Objective: This study aims to determine the structure of anthocyanins of common lilac flowers (Syringa vulgaris L.) of various color intensities and shades of lilac color from nine samples purchased at the Belgorod market by investigation of solute retention regularities of the compounds, peculiarities of their electronic absorption spectra as well as mass-spectra fixtures. Methods: Reversed-phase HPLC with a diode array as well as mass spectra detection by electrospray ionization with partial fragmentation were used. Results and Discussion: It was found that in all the studied samples the main component was delphinidin-3-rutinoside (84–90

It is shown that the column dead time determined from the retention of uracil is longer than the one determined from the retention of homologs—lutein diesters—for C16 reversed phases on three columns with sorbents with different pore diameters (80, 110, and 160 Å). This finding confirms the familiar assumption that the sorbate molecules 10% larger than the pore diameter can cause problems with the retention of the sorbate. It is found for smaller molecules (of alkyl benzoates) that the dead time determined from the retention of uracil is less than the one determined from the retention of the homologs on the same stationary phases, confirming that the main cause of the observed effect is the size factor.

For the first time, a new version of the separation of 3-glucosides and 3,5-diglucosides of the five main anthocyanidins for grape fruits -delphinidin, cyanidin, petunidin, peonidin and mal-vidin under reverse-phase HPLC conditions was proposed. It has been shown that the replacement of the traditionally used acetonitrile with environmentally more acceptable ethanol upon acidifica-tion with ortho-phosphoric rather than formic acid makes it possible to significantly change the selectivity of the separation of two types of glucosides at their joint presence. The proposed separa-tion option allows differentiating between Vitis vinifera grapes, in the fruit peel of which only 3-glucosides of the above anthocyanidins are synthesized, and grapes of other species or hybrid grape varieties. For a complete review of the anthocyanin composition, it is necessary to use a gradient mode, since the acylation of anthocyanins with acetic and p-coumaric acids significantly changes the lipophilicity of anthocyanins. The type of anthocyanins was analyzed according to the previously proposed system, which takes into account the activity of three types of enzymes: 1) 5-O-glucosyltransferase that leads to 3,5-diglucosides synthesis; 2) 3',5'-hydroxylase responsible for ring B hydroxylation; 3) anthocyanin O-methyltransferase for converting cyanidin into peonidin derivatives as well as delphinidine into petunidin and malvidin glycosides. The method was used to determine anthocyanins in 43 grape varieties grown in Belgorod on farms and private farms. Among the studied grapes, varieties were found with grape fruits with the accumulation of only 3-glucosides, as well as 3-glucosides and 3,5-diglucosides simultaneously, with delphinidin and cyanidin types of anthocyanins, and with different degrees of methylation. The calculated parameters for all grape varieties are presented in two tables and a brief discussion of some varieties is given.

A variant of simultaneous determination of chlorogenic (monocaffeoylquinic and dicaffeoylquinic) acids and caffeine in coffee (drink) using reversed-phase chromatography with spectrophotometric detection has been developed. For separation, a gradient mode is proposed explor-ing of two components of the mobile phase containing 3 vol.% of formic acid and 6 and 20 vol.% ace-tonitrile in distilled water and chromatographic column 150x4.6 mm Symmetry C18, 3.5 mu m. Col-umn thermostat temperature is 30 degrees C. The choice of the type and concentration of the acidifier of the mobile phase is justified in the work. It has been established that the acidity of the mobile phases should not be lower than pH 2 in order to transfer most of the chlorogenic acids to the non-ionized state. The dependences of the retention of monocaffeoylquinic acids and caffeine on the concen-tration of acetonitrile and formic acid, which is more effective in the selectivity of separation com-pared to orthophosphoric acid in the mobile phase, are determined. The selectivity of the separation of these compounds depends more on the concentration of formic acid than on the concentration of acetonitrile. Concentrations of chlorogenic acids and caffeine were determined in drinks from two varieties of coffee, coffee husk (dried pericarp of coffee fruits) (produced in Vietnam) and for comparison of two more variants - Arabica coffee from roasted grains and from green decaffein-ated grains from other manufacturers. It is shown that coffee husk is a valuable raw material for the preparation of an alternative drink. Thus, when using 1 g of coffee husk when brewing 100 ml of boiling water, a drink containing about 10 mg (per 100 ml) of monocaffeoylquinic acids, about 6 mg of dicaffeoylquinic acids and about 3 mg of caffeine was obtained.

It was found experimentally that the replacement of acetonitrile with ethanol upon the acidification of the mobile phase with phosphoric acid is an effective version for replacing acetonitrile. At that, the order of elution of glycosides of the same type of the five main anthocyanidins (delphinidin, cyanidin, petunidin, peonidin, and malvidin) does not change in eluents convenient for analysis, and the range of retention times for a complete set of such anthocyanins becomes noticeably narrower in going from acetonitrile to ethanol. To determine the effect of aglycone structure on the retention of derivatives of the same anthocyanidin (cyanidin), a mixture of six glycosides was used: one monoglycoside (3-glucoside), two 3-diglycosides (sophoroside and sambubioside), and two 3-triglycosides (2"-glucosylrutinoside and 2"-xylosylrutinoside). This mixture is a real set of anthocyanins, requiring the careful selection of the mobile phase for separating all components. In this work, based on an analysis of separation maps, the composition of a mobile phase based on ethanol is determined that ensures the achievement of the effective separation of these anthocyanins.

В работе предлагаются карты эффективности, как графики зависимости ширины пиков на половине высоты, Δ 1/2 , от функции фактора удерживания сорбатов, полученной из исправленного выражения для расчета эффективности хроматографических систем для жидкостной хроматографии с добавлением параметра, учитывающего динамические факторы уширения по формуле: \(\Delta _{{1{\text{/}}2}}^{2} = {{a}^{2}} + {{b}^{2}}k(k + 1)\) . Показано, что карты эффективности могут быть использованы для сравнения различных хроматографических систем без необходимости необоснованного выбора случайных конкретных условий хроматографирования – во всем диапазоне составов подвижных фаз. Показано, что карты эффективности чувствительны к механизмам удерживания сорбатов.

Efficiency maps are proposed as plots of the dependence of the peak width at half height Δ 1/2 on the function of the sorbate retention factor, obtained from a corrected expression for the efficiency of chromatographic systems for liquid chromatography with the addition of a parameter that takes into account dynamic broadening factors according to the formula Δ _1/2^2 = a^2 + b^2k(k + 1) . The efficiency maps can be used to compare various chromatographic systems avoiding the need for an unreasonable choice of random specific conditions of chromatography over the entire range of mobile phase compositions. The efficiency maps are shown to be sensitive to sorbate retention mechanisms.

Specific features of chromatographic determination of xanthophyll esters are studied using an example of marigold flower lutein diesters under reversed-phase HPLC conditions. The developed two-column method made it possible to establish that, in samples with a low solubility of carotenoids in the used solvent and on a chromatography column in using a mobile phase with a low solubility of carotenoids, the precipitation of diesters is possible, which detrimentally affects the accuracy of the chromatographic determination. A critical factor in this case isì temperature: the storage of samples (solutions) in a refrigerator is not always advisable, because freezing of the main components is possible. It was shown that the use of a mobile phase containing from 0 to 10 vol % acetonitrile in acetone at a temperature not lower than 20°C is acceptable for the separation of all- trans -lutein diesters from cis -derivatives of lutein and zeaxanthin derivatives on “monomeric” C18-phases.

Anthocyanins from flowers of common lilac (Syringa vulgaris L.) of various color intensities and shades of lilac color from nine samples purchased at the Belgorod market were studied using reverse-phase high-performance liquid chromatography. To determine the structure of anthocyanins, we used the analysis of electronic absorption spectra recorded in the cuvette of a diode array detector and the analysis of mass spectra obtained by electrospray ionization with partial fragmentation. As a result, it was found that in all the studied samples the main component was delphinidin-3-rutinoside (84–90% by peak areas in the chromatogram). The level of cyanidin-3-rutinoside biosynthesis was significantly lower (6–19.6%). Among the minor compounds, delphinidin-3-glucoside and petunidin-3-glucoside were found. Among the unusual compounds, pyranoanthocyanin, built on the basis of delphinidin-3-rutinoside due to condensation with pyruvic acid, was found in a number of studied samples, but the reasons for its appearance have not yet been established. The total content of anthocyanins is low and amounts to 0.020–0.120 g per 100 g of fresh material (depending on the color intensity of the original plant material) in terms of cyanidin-3-glucoside. By drying flowers on a cut branch, air-dried material was obtained containing 0.100–0.300 g per 100 g of anthocyanins.

Two types of separation maps are compared for the first time, based on (1) the dependence of the logarithm of the retention factor on the volume fraction of the organic modifier and (2) the means of relative retention analysis. The two types of map agree with each other. Parameters of quadratic equations for the dependence of retention on the volume fraction of the organic modifier, extrapolated to the zero content of the organic modifier in the mobile phase, must be used in order to compare the energies of interaction between sorbates and sorbent. It is established that separation maps of the second type with linear trend lines are convenient for comparing the interaction energies of sorbates with organic modifiers of mobile phases. Additional possibilities of using this approach in special cases are shown. It is concluded that environmentally harmful acetonitrile can be replaced with more benign acetone to separate 3-glucosides of the main six anthocyanidins.

The paper investigates the change in the separation selectivity of the chlorogenic (mono- and dicaffeoylquinic) acids under conditions of reverse-phase HPLC with the addition of beta-cyclodextrin into the mobile phase. To exclude the effect of in situ modification of the stationary phase by cyclodextrin, the usual C18 phase was replaced by the C4 phase. It was found that as the concentration of beta-cyclodextrin in the mobile phase increases, the retention of all mono- and dicaffeoylquinic acids decreases due to the formation of inclusion complexes in the mobile phase. At the same time, the decrease in retention times is not the same for all the studied isomers, which indicates different values of the constants of the "host-guest" complex formation. The simplified scheme proposed in the paper for calculating the constants of complexation, taking into account the possible formation of complexes of composition 1:2, allowed us to calculate the constants for monocaffeoylquinic acids and the sum of the constants of possible isomeric compounds of composition 1:1 for dicaffeoylquinic acids and showed that the formation of complexes of composition 1:2 is unlikely. It is shown that qualitatively the sum of the constants for dicaffeoylquinic acids correlates with the constants for the corresponding monocaffeoylquinic acids, but taking into account the steric restrictions in the formation of 1:1 inclusion complexes for dicaffeoylquinic acids. For a qualitative assessment of steric restrictions, the analysis of some conformations of dicaffeiolquinic acids optimized by the AM1 method in the HyperChem 8.0.8 package and the complex of inclusion of 4-caffeoylquinic acid in beta-cyclodextrin was used.

A study is performed of the effect of composition of the mobile phases of acetonitrile–formic acid/phosphoric acid–water systems and the concentrations of acetonitrile and acid have on the chromatographic behavior of anthocyanins under the conditions of reversed phase HPLC.

Исследовано влияние различных изменений в составах подвижных фаз систем ацетонирил–муравьиная/фосфорная кислота–вода концентрации ацетонитрила и кислоты на хроматографическое поведение антоцианов в условиях обращенно-фазовой ВЭЖХ.

A method for the determination of the composition of carotenoids of tomato fruits of different colors is proposed using a combination of spectrophotometry and chromatography. An explanation of the elution order of mono- cis -isomers of lycopene under the conditions of reversed-phase chromatography on traditional “monomeric” reversed C18-phases is proposed for the first time. It was found that, for prolycopene, (7Z, 9Z, 7'Z, 9'Z)-lycopene, which is responsible for the orange color of fruits, there is an almost imperceptible transition in normal examination in the vibronic structure of the electronic absorption spectrum with the lowest energy, λ max (1) = 486.2 nm. The main carotenoids of tomato fruits of different colors were determined: trans -lycopene and its cis -isomers for fruits of red and pink colors, protolycopene and other carotenes preceding its biosynthesis for fruits of orange color, and the carotenoid composition of yellow tomatoes significantly differing from that of tomatoes of first two colors by a considerable accumulation of lutein. For the quantitative assessment of the concentration of carotenoids with different chromophores, a calculation system ensuring the determination of the contribution of each of the components of complex mixtures was proposed.

В работе определен видовой состав антоцианов цветков гвоздики традиционной красной и необычных темно-красной и фиолетовой окрасок, а также определен вклад заместителей яблочной кислоты в общее удерживание антоцианов при разделении в условиях обращенно-фазовой ВЭЖХ с использованием диодно-матричного и масс-спектрометрического детектирования. Установлены основные компоненты экстрактов цветков двух сортов гвоздики садовой – алой и темно-красной окраски, – как 3-гликозиды пеларгонидина и цианидина (Pg3Glu и Cy3Glu), соответственно, в основном ацилированные яблочной кислотой в положении 6"-Pg3(6"MalylGlu) и Сy3(6"MalylGlu) При этом антоцианы гвоздики садовой третьего сорта (с фиолетовой окраской) были построены на иной основе и состояли в основном из производных цианидин-3,5-диглюкозида (Cy3,5diGlu), также главным образом ацилированных яблочной кислотой, но уже в иной форме, соединяющей оба глюкозидных радикала в цикл. Во всех случаях электронные спектры поглощения соединений лишь незначительно (на 0.5-1 нм) батохромно сдвигаются при ацилировании. Антоцианы сухих цветков сорта «Мираж» гвоздики турецкой состояли из производных Cy3Glu и Cy3,5diGlu, также ацилированных яблочной кислотой. Последовательность элюирования всех соединений не зависит от составов подвижных фаз системы «ацетонитрил-муравьиная кислота-вода», приемлемых для ВЭЖХ-анализа. Отмечено, что фактор удерживания, k(i), антоцианов увеличивается в 2.5-3.5 раза в зависимости от состава подвижной фазы при ацилировании яблочной кислотой в положение 6" Cy3Glu и Pg3Glu и только на 25-40% для Cy3,5diGlu, что позволяет анализировать состав антоцианов в изократическом режиме элюирования. Впервые сравниваются два варианта карт разделения как logk(i) vs ϕ (объемная доля CH3CN) с использованием квадратичной зависимости и как logk(i) vs logk(Cy3Glu). Показано, что экстраполяция удерживания на подвижные фазы с нулевым содержанием воды приводит к сходным результатам в обоих случаях.

Разработан простой вариант градиентного разделения всех 3-глюкозидов и 3,5-диглюкозидов пяти основных антоцианидинов (дельфинидина, цианидина, петунидина, пеонидина и мальвидина), характеризующих биосинтез антоцианов в плодах различных сортов винограда вида Vitis vinifera (первая группа), а также в других видах винограда (Североамериканского или Дальневосточного происхождения) и в их гибридах. Для разделения использована ВЭЖХ со стационарной фазой Symmetry C18 и элюентами, составленными из 10 об.% муравьиной кислоты и двух различных составов по ацетонитрилу 6 об.% и 20/30 об.% в воде. Межвидовые гибриды отличаются появлением в экстрактах кроме 3-глюкозидов 3,5-диглюкозидов. Как 3-глюкозиды, так и 3,5-диглюкозиды легко разделяются в условиях обращенно-фазовой хроматографии, и проблема разделения возникает только при их совместном присутствии, особенно это касается пары: петунидин-3-глюкозид и мальвидин-3,5-диглюкозид. Для решения задачи разделения указанных антоцианов при совместном присутствии были определены энтальпии переноса этих антоцианов с подвижной фазы на стационарную. При этом было экспериментально установлено, что все процессы сорбции экзотермические, при этом энтальпия переноса для 3-глюкозидов и 3,5-диглюкозидов различна - уменьшается по абсолютной величине при добавлении глюкозидного радикала в положение 5. При этом различие этого параметра для петунидин-3-глюкозида и мальвидин-3,5-диглюкозида позволяет изменять селективность разделения аналитов за счет увеличения температуры разделения с 40 до 55оС. Это позволяет выполнить исследование сортов винограда и вин на содержание маркеров скрещивания винограда и видами, не относящимися к Vitis vinifera. В работе на примере ряда вин, приобретенных в супермаркете, изготовленных из виноградов Vitis vinifera сорта Каберне или Каберне Совиньон, было установлено, что в исследованных винах, произведенных в Чили и во Франции, 3,5-диглюкозидов нет. Это указывает на использование только виноградов Vitis vinifera при их производстве. Однако во всех исследованных в работе винах, произведенных в России, содержание 3,5-диглюкозидов оказалось значительным, указывая на использование при производстве гибридных сортов винограда (типа популярного в России сорта Молдова).

The effect of acylation of anthocyanins with acetic acid on the chromatographic retention is evaluated. It is shown that the absence of correlations between the experimental data on the retention of substances and the CLOGP data is explained, in some cases of structural changes, by the specificity of the float mechanism of the retention of anthocyanins. In this case, the conformational analysis of anthocyanins (in the Chem3D software package) can explain a number of features peculiar to the effect of acylation on the retention in accordance with the Horváth–Melander model used for glycoside substituents. It is shown that the contribution of the acetyl group to the total retention value of anthocyanins depends on the composition of the mobile phase, the structures of glycosidic substituents and aglycone for a given stationary phase, and the temperature of separation. In general, acetylation leads to a substantial increase in the retention of anthocyanins, namely, to an increase by a factor of about 10 for 3-(6-acetylglucosides), and this increase is about 1.5 times lower in the case of similar acetylation of 3,5-diglucosides.