for the calibration mixtures. The concentrations of chlorophyll and astaxanthin were determined by measuring 1 Ds at 477 and 455 nm, respectively. Calibration graphs for chlorophyll and astaxanthin were constructed by plotting the amplitudes of peaks at 477 nm (h1) for chlorophyll and
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FIG. 6. Calibration diagram for chlorophyll determination by firstorder derivative spectrophotometry in the presence of astaxanthin. Cas = 3.0 µg⋅ ml–1 and curve: 4.0–20.0 µg⋅ ml–1 chlorophyll.
FIG. 5. (a) Zero- and (b) first-derivative spectra of chlorophyll and astaxanthin mixtures. Cchl = 4.0 µg⋅ml–1 and Cas = 0.0, 2.0, 4.0, 6.0, 9.0, and 12.0 µg⋅ml–1. These concentrations correspond to lines 1, 2, 3, 4, 5, and 6, respectively.
455 nm (h2) for astaxanthin of the first-derivative signal (dA/dλ) against the different concentrations of chlorophyll and astaxanthin. The concentrations of astaxanthin and chlorophyll were estimated from the linear calibration curves of 1 Ds versus concentration (Figs. 6 and 7). The calibration curve for chlorophyll using 1Ds can be represented by the following equation for concentrations between 0 and 20.0 µg ⋅ml–1 chlorophyll: D477 = 1.9875Cchl − 0.0991
1
(R2 = 0.9958)
(1) 1
The calibration curve for astaxanthin using Ds can be represented by the following equation for concentrations between 0 and 12.0 µg ⋅ml–1 astaxanthin: D455 = 0.5789Cas − 0.5872
1
(R2 = 0.9975)
(2)
The precision and reproducibility of the method were determined using 10 identical samples in a mixture containing 7.0 µg ⋅ cm–3 chlorophyll and 5.0 µg ⋅ cm–3 astaxanthin. The detection limits for the analytical procedure were 0.25 and 0.35 µg⋅ ml–1 for astaxanthin and chlorophyll (signal-to-noise ratio = 2), respectively. The relative standard deviations (n = 10) for the determination of 7.0 µg ⋅ml–1 chlorophyll and 5.0 µg ⋅ ml–1 astaxanthin were 1.2 and 1.1%, respectively.
FIG. 7. Calibration diagram for astaxanthin determination by firstorder derivative spectrophotometry in the presence of chlorophyll. Cchl =4.0 µg⋅ ml–1 and curve: 2.0–12.0 µg⋅ ml–1 astaxanthin.
Simultaneous determination of astaxanthin and chlorophyll in H. pluvialis cells Tables 1 and 2 show a comparison of the results of analyses using the developed method and conventional HPLC for the green, green-brown, brownred, and red H. pluvialis cells. After the calculation of the pooled estimate of standard deviation, the null hypothesis was tested using the equation: X1 − X2 = ±Spooled[(N1+N2)/N1N2]1/2
(3)
obtaining t = 1.75, which was less than the critical value of (t0.95). This finding showed that the null hypothesis could not be rejected. Thus, the two population means are of comparable precision, meaning that the two methods gave similar results with no significant differences in means at a 95% confidence level. Table 3 shows relative changes in chlorophyll and astaxanthin concentrations during the morphological changes of H. pluvialis cells. Conclusion A simple, rapid derivative spectrophotometric method has been developed for the simultaneous analysis of chlorophyll and astaxanthin mixtures and samples of H. pluvialis cells using acetone as the solvent. The
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TABLE 1. Comparison of the precisions of the HPLC and developed method in the measurement of chlorophyll in 1.0 ml of H. pluvialis culture broth Parameters
HPLC G 46.6 2.00 43.6 49.4 1.60 10
GB 30.6 1.02 28.8 32.0 0.817 10
BR 18.1 1.76 16.2 21.3 1.25 10
Mean SD Minimum value Maximum value Variance Number of measurements (N) t-test (95% confidence level) G, Green; GB, green-brown; BR, brown-red; R, red.
R 1.70 1.21 0.467 4.15 0.776 10
G 46.7 1.48 44.7 48.7 1.01 10 0.137
Developed method GB BR 30.8 18.2 1.37 1.68 28.4 16.7 32.7 21.3 0.966 1.14 10 10 0.317 0.100
R 1.68 1.19 0.459 4.09 0.759 10 −0.0336
TABLE 2. Comparison of the precisions of the HPLC and developed method in the measurement of astaxanthin in 1.0 ml of H. pluvialis culture broth Parameters
HPLC G 2.92 1.18 1.57 4.52 0.980 10
Mean SD Minimum value Maximum value Variance Number of measurements (N) t-test (95% confidence level) G, Green; GB, green-brown; BR, brown-red; R, red.
GB 5.95 1.50 3.98 8.43 1.13 10
BR 29.1 1.72 27.1 32.6 1.32 10
TABLE 3. The percentage of chlorophyll and astaxanthin in different morphological conditions of H. pluvialis cells Cells Green Green-brown Brown-red Red
HPLC Chlorophyll Astaxanthin 15.96 0.06 5.14 0.19 0.62 16.1 0.03 36.56
Developed method Chlorophyll Astaxanthin 16.06 0.09 5.18 0.33 0.63 17.24 0.03 36.66
method does not require a separation step conventionally used in HPLC. Beer’s law was satisfied in terms of the concentration ranges, 0–12.0 µg ⋅ml–1 for astaxanthin and 0–20.0 µg ⋅ ml–1 for chlorophyll. The molar absorptivities for astaxanthin and chlorophyll at λmax = 477 nm and λmax = 455 nm were 69513.65 l ⋅ mol–1 ⋅cm–1 and 8873.55 l ⋅mol–1 ⋅ cm–1, respectively. The analytical characteristics of the proposed method, such as sensitivity, detection limit, and coefficient of variation were determined. The method was applied to the determination of astaxanthin and chlorophyll in H. pluvialis cells. The proposed method is suitable for routine analysis. The procedure is simple, rapid, and reliable. A good agreement was achieved between the results obtained by the proposed method and the conventional HPLC. ACKNOWLEDGMENTS This study was supported by the Korea Research Foundation Grant (KRF-2004-005-D00002), for which the authors are grateful.
R 62.2 1.95 59.5 64.9 1.45 10
G 2.91 1.17 1.58 4.48 0.964 10 −0.0219
Developed method GB BR 5.94 29.0 1.48 1.76 4.03 27.5 8.32 33.1 1.13 1.29 10 10 −0.0191 −0.0759
R 61.7 1.56 60.6 64.3 1.09 10 −0.623
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