RU2031399C1 - Method of analyzing phytoplankton in aqueous medium - Google Patents

Abstract

FIELD: analysis of aqueous masses of ponds. SUBSTANCE: medium to be analyzed is supplied into running-through dish of fluorimeter. Intensity of fluorescence of tested medium is indicated. Correction factor is determined which takes pollution of walls of the dish into account. After the medium is supplied into the dish and fluorescence of the medium is measured, standard medium is fed into the dish and light signals from it is measured. Then standard medium is fed into pure dish and light signal is measured from standard medium in pure dish. Correction factor is calculated from the values of light signals mentioned. EFFECT: improved precision; improved reliability. 4 cl, 1 dwg

Description

 The invention relates to techniques for the study of water masses of water bodies and can be used in limnology and oceanology when measuring the concentration of dissolved substances and suspended particles in an aqueous medium, as well as in any other field of science, technology and environmental protection, where it is necessary to conduct a continuous analysis of the aqueous medium with using fluorimeters with flowing ditches.

 Taking into account the existing experience in operating fluorimeters with flow cells and various pumping systems, it can be argued that plaque on the walls of the cell is often formed not only and not so much due to the rusting parts of the pump and pumping system. The main reason for this is the dissolved organic matter present in the aquatic environment. At the same time, in very dirty waters (even natural), such a plaque forms after 0.5-1.0 hours and can significantly affect the measurement results, since as a result the readings fall by 10% or more.

 A known method for the determination of chlorophyll in an aqueous medium [1], which consists in conducting the excitation of fluorescence of the aqueous medium and measuring the fluorescence intensity signal, which is determined. The method does not allow for the contamination of the walls of the fluorimeter cell.

 There is also a method of determining the chlorophyll of phytoplankton in an aqueous medium [2], which takes into account the pollution of the walls of the cell. In the course of work to determine the chlorophyll content of phytoplankton in an aqueous medium, it was shown that the walls of the flow cell of the fluorimeter were coated with plaque, which continuously impaired its optical properties.

This method has the following disadvantages: the long duration and complexity of the process of obtaining an estimate of the influence of plaque on the measurement results, since it takes several hours to one to two days to perform the necessary measurements of fluorescence intensity; low accuracy of accounting for the effect of pollution on the final result. Cases in that between the native fluorescence of chlorophyll and its concentration there is no rigid unambiguous relationship; The ratio of native fluorescence intensity / chlorophyll concentration is influenced by many factors, including species composition, illumination, and mineral nutrition of phytoplankton cells. Therefore, the true value of the correction factor due to the plaque in the cuvette can be greatly distorted by these factors;
the introduction of one correction factor for a group of measurements, while in fact there is a continuous formation of plaque and, therefore, for each individual measurement it is necessary to introduce its own correction.

 The purpose of the invention is to increase accuracy and reduce the time taken into account contamination of the walls of the cell.

 This goal is achieved by the fact that in the method, based on the measurement of the light signal of the fluorescence intensity from the contents of the cuvette, followed by the introduction of a correction coefficient on the result of measuring the fluorescence intensity of the test medium according to the invention, a standard is placed in a clean and contaminated cuvette, both times the light signal intensity from it for the same conditions of irradiation and registration as for the studied medium, and the correction factor is determined based on the data obtained. As a reference, either a substance similar in spectral properties to the test medium is used, or distilled water with the same temperature for both measurements, or a mirror with the possibility of reflection of light by it in the direction of measuring the fluorescence intensity of the test medium. Moreover, if the last two standards are used, then the cuvette is irradiated for the first time in the spectral band, which is the excitation band for the medium under study, and light is recorded in this band, the second time is irradiated in the spectral band, which is the registration band for the studied medium, and light is recorded in the same strip.

The determination of the correction coefficient on the basis of the results of measuring the intensity of the light signal from the standard, in comparison with the prototype, provides an increase in the accuracy of accounting for contamination of the walls of the cell, since it can be determined for each measurement, which is necessary due to the continuous formation of plaque. For a dirty cuvette
K (t _a) = Φ _{fl to} t / Φ _fl (t _o) (2)
where K (t _to ) - correction factor for the measurement time t _to ;
Φ _et (t _to ) - the intensity of the light signal (fluorescence) from the standard for the measurement time t _to ;
Φ _et (t _o ) - the intensity of the light signal at the initial moment of measurement t _o from the standard, placed in a clean cell.

Correction factor for the measurement time t _o , i.e. To (t _o ), respectively, equal to one. In this case, the values of K (t) for the measured signals at t _o <t <t _{k are} determined by interpolation using the values of K (t _o ) and K (t _k ).

где Φ^{λ I} ( t_o) , Φ^{λ I} (t _k) - интенсивность светового сигнала в момент времени t_o или t_к cоответственно ( λI означает, что измерения выполняются в полосе возбуждения флуоресценции анализируемой среды;
Φ^{λ II} ( t_o) , Φ^{λ II} (t _k) - то же, но для полосы регистрации анализируемой жидкости, что обозначено как λII.When used as a reference, mirrors or distilled water
K (t _to ) =

where Φ ^{λ I} (t _o ), Φ ^{λ I} (t _k ) is the intensity of the light signal at time t _o or t _to, respectively (λI means that the measurements are performed in the fluorescence excitation band of the analyzed medium;
Φ ^{λ II} (t _o ), Φ ^{λ II} (t _k ) - the same, but for the registration band of the analyzed liquid, which is indicated as λII.

 Compared to the prototype, the accounting time is also reduced, since in order to determine the correction coefficient for each measurement of the parameters of the medium under study, it is necessary to perform only two measurements of the light signal intensity from the standard placed in the cuvette. In addition, accounting in accordance with the proposed solution is simpler, since there is no need to perform a parallel series of measurements in an independent way.

 The drawing shows the recording of the fluorescence intensity signal of phytoplankton chlorophyll obtained on a Quantum-4 fluorimeter in the flight of the R / V “Professor Marty” in the summer of 1987 (dashed line), and the true curve calculated on the basis of correction factors (solid line), where abscissa - time in hours; along the ordinate axis is the fluorescence intensity of the studied surface of the water in relative units.

 The method is as follows.

After finishing the measurement of the analyzed fluid, carried out over a period of time from t _o to t _to drain it from the flow cell. Then, a reference substance, similar in spectral properties to the test medium, is placed in the cuvette, liquid (for example, a solution of rhodamine, fluorescein, thionine) or solid (a solution of rhodamine in polymethyl methacrylate).

Next, determine the intensity of the light signal (fluorescence intensity) of the standard for the same irradiation and registration conditions as for the medium under investigation Φ _et (t _k ), here t _k is the time the measurement of the analyzed liquid is completed. Next, the cuvette is washed thoroughly, the standard is again placed in it and Φ _et (t _o ) is measured, i.e. the fluorescence intensity of the standard in a clean cell, here t _o corresponds to the start time of the measurement of the analyzed fluid. It can be at 1, 2, 24 hours and earlier than t _to . A standard placed in a clean cuvette always gives a value of Φ _et (t _o ) equal to the same value, and a value of Φ _et (t _k ) is new every time. Since the measurement conditions are the same standard as the analyzed liquid, if the _floor Φ (t _k) less _et Φ (t _o) of 5-10% or more, hence, all the values of the analyzed liquid of the fluorescence intensity obtained in the interval from t _o to t _to require adjustment, as this means that the registered signal was actually partially weakened. To recalculate it to its true value, the value of the recorded signal of the fluorescence intensity of the analyzed liquid is corrected exactly by how much it was weakened by the plaque on the walls of the cell: divide the useful signal for the moment t = t _k by the coefficient K (t _k ) = Φ _et (t _k ) / Φ _et (t _o ). For t = t _o K (t _o ) = 1, since the cell was clean. The intermediate values of the useful signal for the moment t _o <t <t _{k are} divided by the values of K (t) obtained by interpolation of K (t) taking into account the course of the dependence of K on t and the values of K (t _o ) and K (t _k ). Due to the fact that usually the measurement results are recorded on a magnetic medium, the introduction of such a correction is not very difficult. Taking into account the fact that both primary (by excitation) and secondary (by registration) filters are the same when measuring the standard as when measuring the analyzed liquid, this correction takes into account the attenuation of exciting light in the corresponding range with a wavelength of maximum intensity or characteristic wavelength λ I, and the attenuation of the intensity of fluorescence light passing through the other wall of the cuvette and attenuating by a different value, since the fluorescence radiation is recorded in another spectral region, determined by the registration filters and having a different wavelength of maximum intensity or a characteristic wavelength denoted by λII.

The method can also be carried out with the following sequence of operations: before starting the measurement of the parameters of the test medium, a standard is placed in a clean cuvette and Φ _et (t _о ) is measured. Then, studies are performed and, upon completion, the standard is placed in a contaminated cuvette without the test liquid, after which Φ _et (t _{k) is} measured.

а для t=t_o он также равен 1.A mirror that reflects a beam incident on a cuvette at an angle equal to the angle of measurement of the fluorescence intensity is also used as a reference for achieving the purpose of the invention. In this case, the light intensity Φ _zer (t) reflected from the mirror in series under two conditions is measured. Once, provided that the cuvette with the mirror is irradiated with light in the spectral band, which is the excitation band for working fluorimetric measurements (with a wavelength of maximum intensity or characteristic wavelength λI) and registration of reflected light in the same band, and immediately the second time, when the spectral bands are the excitation and registration channels are set equal to the registration bands (with a wavelength of maximum intensity or a characteristic wavelength of λII) during working fluorimetric measurements for which linked account. In this case, the correction coefficient K (t) for t = t _{k is} calculated as
K (t _to ) =

and for t = t _o it is also equal to 1.

 As a reference, you can also use a clean test liquid (not containing the studied impurities and any suspension). For seawater, this may be ordinary distilled water.

, алгоритм использования К(t) прежний.The correction factor is then determined by the same formula, but instead of the intensity of the light signal reflected by the mirror Φ _zer ^{λ I} (t) and Φ _zer ^{λ II} , the elastic light scattering intensity Φ _ras ^{λ I} (t) and Φ _ras ^{λ II are used} , which substitute similar formula
K (t _to ) =

, the algorithm for using K (t) is the same.

For the latter case, it should be noted that the elastic scattering is directly proportional to the temperature of the liquid, therefore, in such measurements, the temperature of the liquid at times t _o and t _k when measuring the correction coefficient K (t) by elastic light scattering should be the same or vary slightly.

 During the cruise of the R / V "Professor Marty" in the summer of 1987, the determination of the correction factor was made on the basis of measurements of the intensity of elastic light scattering for distilled water poured into a contaminated cell. The measurements were carried out on a “Quant-4” fluorimeter manufactured by the Barnaul Design Bureau of NPO Himavtomatika using a standard ship pumping system.

= 0,76
Φ_рас ^{λ I} (t₂)=53 K(t₂)=0,63
Φ_рас ^{λ I} (t₃)=40 K(t₃)=0,55
Φ_рас ^{λ I} (t₄)=33 K(t₄)=0,48
В течение всего 5- дневного измерения кювета не мылась. Анализ полученных данных показал, что в данное время в этом районе проточную кювету нужно либо обязательно мыть через каждые 8 ч (только в этом случае поправка составляет менее 10% и ее можно не учитывать), либо необходимо вводить соответствующую поправку.The fluorescence intensity of phytoplankton chlorophyll was measured upon excitation through SZS-22 plus SS-5 filters; registered through the filter KS-15. To estimate the correction coefficient for elastic scattering in the blue region (in the excitation band), the NS-11 neutral filter was additionally used, and the NS-13 neutral filter was used in the red region (registration band). A fluorimeter with a pumping system worked continuously for 5 days. Correction factor measurements were performed daily. For the red region of the spectrum (recording band λ II), its values remained almost unchanged and equal to 1 for all 5 days. For the blue region (excitation band λ I), the following values of elastic scattering intensity and correction coefficients were obtained
Φ _races ^{λ I} (t _o ) = 133 K (t _o ) = 1
Φ $\genfrac{}{}{0ex}{}{\text{λI}}{\text{ra}}$ _s (t ₁ ) = 77 K (t ₁ ) =

= 0.76
Φ _races ^{λ I} (t ₂ ) = 53 K (t ₂ ) = 0.63
Φ _races ^{λ I} (t ₃ ) = 40 K (t ₃ ) = 0.55
Φ _races ^{λ I} (t ₄ ) = 33 K (t ₄ ) = 0.48
During the whole 5-day measurement, the cuvette did not wash. An analysis of the data showed that at this time in this area the flow cell must either be washed every 8 hours (only in this case the correction is less than 10% and it can be ignored), or an appropriate correction must be introduced.

 The proposed method is more accurate compared to the prototype, as it allows you to determine the correction factors for each measurement, taking into account the constantly formed plaque.

Claims (4)

1. METHOD FOR ANALYSIS OF PHYTOPLANKTON CHLOROPHYLL IN AQUEOUS ENVIRONMENT, including feeding the analyzed medium into a flow cell of a fluorimeter, irradiating the cell with light in the excitation band of the analyzed medium, measuring the fluorescence intensity of the medium, determining a correction factor that takes into account the pollution of the walls of the flow cell color of the cell color fluorescence corrected for the correction factor, characterized in that, in order to improve the accuracy of the analysis of the path determining a correction factor taking into account the changing time of contamination of the cell walls and reduce analysis time measured intensity of the environment of the fluorescence F (t _k) in the fixed time t _to then feed the medium in a cuvette, and then supplied into a cell reference, a similar spectral properties to the analyte medium and containing no chlorophyll phytoplankton and suspended impurities, keeping the conditions of excitation and registration of the light signal at time t _k, is irradiated with light and measure the intensity of the light signal from floor womb in contaminated cuvette F _{e t} t _k, then fed the standard in uncontaminated cuvette, irradiated with it, and measured the intensity of the light signal from the sample in a clean cuvette F _{e m} with the same spectral conditions for the moment t of time _to calculate the correction factor K (t _k) = F _{e t} (t _k) / F _{e t} is determined and the corrected fluorescence intensity of the analyzed sample
Ф '(t _к ) = Ф (t _к ) / Ф' (t _к ).  2. The method according to p. 1, characterized in that a mirror is placed in the flow cell to reflect light in the direction of measuring the fluorescence intensity of the analyzed medium. 3. A method according to claim 1, characterized in that as reference is used, distilled water to the same temperature during registration F _{e T} (t _k) and F _{e t,} respectively.  4. The method according to claim 1, characterized in that when the cuvette with the standard is irradiated, the light signal from the standard is recorded in the spectral region of the medium excitation, and when measuring the signal from the standard, the radiation is carried out with light with a wavelength from the medium fluorescence detection region.

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