The preparation and characterization of bile pigments.

Brief reduction of bilirubin with dilute sodium amalgam was shown to give chromogens containing both vinyl and ethylidene beta-substituents. Acid-catalysed rearrangement of these chromogens with bilirubin or mesobilirubin and subsequent dehydrogenation gave a range of new violins containing unconjugated ethylidene and vinyl substitutents. Rearrangements between mesobilirubinogen, bilirubin and mesobilirubin gave dihydrobiliviolins, mesobiliviolins, biliverdins, dihydrobiliverdins and mesobiliverdins of the IIIalpha, IXalpha and XIIIalpha series. In this way 30 compounds were prepared, purified by t.l.c. as dimethyl esters, and characterized by n.m.r., mass and electronic spectroscopy, and by chemical interconversion and degradation.     

Understanding bilirubin conformation and binding. Circular dichroism of human serum albumin complexes with bilirubin and its esters

Human serum albumin binds tightly and noncovalently to a wide variety of hydrophobic bilirubins, including (4Z,15Z)-bilirubin-IX alpha, its dimethyl ester and mono methyl esters, its mono 2-butyl esters and amides, the dimethyl ester of (4Z,15Z)-mesobilirubin-IV alpha, and even (4Z,15Z)-etiobilirubin-IV gamma. The heteroassociation complexes formed from these highly water-insoluble pigments and the protein can be prepared in pH 7.4 aqueous by using a small quantity of dimethyl sulfoxide as amphiphilic carrier. In those solutions the protein acts as a water-soluble chiral complexation agent to induce an asymmetric transformation of the bound pigment. This is recognized by positive chirality, bisignate induced circular dichroism (CD) Cotton effects that fall in the region of the bichromophoric pigment's long wavelength UV-visible absorption band and are characteristic of intramolecular exciton coupling of the bilirubin component pyrromethenone chromophores. The same-signed CD spectra shared by all the pigments of this work indicate selection at the protein binding site for a positive chirality conformer and suggest a common binding site. The CD intensities, which are greatest ([delta epsilon[ congruent to 50) for pigments with one or two free carboxyl groups, are consistent with a binding model where one salt linkage plays a major role in the enantioselectivity of the right-handed folded conformation stabilized by inter- and intramolecular hydrogen bonds.     

The isolation and characterization of bilirubin diglucuronide, the major bilirubin conjugate in dog and human bile.

The chemical structure of the major conjugate of bilirubin was unequivocally elucidated by structural analysis. The conjugated bilirubins were first separated from the lipid components of human duodenal aspirates or dog gall-bladder bile, and then resolved by t.l.c. into a series of tetrapyrroles. The major tetrapyrrole was then converted into its more stable dipyrrolic azo derivative for further analysis. The conjugated moiety of the azopigment was characterized after methanolysis with sodium methoxide. This reaction yields two types of product, those soluble in water and those soluble in organic solvents. The organic-soluble fraction was shown by t.l.c. and mass spectrometry to contain the methyl esters of the dipyrrolic azo derivatives of bilirubin. The water-soluble materials were analysed by enzymic procedures, t.l.c., n.m.r. spectrometry and combined g.l.c. and mass spectrometry. This analysis showed that the only water-soluble product resulting from the methanolysis was glucuronic acid. The structure was identical with that of pure standards, on both mass spectrometry and n.m.r. spectroscopy. No contaminating moieties were found. Quantitative measurement indicated that the glucuronic acid had been released in a 1:1 molar ratio with the resulting methyl esters of the dipyrrolic azo derivatives of bilirubin. This unequivocally establishes bilirubin diglucuronide as the major pigment present in bile. Past problems with identification of bilirubin diglucuronide were shown to originate from procedures which resulted in incomplete separation and isolation of the azopigments of the conjugated bilirubins, owing to contamination by biliary lipids.     

Isolation of oligomers of 5,6-dihydroxyindole-2-carboxylic acid from the eye of the catfish

The reflecting material of the tapetum lucidum of the sea catfish (Arius felis) was chromatographed on Sephadex LH-20 in methanol–dimethyl sulphoxide–formic acid. Two components were present: one, showing an absorption maximum at 330nm, was tapetal pigment; the other, at 257nm, was an associated nucleoside. The tapetal pigment was extracted in methanol–HCl and isolated by adsorption chromatography on Sephadex LH-20. It yielded a methoxy methyl ester on treatment with diazomethane, and permanganate oxidation gave pyrrole-2,3,5-tricarboxylic acid. From the information provided by u.v. and i.r. spectra of the pigment and its methoxy methyl ester, from elemental analyses and from the oxidation products, we suggest that the tapetal pigment is derived from oxidative coupling of 5,6-dihydroxyindole-2-carboxylic acid. A molecular-weight determination and chromatography of the methoxy methyl ester indicate that the pigment is a mixture of oligomers, among which the tetramers probably predominate. We consider that the monomers are joined mainly by C-C linkages at positions 4 and 7. A synthetic pigment having spectral properties nearly identical with those of the natural pigment was prepared by enzymic oxidation of 5,6-dihydroxyindole-2-carboxylic acid with mushroom tyrosinase. The identity of the tapetal pigment with the synthetic pigment was further confirmed by comparing u.v. and i.r. spectra of their methoxy methyl esters. Formation of the tapetal pigment from tyrosine and relationships of the tapetal pigment to melanin are discussed.     

Free ceramide, sphingomyelin, and glucosylceramide of isolated rat intestinal cells.

Free ceramide, glucosylceramide, and sphingomyelin were isolated from mature cells of adult rat small intestine. Free ceramide and ceramide cleaved from sphingomyelin by enzymatic hydrolysis were fractionated by thin-layer chromatography on borate-impregnated silica gel plates. Sphingoid bases were characterized by gas-liquid chromatography of aldehydes formed upon periodate oxidation. Fatty acids were quantified as methyl esters. Ceramide structures were confirmed by direct-inlet mass spectrometry. Free ceramide was found to contain two major long-chain bases in nearly equal quantity: sphingosine, mainly linked to palmitic acid, and 4D-hydroxysphinganine associated with C20 to C24 fatty acids, 22% being hydroxylated. Sphinganine occurred as a minor component linked to nonhydroxy fatty acids. Sphingomyelin contained the three long-chain bases and 63% of its ceramide was N-palmitoyl-sphingosine. Mass spectrometry of glucosylceramide confirmed 4D-hydroxyshingamine as the major sphingoid base associated preferentially with longer chain hydroxy fatty acids.     

Analysis of bilirubin and bilirubin mono- and di-conjugates. Determination of their relative amounts in biological samples.

1. A novel method for determination of the relative amounts of unconjugated bilirubin and sugar mono- and di-conjugates of bilirubin in biological samples, including serum, is described and illustrated by its application to the analysis of bilinoids in rat bile. 2. The method is based on specific conversion of the carbohydrate conjugates of bilirubin into the corresponding mono- or di-methyl esters by base-catalysed transesterification in methanol. Under the selected reaction conditions, unconjugated biliru-in remains intact and no dipyrrole exchange in the bilinoids is detectable; transesterification of bilirubin mono- or di-glucuronide is virtually complete (approx. 99%), and sponification is negligible (less than 1%); recovery of the pigments is approx. 95%. 3. The reaction products bilirubin and its methyl esters are separated by t.l.c. and determined spectrophotometrically; the two isomeric bilirubin-IX alpha monomethyl esters are separated and therefore can be determined individually. 4. Reference bilirubin mono- and di-methyl esters have been synthesized and characterized, and the two isomers of bilirubin-IX alpha monomethyl ester and bilirubin dimethyl ester were obtained individually, in crystalline form. 5. With this new method, virtually all bilinoids (over 99%) in normal rat bile have been found to be conjugated, with diconjugates (71%) predominating. A significantly increased proportion of monoconjugates is present in bile collected from heterozygous Gunn rats or from normal rats that were refused with large amounts of bilirubin.     

OCCURRENCE OF LOROXANTHIN,LOROXANTHIN DECENOATE,AND LOROXANTHIN DODECENOATE IN TETRASELMIS SPECIES (PRASINOPHYCEAE,CHLOROPHYTA)1

The pigment composition of six species of Tetraselmis (Prasinophyceae) was analyzed using improved HPLC methods. All pigment extracts showed three peaks corresponding to unknown carotenoids. The isolated pigments were analyzed using UV–Vis spectroscopy, electrospray ionization–mass spectrometry (ESI–MS), and when carotenoid esters were suspected, gas chromatography–mass spectrometry (GC–MS) of the methyl ester and dimethyloxazoline derivative of the corresponding fatty acid. The new pigments were determined to be loroxanthin, loroxanthin 19‐(2‐decenoate), and loroxanthin 19‐(2‐dodecenoate); this is the first time these pigments have been described in the genus Tetraselmis. Moreover, this is the first report of esterification of 2‐decenoic acid to loroxanthin. The relative contents of these pigments depended on the light regime, with the lowest proportions measured at the highest photon flux density assayed. The implications of the identification of these pigments in the genus Tetraselmis for the pigment types previously described in the class Prasinophyceae are discussed.     

Bilirubin conjugates of human bile. Isolation of phenylazo derivatives of bile bilirubin

A method is presented that allows the isolation of eight different phenylazo derivatives of bile bilirubin. In step I of the isolation procedure, three bilirubin fractions (bilirubin fractions 1, 2 and 3) from human hepatic bile are separated by reverse-phase partition chromatography on silicone-treated Celite with the use of a solvent system prepared from butan-1-ol and 5mm-phosphate buffer, pH6.0. Azo coupling is then performed with diazotized aniline. The three azo pigment mixtures are subjected to step II, in which the above chromatography system is used again. With each azo pigment mixture this step brings about the separation of a non-polar and a polar azo pigment fraction (azo 1A and azo 1B, azo 2A and azo 2B, and azo 3A and azo 3B from bilirubin fractions 1, 2 and 3 respectively). Approximately equal amounts of non-polar and polar pigments are obtained from bilirubin fractions 1 and 2, whereas bilirubin fraction 3 yields azo 3B almost exclusively. In step IIIA the non-polar azo pigment fractions are fractionated further by adsorption chromatography on anhydrous sodium sulphate with the use of chloroform followed by a gradient of ethyl acetate in chloroform. Three azo pigments are thus obtained from both azo 2A (azo 2A(1), azo 2A(2) and azo 2A(3)) and azo 3A (azo 3A(1), azo 3A(2) and azo 3A(3)). The 2A pigments occur in approximately the following proportions: azo 2A(1), 90%; azo 2A(2), 10%; azo 2A(3), traces. The pigments are purified by crystallization, except for the A(3) pigments, which are probably degradation products arising from the corresponding A(2) pigments. In step IIIB the polar azo pigment fractions are subjected to reverse-phase partition chromatography on silicone-treated Celite with the use of a solvent system prepared from octan-1-ol-di-isopropyl ether-ethyl acetate-methanol-0.2m-acetic acid (1:2:2:3:4, by vol.). Azo pigment fractions 2B and 3B each yield six azo pigments (azo 2B(1) to azo 2B(6) and azo 3B(1) to azo 3B(6) respectively) together with small amounts of products of hydrolysis (azo 2A(B) and azo 3A(B)). Only one azo B pigment is obtained from bilirubin fraction 1, and this azo pigment is probably of the B(2) type. The yields of the azo 3B pigments suggest that these pigments are present in approximately the following proportions: azo 3B(1), 0-0.4%; azo 3B(2), traces; azo 3B(3), traces; azo 3B(4), 10%; azo 3B(5), 50%; azo 3B(6), 40%. Azo pigments 2B(1) to 2B(6) are estimated to occur in similar proportions. Since pairs of correspondingly numbered azo pigments from bilirubin fractions 1, 2 and 3 do not separate on rechromatography together (e.g. azo 2A(1) co-chromatographs with azo 3A(1), and azo 2B(6) co-chromatographs with azo 3B(6)), it is concluded that such pigments are chemically identical. The structures of the isolated phenylazo derivatives are discussed in an accompanying paper (Kuenzle 1970c).     

Carotenoid pigments of facultatively anaerobic spirochetes.

Carotenoid pigments were purified from a previously undescribed, red, halophilic spirochete (spirochete RS1), and from Spirochaeta aurantia strain J1. Both spirochetes are facultative anaerobes and produce pigments when growing aerobically. The major pigments of the two spirochetes were identified by means of chromatographic analysis, absorption spectroscopy, hydride reduction, acetylation and silylation experiments, and mass spectrometry. It was concluded that the major pigment from spirochete RS1 was 4-keto-1',2'-dihydro-1'-hydroxytorulene. This conclusion was further supported by infrared spectroscopy and additional analytical data. The evidence showed that the major pigment from S. aurantia was 1',2'-dihydro-1'-hydroxytorulene. Chromatographic and spectrophotometric evidence indicated that this pigment was also present, as a minor carotenoid component, in spirochete RS1. These pigments have been previously detected almost exclusively in gliding bacteria, such as species of Flexibacter, Stigmatella, and Myxococcus. The occurrence of 4-keto-1',2'-dihydro-1'-hydroxytorulene and 1',2'-dihydro-1'-hydroxytorulene in both spirochetes and gliding bacteria may have significance with respect to the evolutionary development of these organisms.     

The synthesis of esters of bilirubin

1-Alkyl-3-p-tolytriazenes were used to synthesize the methyl, ethyl, isopropyl and benzyl esters of bilirubin. Treatment of a chloroform solution of bilirubin with the triazene at room temperature gave high yields of the corresponding esters. These were identified by n.m.r. and mass spectroscopy together with elemental analysis. N.m.r. studies also suggest that bilirubin dimethyl ester is in the lactam rather than the lactim form.     

Synthesis, chromatographic purification, and analysis of isomers of biliverdin IX and bilirubin IX.

Neutral solvent systems were developed to isolate the alpha, beta, gamma, and delta isomers of biliverdin IX dimethyl ester by TLC. The individual free acids of biliverdin IX were obtained by saponification of the corresponding dimethyl esters. The bilirubin IX isomers were prepared by reducing the corresponding biliverdin IX isomers with NaBH3CN. Starting from a pure biliverdin IX dimethyl ester, the corresponding free acid of biliverdin IX or bilirubin IX was available within 3-4 h. Preparation of spectrally pure bile pigment required final TLC on acid-cleaned neutral TLC plates. The absorption spectra of the free acids and dimethyl esters of biliverdin IX in methanol showed a broad band at about 650 nm and a sharp band at about 375 nm. The long-wave-length band was extremely sensitive to the presence of strong acid. A 10-fold molar excess of HCl caused a 35- to 50-nm shift of the absorption maximum to longer wavelengths and near doubling of the maximum absorption. The molar absorption coefficients of biliverdins were identical for each free acid and dimethyl ester pair. In each case, Beer's law was followed in both methanol and acidified methanol. Methanol also proved to be a suitable solvent for spectroscopic determination of the non-alpha isomers of bilirubin IX. The wavelength of maximum absorption and molar absorption coefficient of each dipyrrolic ethyl anthranilate azo pigment derived from the various bilirubin IX isomers are also reported.     

Preparation and properties of crystalline biliverdin IX alpha. Simple methods for preparing isomerically homogeneous biliverdin and [14C[biliverdin by using 2,3-dichloro-5,6-dicyanobenzoquinone.

Amorphous isomerically pure biliverdin IX alpha is readily prepared in more than 70% yield by dehydrogenation of bilirubin with 2,3-dichloro-5,6-dicyanobenzoquinone in dimethyl sulphoxide under carefully controlled conditions. Crystalline biliverdin IX alpha and amorphous [14C]biliverdin can be obtained similarly in more than 40+ yield. The pure crystalline pigment was characterized by elemental analysis, methylation, chemical and enzymic reduction to bilirubin, i.r.- and u.v.-visible-absorption spectroscopy, n.m.r. spectroscopy and field-desorption mass spectrometry, and its solubility was determined. Under certain conditions, dehydrogenation, gave biliverdin contaminated with III alpha and XIII alpha isomers as a result of disproporationation of bilirubin. Formation of non-IX alpha isomers depends on the concentrations of the reagents and the order in which they are mixed, and occurs under neutral anaerobic conditions. Free-radical reactions probably are responsible, suggesting that the first step in the deydrogenation of bilirubin with 2,3-dichloro-5,6-dicyanobenzoquinone in dimethyl sulphoxide is formation of a bilirubin cation radical, rather than hydride ion abstraction.     

Optimized enzymatic synthesis of ascorbyl esters from lard using Novozym 435 in co-solvent mixtures

A mild and efficient method for the conversion of fatty acid methyl esters from lard into ascorbyl esters via lipase-catalyzed transesterification in co-solvent mixture is described. A solvent engineering strategy was firstly applied to improve fatty acid ascorbyl esters production. The co-solvent mixture of 30% t-pentanol:70% isooctane (v/v) was optimal. Response surface methodology (RSM) and central composite design (CCD) were employed to estimate the effects of reaction parameters, such as reaction time (12–36 h), temperature (45–65 °C), enzyme amount (10–20%, w/w, of fat acid methyl esters), and substrate molar ratio of fatty acid methyl esters to ascorbic acid (8:1–12:1) for the synthesis of fatty acid ascorbyl esters in co-solvent mixture. Based on the RSM analysis, the optimal reaction conditions were determined as follows: reaction time 34.32 h, temperature 54.6 °C, enzyme amount 12.5%, substrate molar ratio 10.22:1 and the maximum conversion of fatty acid ascorbyl esters was 69.18%. The method proved to be applicable for the synthesis of ascorbyl esters using Novozym 435 in solvent.     

Transesterification of phospholipids or triglycerides to fatty acid benzyl esters with simultaneous methylation of free fatty acids for gas-liquid chromatographic analysis

A novel method is presented for transesterification of fatty acid esters in phospholipids and triglycerides to benzyl esters while simultaneously recovering free fatty acids as methyl esters. Transesterification is catalyzed by 0.2 M (m-trifluoromethyl phenyl)trimethyl ammonium hydroxide in methylene chloride, 10% (v/v) benzyl alcohol, and 1% (w/v) potassium tert-butoxide, and is complete in 30 min at room temperature. Methyl esters of all common fatty acids separate from the benzyl esters formed from phospholipids. This method has broad utility and is applicable to the formation of esters optimized for detection by absorbance or fluorescence (high performance liquid chromatography), electron capture (gas-liquid chromatography), or negative ion chemical ionization (gas-liquid chromatography-mass spectrometry).     

Assay for polyunsaturated fatty acids by argentation-thin-layer chromatography using commercial thin-layer plates

A simple, rapid, and convenient procedure for silver nitrate impregnation of commercial precoated silica gel plates is described. Silica-gel plates (Silica gel 60, E. Merck) were sprayed with 40% silver nitrate in water, dried in air, and activated at 100°C for 30 min. Samples containing fatty acid methyl esters were applied as 0.5- to 1.0-cm streaks and developed with a solvent system of benzene:ethyl acetate (9:1, v/v). The plates were sprayed with 70% sulfuric acid saturated with potassium dichromate, and the spots were detected by careful heating at 120°C for 90 min. This procedure is useful for separation and isolation of various species of fatty acid methyl esters and for simple, rapid, and reproducible estimation of microgram quantities of materials by spectrodensitometry of the chromatogram.     

Bile Pigments in the Bile of Freshwater Fish,Rainbow Trout,Tilapia, and Pejerrey

Bile pigments in the bile of the rainbow trout, Salmo gairdneri, tilapia, Tilapia nilotica, and pejerrey, Odonthetes bonariensis, were analyzed by HPLC and TLC.

Major bile pigments of rainbow trout and pejerrey were bilirubin glucuronides and ditauro-bilirubin, respectively. Ditaurobilirubin was not detected in rainbow trout and bilirubin glucuronides were scarcely found in pejerrey. Biliverdin seemed to be the sole bile pigment in tilapia, but it was a minor component in the other fish. These results are in accord with the previous reports in which the diversity of bile pigment composition was demonstrated in some fish.     

Reverse-phase h.p.l.c. separation, quantification and preparation of bilirubin and its conjugates from native bile. Quantitative analysis of the intact tetrapyrroles based on h.p.l.c. of their ethyl anthranilate azo derivatives.

We describe a facile and sensitive reverse-phase h.p.l.c. method for analytical separation of biliary bile pigments and direct quantification of unconjugated bilirubin (UCB) and its monoglucuronide (BMG) and diglucuronide (BDG) conjugates in bile. The method can be 'scaled up' for preparative isolation of pure BDG and BMG from pigment-enriched biles. We employed an Altex ultrasphere ODS column in the preparative steps and a Waters mu-Bondapak C18 column in the separatory and analytical procedures. Bile pigments were eluted with ammonium acetate buffer, pH 4.5, and a 20 min linear gradient of 60-100% (v/v) methanol at a flow rate of 2.0 ml/min for the preparative separations and 1.0 ml/min for the analytical separations. Bile pigments were eluted in order of decreasing polarity (glucuronide greater than glucose greater than xylose conjugates greater than UCB) and were chemically identified by t.l.c. of their respective ethyl anthranilate azo derivatives. Quantification of UCB was carried out by using a standard curve relating a range of h.p.l.c. integrated peak areas to concentrations of pure crystalline UCB. A pure crystalline ethyl anthranilate azo derivative of UCB (AZO . UCB) was employed as a single h.p.l.c. reference standard for quantification of BMG and BDG. We demonstrate that: separation and quantification of biliary bile pigments are rapid (approximately 25 min); bile pigment concentrations ranging from 1-500 microM can be determined 'on line' by using 5 microliters of bile without sample pretreatment; bilirubin conjugates can be obtained preparatively in milligram quantities without degradation or contamination by other components of bile. H.p.l.c. analyses of a series of mammalian biles show that biliary UCB concentrations generally range from 1 to 17 microM. These values are considerably lower than those estimated previously by t.l.c. BMG is the predominant, if not exclusive, bilirubin conjugate in the biles of a number of rodents (guinea pig, hamster, mouse, prairie dog) that are experimental models of both pigment and cholesterol gallstone formation. Conjugated bilirubins in the biles of other animals (human, monkey, pony, cat, rat and dog) are chemically more diverse and include mono-, di- and mixed disconjugates of glucuronic acid, xylose and glucose in proportions that give distinct patterns for each species.     

Eumelanin levels in rufous feathers explain plasma testosterone levels and survival in swallows

Pigment‐based plumage coloration and its physiological properties have attracted many researchers to explain the evolution of such ornamental traits. These studies, however, assume the functional importance of the predominant pigment while ignoring that of other minor pigments, and few studies have focused on the composition of these pigments. Using the pheomelanin‐based plumage in two swallow species, we studied the allocation of two pigments (the predominant pigment, pheomelanin, and the minor pigment, eumelanin) in relation to physiological properties and viability in populations under a natural and sexual selection. This is indispensable for studying the evolution of pheomelanin‐based plumage coloration. Pheomelanin and eumelanin share the same pathway only during their initial stages of development, which can be a key to unravel the functional importance of pigment allocation and thus of plumage coloration. Using the barn swallow, Hirundo rustica, a migratory species, we found that plasma testosterone levels increased with increasing the proportion of eumelanin pigments compared with pheomelanin pigments, but not with the amount of pheomelanin pigments, during the mating period. In the Pacific swallow Hirundo tahitica, a nonmigratory congener, we found that, during severe winter weathers, survivors had a proportionally smaller amount of eumelanin pigments compared with pheomelanin pigments than that in nonsurvivors, but no detectable difference was found in the pheomelanin pigmentation itself. These results indicated that a minor pigment, eumelanin, matters at least in some physiological measures and viability. Because the major pigment, pheomelanin, has its own physiological properties, a combination of major and minor pigments provides multiple information to the signal receivers, potentially enhancing the signaling function of pheomelanic coloration and its diversification across habitats.     

In situ and Ex situ adsorption and recovery of betalains from hairy root cultures of Beta vulgaris

Various adsorbents were screened for in situ recovery of betalain pigments effluxed from hairy root cultures of red beet, Beta vulgaris. Alumina/silica (1:1) appeared ideal, showing in situ adsorption of 97% in a unit time of 30 min accounting for in situ recovery of 71.39% of the total betalaine effluxed. Other adsorbents such as Amberlite series (XAD-2 and -4), cyclodextrin, maltodextrin, dextrin white, and starches such as wheat starch and corn starch exhibited very poor in situ adsorption properties. Pretreatment of adsorbents with methanol significantly improved the adsorption capacities of some of the adsorbents, with a highest adsorption of 97.2% for alumina followed by alumina/silica (1:1) and higher adsorption by XAD-2 and -4. Complete in situ adsorption equilibrium was reached in 20 min for a solution containing 2.5 mg mL(-)(1) of betalain in adsorbents alumina, silica, and a mixture of alumina and silica. In situ betalain adsorption parameters for alumina/silica were determined using the Langmuir isotherm model where the adsorption capacity was found to be 0.174 mg g(-)(1) and the adsorption energy was 0.9 at pH 5.5 and 25 degrees C. Desorption of pigments from the adsorbents was invariably highest in poor adsorbents, indicating their poor adsorption energy for betalaines. Similarly, recovery by desorption was low in those adsorbents having high adsorption capacity, indicating that adsorbents such as activated ones with highest adsorption capacity with zero desorption property were unsuitable for the recovery of effluxed pigments. Ex situ recovery of betalain done using various combinations of alumina/silica and processed sand and different column geometries indicated that alumina with processed sand at a 2:1 ratio (w/w) and a minimum column material of 2 cm height and 2 cm diameter was good enough to cause 97% pigment adsorption from a solution containing 1.6 mg mL(-)(1). Desorption and recovery of pigments ex situ from columns were affected by various elution mixtures, where a gradient elution with ascending levels of HCl/ethanol in water resulted in 100% recovery of adsorbed pigments in a significantly lesser volume of eluent in a short period of 1 h. Different pigment flow rates of 0.2, 0.3, and 3.1 mL s(-)(1) through a column of alumina/processed sand indicated that a pigment equilibrium concentration of 0.18 mg mL(-)(1) at flow rates of 0.02 and 0.3 mL s(-)(1) resulted in a breakthrough at 110 and 14 min adsorbing 16.9 and 16.91 mg g(-)(1) betalain, respectively. From the breakthrough curves, the column capacities for respective flow rates were calculated as 8.86 and 9.6 mg g(-)(1), and the higher flow rates resulted in earlier breakthrough with lower capacity. Observations made in the present study are useful to develop a process for the on-line recovery of betalains effluxed from hairy roots.     

Separation and quantification of pigments from natural phototrophic microbial populations

Abstract A simple high-performance liquid chromatography (HPLC) method is described for the rapid (approx. 20 min) simultaneous separation and identification of the major chlorophylls and carotenoids from phytoplankton cells and phototrophic sulfur bacteria. Lyophilized samples were extracted with acetone in the dark at room temperature. Pigments were eluted from a silica column with a hexane-acetone mixture (80: 20, v/v). About 20 algal and bacterial chlorophyll and carotenoid pigments could be separated in one run. The method allowed for the detection of trace amounts of major carotenoids (> approx. 6 ng · 1⁻¹) and of chlorophylls and pheophytins (> approx. 200 ng · 1⁻¹) in natural samples. The method has been applied to samples from the metalimnion of Lake Vechten (The Netherlands) and has proved very useful in estimating algal and bacterial pigments simultaneously with respect to depth distribution and biomass changes of the microbial populations.