CN104596965A - Determination method of concentration of iron ion in algae nutrient - Google Patents
Determination method of concentration of iron ion in algae nutrient Download PDFInfo
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- CN104596965A CN104596965A CN201510056756.2A CN201510056756A CN104596965A CN 104596965 A CN104596965 A CN 104596965A CN 201510056756 A CN201510056756 A CN 201510056756A CN 104596965 A CN104596965 A CN 104596965A
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- concentration
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- ferric
- nutritive salt
- citrate
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Abstract
The invention relates to a determination method of iron ions and in particular relates to a determination method of the concentration of iron ion in an algae nutrient. The determination method comprises the following steps: preparing a ferric citrate standard liquid with ferric ion concentration of 6*10<-3>mol/L; diluting the standard liquid into diluents with different concentrations, measuring the average absorbance of the diluents at 450nm by utilizing an ultraviolet spectrophotometer, and drawing a standard curve; determining an absorbance value of a sample at 450nm, and calculating the concentration of the ferric ion in the sample according to the standard curve; and subtracting the actually measured concentration of the ferric ion from the initial concentration of the ferric ion in the sample to acquire the concentration of the ferrous ion in the sample. The determination method is capable of quantitatively detecting the contents of ferric citrate and ferrous citrate in a stock solution of the algae nutrient, is simple and rapid and is high in accuracy, wide in detection range and little in middle process without addition of other agents and pretreatment of the sample, so that the test cost is low, and the time is short.
Description
Technical field
The present invention relates to a kind of assay method of ferric ion, particularly the assay method of iron concentration in a kind of algae nutritive salt.
Background technology
Nowadays, algae scientific research and production scale grow stronger day by day, and in commercial production and laboratory cultures, ferro element is the critical nutrients in algae nutritive salt always.Different algal species requires different to the price of ferro element, some algae needs ferric iron, and other need ferrous iron.The complex compound of conventional ferro element nutritive salt mainly citric acid and iron, i.e. ferric citrate or ferrous citrate.Ferric citrate is in preparation and to deposit in process part and be reduced to ferrous citrate, ferrous citrate is in preparation and deposit in process and be very easily oxidized to ferric citrate, both storage liquid all can form the potpourri of ferric citrate and ferrous citrate, the ferric ion of different chemical price and variable concentrations then have impact on the normal growth of algae, and how the correct cultivation of content to algae of measuring ferric citrate in algae nutritive salt and ferrous citrate is most important.
But propagate artificially or in laboratory study, in algae nutritive salt, the content of ferric citrate and ferrous citrate creates notable difference due to factors such as reagent preparation, compound method and holding conditions.Only have the method quantitatively detecting ferro element at present, but for the potpourri of ferric citrate with ferrous citrate, there is no the method quantitatively detected, also do not determine the method for the ratio of ferric citrate and ferrous citrate, cause inconvenience to production and scientific research.
Summary of the invention
The object of this invention is to provide the assay method of iron concentration in a kind of algae nutritive salt, the method content that is real-time by ultraviolet spectrophotometry, quantitatively detection ferric citrate, also can calculate the content of ferrous citrate, simply, fast, accuracy rate is high, sensing range is wide, testing cost is low simultaneously.
In order to realize above technique effect, the present invention realizes as follows:
An assay method for iron concentration in algae nutritive salt, its step comprises:
(1) preparing ferric ion concentration is 6 × 10
-3the ferric citrate titer of mol/L, is cooled to 18-25 DEG C; Select this ferric ion concentration to depart from standard little, error is little.
(2) the ferric citrate titer in step (1) is diluted to the dilution of variable concentrations, light absorption microplate reader is utilized to record the absorbance values of ferric citrate dilution at 450nm place, with A450 mean value for ordinate, to should the iron concentration of absorbance be horizontal ordinate, drawing standard working curve; Select to survey absorbance, drawing standard curve at 450nm place, otherwise typical curve is non-linear.
(3) absorbance of algae nutritive salt sample at 450nm place is measured, according to the standard working curve drawn in step (1), the ferric ion concentration in calculation sample; Select to survey absorbance at 450nm place, the light absorption value of ferric citrate is between 0.1-0.5, and ferrous citrate light absorption value is 0, can distinguish ferric citrate and these two kinds of materials of ferrous citrate well.
(4) initial concentration of the ferric ion in algae nutritive salt storage liquid sample deducts the actual measurement ferric ion concentration in step (2), is the ferrous ion concentration in algae nutritive salt sample.
In described step (2), the ferric ion concentration in ferric citrate dilution is 0,0.6 × 10
-3mol/L, 1.2 × 10
-3mol/L, 2.4 × 10
-3mol/L, 4.8 × 10
-3mol/L and 6 × 10
-3mol/L.
The composition of described algae nutritive salt sample is the mixed liquor of ferric citrate and ferrous citrate.
In described step (1), open-assembly time is no more than 3 minutes to the ferric citrate titer after preparing in atmosphere.Avoid ferric citrate to be reduced.
In described step (2), during titer dilution, the dilution of employing is anaerobic distilled water.
In described step (2), mix when dilution standard liquid, can not bubble be produced, and Ambient Operating Temperature is lower than 25 DEG C.Avoid ferric citrate to be reduced.
In described step (3), before algae nutritive salt sample determination, preserved at 4 DEG C of lower seals by sample and can not be mixed with bubble, open-assembly time is no more than 3 minutes to sample in atmosphere.
The composition of described algae nutritive salt sample is ferric citrate.
The beneficial effect of the invention is: 1, in algae nutritive salt provided by the invention, the assay method of iron concentration can the content of citric acid iron-based ferrous citrate in Real_time quantitative detection algae nutritive salt storage liquid, simple, fast, accuracy rate is high, sensing range is wide.2, this assay method due to pilot process few, without the need to adding other reagent, sample without the need to pre-service, thus than National Standard Method more close to actual value, testing cost is low, the time is short.3, this detection method directly can measure ferric citrate, uses in micro-algae field, with strong points, highly sensitive,
During the error of calculation, because National Standard Method can not directly measure ferric citrate content, thus measure ferrous citrate by National Standard Method and calculate ferric citrate content, and compare with the ferric citrate that this method directly measures.
Accompanying drawing explanation
Fig. 1 is brand-new ferric citrate and ferrous citrate spectrum correlation curve in embodiment 1.
Fig. 2 is ferric citrate canonical plotting in embodiment 1.
Fig. 3 is in embodiment 3, ferric citrate canonical plotting when adopting National Standard Method to detect.
Embodiment
Below in conjunction with embodiment, the invention will be further described:
Equipment used by experiment: uv analyzer UV-1800.
Reagent used by experiment: Citric Acid Mono (C
6h
8o
7h
20), Ferric Ammonium Citrate (C
6h
10feNO
8), ferrous sulphate (FeSO
47H
2o), anaerobic distilled water;
National Standard Method reagent: oxammonium hydrochloride (HO-NH
2hCl), Phen (C
12h
8n
2h
2o), 1mol/L sodium acetate (CH
3cOONa), metallic iron, hydrochloric acid, distilled water.
1, the preparation of titer
Get citric acid 0.3g, Ferric Ammonium Citrate 0.3g, citric acid and Ferric Ammonium Citrate are dissolved with the distilled water boiled, be settled to 100mL after mixing, be cooled to 18-25 DEG C, ferric ion final concentration is 6 × 10
-3the ferric citrate titer of mol/L.This ferric citrate is now with the current, and after preparing, open-assembly time is no more than 3 minutes in atmosphere.
Get citric acid 0.3g, ferrous sulphate (FeSO
47H
2o) 0.17g, first dissolves citric acid with the distilled water boiled, then is dissolved by ferrous sulphate, and be settled to 100mL after mixing, be cooled to 18-25 DEG C, ferrous ion final concentration is 6 × 10
-3the ferrous citrate titer of mol/L.
2, the drafting of standard working curve
Get ferric citrate titer and the ferrous citrate titer of the preparation of 3mL above-mentioned steps respectively, absorption spectrum (190nm-750nm) is measured with ultraviolet spectrophotometer, find at 450nm place, the light absorption value of ferric citrate is between 0.1-0.5, and ferrous citrate light absorption value is 0, specifically as shown in Figure 1, so choosing A450 is measured value.
Get the centrifuge tube of 6 1.5mL, according to the additional proportion of following table, the citric acid solution (unit: μ L) of preparation variable concentrations
| 1 | 2 | 3 | 4 | 5 | 6 | |
| Anaerobic distilled water | 1000 | 900 | 800 | 600 | 200 | 0 |
| Titer | 0 | 100 | 200 | 400 | 800 | 1000 |
ELISA Plate is got some holes, add the citric acid solution 200 μ L of each concentration in 1-6 pipe in table respectively, microplate reader measures the A450 value in each hole, repeatedly, and calculate the mean value of A450 in the identical holes of each group # respectively, and with A450 mean value for ordinate, to should the Ferric Citrate concentration of absorbance be horizontal ordinate, drawing standard working curve, is shown in Fig. 2.
3, sample determination
Get each 250mL of algae nutritive salt sample solution of new preparation, this sample is protected 4 DEG C of sealings.The composition of this sample solution is ferrous citrate, and the theoretical concentration of its ferrous ion is 6 × 10
-3mol/L, tests it and places ferric ion between 7 days and ferrous ion concentration change.
When placing 1 day, by algae nutritive salt sample solution in ELISA Plate, measure its A450 value, substitute into standard working curve, calculate Ferric Citrate concentration, show that the concentration of ferric ion is 1.6 × 10
-3mol/L, the concentration of ferrous ion is 4.4 × 10
-3mol/L.
According to the method described above, ferric citrate when detecting placement 1 day, 2 days, 3 days, 4 days, 5 days, 6 days and 7 days and the concentration of ferrous citrate, shown in table 1 specific as follows:
Iron concentration change list in table 1 ferrous citrate storage liquid
As can be seen from Table 1, after placing one week, ferrous citrate is oxidized to ferric citrate gradually.
Embodiment 2
1, the drafting of typical curve is with embodiment 1.
2, get brand-new and sealing deposits each 250mL of ferric citrate titer of a week in ELISA Plate, measure A450 value, substitute in typical curve, calculating Ferric Citrate concentration, result is as following table 2:
Ferric citrate content in table 2 ferric citrate storage liquid
| Brand-new ferric citrate | 0.4 times of brand-new ferric citrate | Place the ferric citrate of a week |
| 5.9×10 -3mol/L | 3.58×10 -3mol/L | 4.2×10 -3mol/L |
After placing one week, ferric citrate partial reduction is ferrous citrate, so the concentration of ferrous citrate is 1.7 × 10
-3mol/L.
Embodiment 3
Accuracy rate is verified: measure ferric citrate according to national standard method (GB7873-87,3).
Principle: be ferrous iron by ferric iron hydrochloric acid hydrogen amine Restore All, then measure total iron with Phen reacting generating complex; Phen and ferrous iron reacting generating complex is utilized to measure ferrous iron content.
1, reagent: 10% hydroxylamine hydrochloride solution (newly joining);
O-phenanthrolin developer: 0.15% Phen aqueous solution;
Sodium acetate solution (1mol/L);
Iron (Fe) standard solution (0.2 × 10
-3mol/L): accurately take simple metal iron powder or pure iron silk (first washing away oxide on surface with hydrochloric acid), be dissolved in watery hydrochloric acid, heating for dissolving.
2 Specification Curve of Increasings
2.1 standards systems/5ml
Standards system is prepared according to following table 3
Table 3
| Composition | Fe titer | 10% oxammonium hydrochloride | 0.15% Phen | Sodium acetate | Distilled water |
| Mark 1 (ml) | 0 | 0.5 | 1 | 2.5 | 1 |
| Mark 2 (ml) | 0.2 | 0.5 | 1 | 2.5 | 0.8 |
| Mark 3 (ml) | 0.4 | 0.5 | 1 | 2.5 | 0.6 |
| Mark 4 (ml) | 0.6 | 0.5 | 1 | 2.5 | 0.4 |
| Mark 5 (ml) | 0.8 | 0.5 | 1 | 2.5 | 0.2 |
| Mark 6 (ml) | 1 | 0.5 | 1 | 2.5 | 0 |
2.2 microplate reader measure
Getting each 250ul of standards system 1-6 respectively drips in ELISA Plate, measures A510 and drawing standard curve, as shown in Figure 3.
Table 4
3 sample determinations
3.1 sample standard liquid
The ferric citrate titer of brand-new in example 2 is diluted, makes ferric iron final concentration be 0.2 × 10
-3mol/l.
3.2 sample system/5ml
Prepare according to following table 5
Table 5
3.3 sample determination
Get 250ul ELISA Plate method or cuvette method mensuration A510, substitute into typical curve, acquired results is as shown in table 6 below:
Table 6
From shown in table 6, National Standard Method due to pilot process more, sample pretreatment is difficult to avoid loss, and thus gap is larger.When this method directly measures ferric citrate content, without the need to adding other reagent, sample without the need to pre-service, thus more close to actual value.
Embodiment 4
National Standard Method (GB7873-87,3) is adopted to measure ferrous citrate
4.1 sample standard liquid
By brand-new in embodiment 1 and deposit ferrous citrate titer dilution, make ferrous iron final concentration be 0.2 × 10
-3mol/L.
4.2 sample system/5ml
Prepare according to following table 7:
Table 7
| Numbering | Deposit number of days (my god) | Sample liquid (ml) | 0.15% Phen (ml) | Sodium acetate (ml) | Distilled water (ml) |
| 1 | 1 | 1 | 1 | 2.5 | 0.5 |
| 2 | 2 | 1 | 1 | 2.5 | 0.5 |
| 3 | 3 | 1 | 1 | 2.5 | 0.5 |
| 4 | 4 | 1 | 1 | 2.5 | 0.5 |
| 5 | 5 | 1 | 1 | 2.5 | 0.5 |
| 6 | 6 | 1 | 1 | 2.5 | 0.5 |
| 7 | 7 | 1 | 1 | 2.5 | 0.5 |
4.3 sample determination
The algae nutritive salt sample solution of new preparation in Example 1, gets 250ul ELISA Plate method or cuvette method measures A510, and substitute into the National Standard Method typical curve of example 3 gained, acquired results is as shown in table 8 below: (unit: 10
-3mol/L)
Table 8
During the error of calculation, because National Standard Method can not directly measure ferric citrate content, thus measure ferrous citrate by National Standard Method and calculate ferric citrate content, and compare with the ferric citrate that this method directly measures.
Although the overall ferrous measurement result of National Standard Method rule compared with this method is identical, content is lower; When National Standard Method measures brand-new ferrous citrate, because the oxidation in process is difficult to control, occurred comparatively big error, average error has exceeded 25%.And use more poisonous and harmful reagent, process is loaded down with trivial details wayward.And this method directly can measure ferric citrate, with strong points, highly sensitive in micro-algae field.
When measuring ferrous citrate by National Standard Method reluctantly, because operating process is loaded down with trivial details, in sample pretreatment process, ferrous iron will be oxidized to ferric iron, and the more difficult control of practical measurement, error is larger.And this method directly measures in potpourri, particularly in micro-algal nutrient during ferric iron content, because process is simple, agents useful for same is less, and the time is shorter, and ferrous iron to greatest extent result avoids oxidation, can be widely used in high precision scientific experiment and production practices.
Claims (7)
1. the assay method of iron concentration in algae nutritive salt, its step comprises:
(1) preparing ferric ion concentration is 6 × 10
-3the ferric citrate titer of mol/L, is cooled to 18-25 DEG C;
(2) the ferric citrate titer in step (1) is diluted to the dilution of variable concentrations, light absorption microplate reader is utilized to record the absorbance values of ferric citrate dilution at 450nm place, with A450 mean value for ordinate, to should the iron concentration of absorbance be horizontal ordinate, drawing standard working curve;
(3) absorbance of algae nutritive salt sample at 450nm place is measured, according to the standard working curve drawn in step (1), the ferric ion concentration in calculation sample;
(4) initial concentration of the ferric ion in algae nutritive salt storage liquid sample deducts the actual measurement ferric ion concentration in step (2), is the ferrous ion concentration in algae nutritive salt sample.
2. the assay method of iron concentration in algae nutritive salt according to claim 1, it is characterized in that: in described step (2), the ferric ion concentration in ferric citrate dilution is 0,0.6 × 10
-3mol/L, 1.2 × 10
-3mol/L, 2.4 × 10
-3mol/L, 4.8 × 10
-3mol/L and 6 × 10
-3mol/L.
3. the assay method of iron concentration in algae nutritive salt according to claim 1, is characterized in that: the composition of described algae nutritive salt sample is the mixed liquor of ferric citrate and ferrous citrate.
4. the assay method of iron concentration in algae nutritive salt according to claim 1, it is characterized in that: in described step (1), open-assembly time is no more than 3 minutes to the ferric citrate titer after preparing in atmosphere.
5. the assay method of iron concentration in algae nutritive salt according to claim 1, is characterized in that: in described step (2), and during titer dilution, the dilution of employing is anaerobic distilled water.
6. the assay method of iron concentration in algae nutritive salt according to claim 1, is characterized in that: in described step (2), mix, can not produce bubble, and Ambient Operating Temperature is lower than 25 DEG C when dilution standard liquid.
7. the assay method of iron concentration in algae nutritive salt according to claim 1, it is characterized in that, in described step (3), before algae nutritive salt sample determination, preserved at 4 DEG C of lower seals by sample and can not be mixed with bubble, open-assembly time is no more than 3 minutes to sample in atmosphere.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101782508A (en) * | 2010-02-05 | 2010-07-21 | 中国科学院海洋研究所 | Method for measuring contents of ferrous, ferric iron and total iron in seawater |
| CN103048296A (en) * | 2012-12-19 | 2013-04-17 | 浙江大学 | Method for detecting iron content of chlorella |
| CN103913421A (en) * | 2012-12-28 | 2014-07-09 | 中国科学院沈阳应用生态研究所 | Method for determining water-soluble Fe content of eutrophic lake |
-
2015
- 2015-02-03 CN CN201510056756.2A patent/CN104596965B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101782508A (en) * | 2010-02-05 | 2010-07-21 | 中国科学院海洋研究所 | Method for measuring contents of ferrous, ferric iron and total iron in seawater |
| CN103048296A (en) * | 2012-12-19 | 2013-04-17 | 浙江大学 | Method for detecting iron content of chlorella |
| CN103913421A (en) * | 2012-12-28 | 2014-07-09 | 中国科学院沈阳应用生态研究所 | Method for determining water-soluble Fe content of eutrophic lake |
Non-Patent Citations (1)
| Title |
|---|
| 贾翠莉 等: "硅藻土中可溶性铁离子的测定", 《啤酒科技》 * |
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