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Rhena Schumann, Dana Zimmer

Principle

Phosphate ions react in acid solution with molybdate to yellow phosphorus molybdic acid, which can be reduced with ascorbic acid to molybdenum blue (Denigès 1921, in the variant of Murphy & Riley 1962, as textbook chapter by Hansen & Koroleff 1999). Antimonyl tartrate stabilises the dye. Molybdenum blue is a colloidal mixed oxide, in which the molybdenum has oxidation states between V and VII. Molybdenum blue is quantified photometrically.

Interfering ions, such as arsenate (chapter 5.2.1, cf. Review: Blomqvist et al. 1993) e.g. in waste water treatment plants with industrial waste water, have to be eliminated. A further and serious disturbance is silicate, an omnipresent ion in sand and glass. Very acidic reaction conditions hinder the reaction of silicate with molybdate (Gripenberg 1929). However, such acidic condition may cause decomposition of acid-labile organic phosphate compounds such as glucose-6-phosphate, which reacts to phosphate and is measured as phosphate as well. For this reason, the result of the molybdenum blue reaction under these conditions is not called (ortho-)phosphate but soluble reactive phosphorus (SRP). Probably, it is a bit overestimated in comparison to phosphate. 

Reaction equation

Concentration range

The disadvantage of this method in comparison to vanado-molybdenum-yellow and probably the malachite-green (both contain molybdate as well in the reagent) is the low stability of the used reagents. The ascorbic acid as well as the molybdenum mixed reagent have a short stability time. The ascorbic acid solution changed to a greenish solution (after around 10 days) or loses its effect and the mixed reagent is extremely sensitive to impurities. These impurities originated from ultra-pure water for the reagent or the vessels, e.g. dishwasher-cleaned vessels. Additionally, pipetting errors can transfer discolorations. That means that it is better to pipette from small vessels of 5 to 20 ml (for around 20…30 samples) than the original 100 to 200 ml reagent vessel. Reagents are ageing by light (change to blue-green within 2 to 3 days) as well, causing false-positive results.

The linear range of the method at a wavelength of 885 nm and a cuvette length of 5 cm is between 0.05 µmol l-1 (limit of quantification) and 10 µmol l-1 (around 0.002 and 0.31 mg P l-1). For this reason, the method is well suited for water samples, but for P-rich material it has to be adjusted to the measurement range (strong or stepwise dilution).

The molybdenum blue reaction is one of the most sensitive methods, even though it is not free from matrix effects, which are caused, for example, by salt in seawater. For this reason, standards for seawater samples have to be set in seawater salinity (chapter 5.2.2). The limit of detection is normally 0.05 µmol l-1 (1.6 µg l-1), if a 5 cm long cuvette is used. Two options exist to bring down the limit of detection into the nano-molar range: concentration of the analyte (chapter 5.5.1) and the improvement of detection, e.g. by longer cuvettes (Review: Patey et al. 2008, chapter 5.5.2).

Procedure

► Calibration line: The samples volume has to be at least 15 ml to fill a  5 cm cuvette. 20 or 25 ml are possible as well.

► A further miniaturisation is only possible by a higher limit of detection (4 ml for a 1 cm cuvette). Impurities and errors of pipetting have a much larger effect for smaller sample volumes.
► Vessels can be 25 or 50 ml Erlenmeyer flasks (best wide-neck flasks) or 50 ml centrifuge tubes (multiple reusable) (Fig. 5.2.3-1).
► In water analytics equidistant calibration lines with 10 measurement points are usual (chapter 6.1). The coefficient of determination should be r2 > 0,995.

► Setting and measuring of samples

► Samples with high turbidity (seston, milled sediment material) have to be filtrated with vacuum filtration by GF 6 (nominal pore size 1-2 µm) or if containing very small particles by cellulose acetate (nominal pore size 0.45 µm). Without negative pressure folded phosphate-free filters (e. g. MN 616 G) can be used (Fig. 5.2.3-2).
► If necessary, measure turbidity or discolouration of the filtrates at 885 nm (not necessary in persulfate digestions),
► add 0.25 ml ascorbic acid solution to 25 ml of the filtrated sample,
► add 0.5 ml Mo-mix-reagent, wait 20 minutes,
► make reagent blank RBW analogous to samples with 25 ml of ultra-pure water and
► measure all samples at 885 nm in a 5 cm cuvette.

► Calibration line: The samples volume has to be at least 15 ml to fill a  5 cm cuvette. 20 or 25 ml are possible as well.
► A further miniaturisation is only possible by a higher limit of detection (4 ml for a 1 cm cuvette). Impurities and errors of pipetting have a much larger effect for smaller sample volumes.
► Vessels can be 25 or 50 ml Erlenmeyer flasks (best wide-neck flasks) or 50 ml centrifuge tubes (multiple reusable) (Fig. 5.2.3-1).
► In water analytics equidistant calibration lines with 10 measurement points are usual (chapter 6.1). The coefficient of determination should be r2 > 0,995.

Quality management

► Per 10 samples at least 1 blank
► If no real samples repetitions are planned, set 2 measurement repetitions for every 10th sample.
► Measure only in the calibrated range.
► Make control cards such as in chapters 6.3-6.5 explained.

Calculation

► The RBW can be subtracted, if the signal ("noise") originates from the reagent.
► FBW can only be subtracted, if the sample is turbid and cannot be filtrated or if an appreciable discolouration is visible.

Dana Zimmer, Rhena Schumann

Principle

Similar to the molybdenum blue method (chapter 5.2.3) a green colour complex is formed under acidic conditions by molybdate, malachite green and phosphate. The extinction of the blue green colour complex is measured at a wavelength of 623 nm and used for determination of P concentrations. The method of Altmann et al. (1971) is used in the working group Soil Science to measure P concentrations in NaHCO₃- and NaOH-extracts of soil samples. The calibration line in table 5.2.4-1 was created in a water matrix to test the linear range of the method.

Concentration range

The linear range of the method is between 20 µg or 0.6 µmol and 250 µg or 8 µmol l^-1. For this range, the limit of quantification was with 39 µg P l^-1 (1.3 µmol l^-1) very high, even if the reagent blank was subtracted from all blanks. Here, the limit of quantification should be calculated according to the calibration line method (chapter 6.2).

Procedure

► Preparation:

► Fill graduated 25 ml test tubes or other volumetric flaks with ultra-pure water (RW) and leave to stand overnight for emaciation.
► If necessary, dry KH₂PO₄ for 2 hours at 40 °C and cool down in desiccator, alternatively use commercial P standards.

► Calibration line:

► Pipette from P working solution in table 5.2.4-1 specified ml in 25 ml graduated test tubes or volumetric flasks for standards.
► Fill to 25 ml with RW.
► Calibrants contain up to 250 g P l^-1 or 8,1 µmol l^-1.
► For a linear calibration line, it is recommended to set standards between 20 and maximal 250 µg P l^-1 (Tab. 5.2.4-1).

► Setting of samples and measurement:

► Pipette in the standard vessels the same amounts of calibrants as samples are given into the sample vessels (e.g. 10 ml NaHCO₃-extract).
► Calibrants should be dissolved in the same matrix like the samples.
► Add, for example, 10 ml sample or blank into the vessels for samples and blanks.
► Fill all vessels (standards, samples and blanks) to ca. 12 ml with RW.
► Add 1.5 ml of 24 % H₂SO₄ in all vessels and wait 10 min for silicate elimination.
► Add 2.5 ml malachite-PV-solution and swing for mixing (do not shake too strong, to avoid creation of foam). Wait around 5 min.
► Add 2.5 ml molybdate solution, fill vessels with RW to 25 ml and swing for mixing. Wait 1 hour for colour development.
► Fill aliquots into cuvettes and measure extinction at 623 nm.
► Dilute samples with extinctions > 1, set them again in 25 ml test tubes or volumetric flasks with all reagents and measure again.

A very long calibration line in a water matrix shows the restricted linear range (Fig. 5.2.4-2). A concentration higher than 250 µg P l^-1 (extinction 0.62 in 1 cm cuvettes) saturation effects are visible. At a concentration of 1000 µg P l^-1 the malachite green flocculates.

Quality management

Reagents of the malachite green method have a weak green-yellow inherent colour. That means that the extinction of the reagent blank has to be subtracted from all values (samples, blanks and calibrants).

Sometimes it is recommended, and often the software offers it as well, to set this value in the photometer or set it as reference in the photometer. This is not recommended, because simple operating errors can have serious consequences. Errors can be for example: at reference measurement the cuvettes were not completely clean, the blank could be turbid by lint or something similar. Additionally, a drift or a suddenly higher blank is not visible and cannot be corrected later.   

Calculation

According to the inherent colour of the reagents, the subtraction of the reagent blank is urgently required. Another question is the stability of the reagent blank. For the calibration line itself, it is not important for calculation of the conversion factor. The increase is shifted parallelly along the y-axis. In each case, either the reagent blank of the sample or the reagent blank of the calibration line has to be subtracted.

PO₄^3-=F^.(E_sample-E_RBW-E_FBW)         

PO₄^3-   phosphate concentration (mg P l^-1)
F         Factor of calibration line¹
E         Extinction²
RBW    Reagent blank
FBW    Absorption of the filtrates

Dana Zimmer, Theresa Zicker, Rhena Schumann

Principle

In the vanadate-molybdate-procedure, orthophosphate and ammonium vanadate react together with molybdate to yellow ammonium phosphorus vanado-molybdate. Misson (1908) is stated as discoverer of this reaction in older publications. This source was electronically not available, but the method was. This method was analysed more detailed, adjusted and assessed as better suited for P determination in steel than the former gravimetric molybdenum blue method and the titrimetric method by Kitson & Mellon (1944), Hill (1947) and Gerricke & Kurmies (1952), since this method was simpler and faster to handle. A compilation of such older gravimetric and titrimetric methods can be found in Huber von Schönenwerd & Solothurn (1950). Such precipitation methods with molybdenum blue are describe by Lunge (1905) und Neubauer & Lücker (1912) as well.

The vanado-molybdate colour complex has a slight yellow colour already without P. For this reason, the reagent blank has to be subtracted from all measurement values. The reagent is prepared in HNO₃, which means that measurement of P in HNO₃-containing solutions is possible as well. Similar to the molybdenum blue method interferences of for example As(V) have to be taken into consideration (Gee & Deitz 1953). Measurement of the extinction for P determination is done at different wave lengths (e.g. Cavell 1954, Leonard 1946, Quin & Woods 1976, Singh & Ali 1987), since the optimal wave length is between 380 and 450 nm (Gee & Deitz 1953).

Reaction equation

According to the specific method, reagents and end products vary, which additionally affects limit of quantification, necessary time of reaction and suchlike. The reagent ammonium heptamolybdate is very common: (NH₄)₆Mo₇O₂₄·2 H₂O. The general reaction equation and the end products are:

PO₄^3- + 2 VO^3- + 10 MoO₄^2- + 10 H₂O → [PV₂Mo₁₀O₄₀]^5- + 20 OH^- (ZUM.Wiki)

→ [P₂(V₂O₆) (Mo₂O₇)₁₁] (Hanson 1950)

→ H_3+nPMo_{12 n}V_nO₄₀·x H₂O (Zhang et al. 2007)

http://vanadium.atomistry.com/heteropoly_acids_with_vanadium.html
If presented with NH₄: 2 formulas are given:

  and  

which is measurable as PO₄∙(NH₄)₃∙VO₃∙NH₄∙16 MoO₃ (Huber von Schönwerd & Solothurn 1950)

Concentration range

At a wave length of 430 nm the linear range of the method is between 0.3 (limit of detection) and 20 mg P l-1 (9.7…645 µmol l-1) and corresponds to the range presented by Singh & Ali (1987). At a concentration of 20 mg P l-1 the extinction was 0.8. From concentrations of 40 mg P l-1 the extinctions were > 1 (transmission < 10 %) and, therefore, the power range of the photometry is exhausted.

According to the linear concentration range, this method is not suitable for seston (water samples) and needs higher weigh-in for most sediments and soils (compare to chapters 4.1.2 to 4.3.1).

This presented method is used in the working group Agronomy for P determination in plant samples after ashing and digestion with HCl (see chapter 4.3). Currently, phosphorus is determined with other elements at ICP-OES after digestion. The vanadate-molybdate-method is currently only used for P determination in the double-lactate-extract.

The advantage of the vanadate-molybdate-method in comparison to molybdenum blue, instead of much lower sensitivity, is the stability of the used reagents and the colour complex of at least one week (Burns & Hutsby 1986). According to Gerricke & Kurmies (1952), the reagent is longer stable than a week in a brown bottle.

Procedure

► Determination of wave length: Measurement of blanks and standards in a range of 370 to 450 nm.
► Below 370 nm care for optical quality of the cuvette (OS: Optical special glass or plastic cuvettes for specific wave length range)!
► Assessment between high signal-noise ratio (increases in the UV range) and low blanks (increases strongly in the UV range as well (Fig. 5.2.1-1, compare to Ma & McKinley 1953).
► Most suited wave length are around 400 nm. The working group Agronomy decided to measure at 430 nm.

► Calibration lien for high concentrations (1…20 mg P l-1): pipette the specific volume in a 100 ml volumetric flask (Tab. 5.2.5-1).
► Add the corresponding volume of the sample matrix, e.g. 2 ml of   25 % HCl, if plant ash was digested in boiling HCl (chapter 4.3.1) and
► fill flask with RW to calibration mark.
► For the determination of the measurement range only ultra-pure water (without matrix, Fig. 5.2.5-3 a) mixed with working solution (see below) also can be used.

► divergent calibration line for low concentrations (< 1 mg P l-1):
► the increases of the calibration lines until 1 mg P l-1 and those beyond varied by more than 10 % (Fig. 5.2.5-2). For this reason, values for > 1 mg P l-1 have to be calculated with the second factor (Fig. 5.2.5-2 b).
► In dependency to the measurable concentration range, the weigh-in of the samples have to be adjusted.

► Calibration line for other matrices (1-20 mg P l-1):
► The increases of calibration lines in colour-changing matrices (Fig. 5.2.5-3 b compared to Fig. 5.2.5-2) can differ strongly from each other.
► The colour depth of reagents is strongly affected by the pH value of the solution and a bit less from the salt concentration and other ions.

► Set and measure samples

► Set the calibration line (Tab 5.2.5-1) in a 50 ml volumetric flask for each sample, each blank and each standard.
► Pipette in each flask 15 ml of the VM-mixture.
► Fill the flasks with the specific filtrate (sample, blanks) and the standard.
► Close each flask with a plug, swing and wait for 2 hours.
► Swing the solution directly before measurement.
► Measure extinction at spectral photometer in a 1 cm cuvette at 430 nm (Fig. 5.2.5-5).
► If the measurement value is outside the calibration range, the sample extract has to be diluted und mixed again with the VM-Mix reagent and measured again.

Quality management

► Determine optimal wave length for used extraction matrix (see Fig. 5.2.5-1).
► Use at least 1 blank for 10 samples.
► If no real sample repetitions are planned, measure 2 repetitions for every 10th sample.
► Measure only within the calibration range.
► Monitor by control charts according to chapters 6.3 to 6.5.

Calculation

The strong dilution of the samples (35 ml) by the reagent mixture (15 ml) means that both volumes have to be measured exactly. This is less critical in other common reagent amounts (<  5 % of total volume). This mixture-relation has to be stable for all measurements to calculate the corrects concentration according to the calibration line.

Chemicals

The necessary amount of the vanadate-molybdate-mixture depends on the number of samples. In the working group Agronomy, a 12 litre vanadate-molybdate-mixture is prepared, since after harvest of pot and field experiments a lot of samples have to be measured. If a few samples must be measured, less chemicals are necessary.

► Ultra-pure water (RW)
► diluted HNO₃: fill 2776 ml RW in a 15 l glass vessel and subsequently 1224 ml concentrated HNO₃. The solution is warmed up only a bit.

► 4 l molybdate solution: produce 2 x 2 litre.
► Weigh in 100 g ammonium molybdate ((NH₄)₆Mo₇O₂₄·2 H₂O) in a 2000 ml volumetric flask and dissolve in around 1000 ml of warm RW.
► Fill to calibration mark with RW after cooling down.

► VM-reagent (vanadate-molybdate-mixture) for a 12 l stock solution
► Mix 4 litres diluted HNO₃, 4 litres vanadate solution and 4 litres molybdate solution.
► Store the mixture in the dark. Wait at least 3 days before first usage, waiting for a week would be better.
► Mixture can be used for around 3 months.

► Standards
► Dry KH₂PO₄ at 105 °C and cool down in a desiccator.
► P stock solution (1 g P l-1): weigh in 4.394 g of dried KH₂PO₄ in a 1000 ml volumetric flask (molecular mass of KH₂PO₄ = 136,09 g l-1)
► Add 500-700 ml of RW, dissolve KH2PO4 and fill with RW to calibration mark.
► Alternatively, use commercial P standards.
► P working solution (100 mg P l-1) for calibration line from P stock solution (1 g P l-1) dilute 1:10: pipette 10 ml of P stock solution in a 100 ml volumetric flask and fill with RW to calibration mark.

References

Burns IG, Hutsby W (1986) Critical comparison of the vanadomolybdate and the molybdenum blue methods for the analysis of phosphate in plant sap. Comm Soil Sci Plant Anal 17: 839-852, DOI: 10.1080/00103628609367756

Cavell AJ (1954) The colorimetric determination of phosphorus in plant materials. J Sci Food Agric 5: 479-480, DOI: 10.1002/jsfa.2740060814

Gee A, Deitz VR (1953) Determination of phosphate by differential spectrophoto¬metry. Anal Chem 25: 1320-1324, DOI: 10.1021/ac60081a006

Gericke S, Kurmies B (1952) Colorimetrische Bestimmung der Phosphorsäure mit Vanadat-Molybdat. Fresenius' Zeitschr anal Chem 137: 15-22, DOI: 10.1007/978-3-662-11336-3_1

Hanson W (1950) The photometric determination of phosphorus in fertilizers using the phosphovanado‐molybdate complex. J Science of Food Agric 1: 1949–1950, DOI: 10.1002/jsfa.2740010604

Hill UT (1947) Colorimetric Determination of Phosphorus in Steels. Anal Chem 19: 318–319, DOI: 10.1021/ac60005a010

Huber von Schönenwerd und Solothurn AA (1950) Ueber die kolorimetrische Bestimmung von Mangan und Phosphor in Stahl und ihre Verteilung in Elektroschweissungen. Dissertation at ETH Zurich, DOI: 10.3929/ethz-a-000096545

Kitson RE, Mellon MG (1944) Colorimetric determination of phosphorus as molybdivanadophosphoric acid. Industr Engin Chem 16: 379-383, DOI: 10.1021/i560130a017

Leonard JD (1964) The colorimetric determination of phosphorus in fertilizers. J South African Chem Inst 17: 101-113

Lunge G (1905) Eisenerze-Bestimmung von Phosphorsäure. In Chemisch-Technische Untersuchungsmethoden. Vol. 2, 5th ed., 25-28, Verlag von Julius Springer

Ma TS, Mc Kinley, JD (1953) Determination of phosphorus in organic compounds: A new micro procedure. Mikrochim Acta 1-2: 4-13, DOI: 10.1007/BF01215760

Misson G (1908) Colorimetric estimation of phosphorus in steel. Chemiker-Zeitung 32: 633

Neubauer H, Lücker F (1912) Über die v. Lorenz'sche Methode der Phosphorsäurebestimmung. Zeitschr Anal Chem 51: 161-175, DOI: 10.1007/BF01440989

Quin BF, Woods PH (1976) Rapid manual determination of sulfur and phosphorus in plant material. Communications in Soil Science and Plant Analysis 7: 415-426, DOI: 10.1080/00103627609366652

Singh V, Ali SZ (1987) Estimation of phosphorus in native and modified starches. Improvement in the molybdovanadophosphoric acid method. Starch/Stärke 39: 277-279, DOI: 10.1002/star.19870390806

Zhang F, Guo M, Ge H, Wang J (2007) A new method for the synthesis of molybdovanadophosphoric heteropoly acids and their catalytic activities. Front. Chem. Sci. Eng 1 (3), 296-299, DOI: 10.1007/s11705-007-0054-0

For citation: Zimmer D, Zicker T, Schumann R (year of download) Chapter 5.2.5 Vanado-molybdate-yellow: in medium concentration range (Version 1.0) in Zimmer D, Baumann K, Berthold M, Schumann R: Handbook on the Selection of Methods for Digestion and Determination of Total Phosphorus in Environmental Samples. DOI: 10.12754/misc-2020-0001

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Dana Zimmer, Sebastian Strauch, Rhena Schumann

In the working group Aquaculture and Sea-Ranching (AUF, University of Rostock) the discrete analyzer Gallery™ of the producer Thermo Fisher Scientific™ (Fig. 5.4.3-1) is used for photometric P determination. This analyzer uses up to 200 colorimetric and enzymatic reactions to prove elements and (to some extend) organic compounds in sample (extracts). Besides phosphate, aluminum, ammonium, nitrite, nitrate, iron, urea, magnesium, sulphate and tin can be determined in liquid food and different liquid environmental sample (extracts). Photometric measurements are possible between 275 and 880 nm. Additionally, pH values and conductivity can be determined by ion-selective electrodes.

Sample extracts and necessary detection reagents are placed in glass or plastic tubes (0,5; 2; 4; 5; 7 or 10 ml) in specific "segments" (Fig. 5.4.3-2).
Overall, 6 segments with 9 places each for samples and reagents can be set on the “segment plate”. There is a barcode for internal identification on each segment. Specific setting of samples and reagents is recorded in the program previously. The type of analyses as well as the method can be selected individually for each sample. It has to be considered that for each analysis method the reagents (and calibration lines) are placed in the analyzer. Standards for quality control can be placed in the measurement series as well. After the test measurement an automatic dilution of samples can be set. The necessary ultra-pure water has to be placed in the allocated vessel. The analyzer sucks in the sample, the reagents and, if necessary, the water (for dilution) to pipette all in cuvettes (volume 300 µl) according to the selected programs. Filled cuvettes are transported in the incubator and after waiting time the extinction is determined (Fig. 5.4.3-3). Extinctions are converted to concentrations per litre by the calibration lines. Method blanks are measured like the samples and have to be subtracted from samples afterwards. After measurement, the cuvettes are transferred into the waste container automatically.

References

Operation manual Gallery, last accessed 15.05.2018

Product catalogue Gallery of Thermo Fisher Scientific, last accessed 15.05.2018

Product presentation Gallery, last accessed 15.05.2018

For citation: Zimmer D, Strauch SM, Schumann R (year of download) Chapter 5.4.3 Discrete Measurement (Version 1.0) in Zimmer D, Baumann K, Berthold M, Schumann R: Handbook on the Selection of Methods for Digestion and Determination of Total Phosphorus in Environmental Samples. DOI: 10.12754/misc-2020-0001

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Shortened taken from Schneider et al. (2016)

Chromatography is the general term for physical-chemical separation processes, which are based on the distribution of a substance between a mobile and a stationary phase. In ion chromatography, charged particles are being separated. It is based on three different separation mechanisms: ion exchange, ion pair production and ion exclusion. The ion exchange chromatography is simplified referred to as ion chromatography (IC); the ion pair chromatography (IPC) and the ion exclusion chromatography (IEC) are considered as more specific applications. Depending on the column, anions and cations may be separated in the IC.

An IC consists of a storage vessel with the eluents, a pump, the injector for the samples, the separation column, the suppressor system, the detector, the computer for data processing and a waste container (Fig. 5.6-1).

Shortened taken from Schneider et al. (2016)

The solid phase in the separation column normally consists of a polymer resin. Quaternary ammonium salt compounds, which are charged with the eluent (e.g. NaHCO3 in the mobile phase), have been established for the separation of anions. The charging of the separation column before the injection of the sample is crucial, since the exchange of the ions from the sample are proceeded by stoichiometric amounts of the respective ions. In the course of the chromatography process the ions of the sample (e.g. Br^-, Cl^-) displace the counterion of the eluent (e.g. HCO₃^-). By the further adding of the eluent the ions of the sample are being replaced until they reach the detector and get detected. This is a reversible equilibrium process. As a result of the various affinity of the ions for the stationary phase a separation comes about. The constant, which characterizes the equilibrium process, is referred to as partition coefficient K and is defined as relation of the concentration of a substance A in the stationary and mobile phase.

Therefore, substances with a high partition coefficient K are held back stronger than those with a small K. The partition coefficient K is, on the one hand, proportional to the ionic charge (so for an anion Ax-: K(A^3-) > K(A^2-) > K(A^-)) and, on the other hand, proportional to 1 / ionic size (in solvated condition (dissolved ion + associated shell made of ionic solvent)).
Due to the different retention time of the anions at the column the anions are being released with time lag and thus detected (Fig. 5.6-2).

With the appropriate sample preparation, the columns and device settings different phosphates can also be detected. In cheese for example, it was differentiated between open-chained condensed phosphates (P1 to P7) and cyclic phosphates (e.g. trimetaphosphate; P3m) (Jensen 2013, Fig. 5.6-3).

In the working group Soil Physics (AUF, University of Rostock) a new IC, the 930 Compact IC Flex from Metrohm, is available since summer 2018. This instrument consists of two Compact IC Flex with column oven and Degasser (each for anions and cations), with separate auto samplers. That means that anions, cations and polar substances can be determined with and without sequential suppression. The background conductivity is reduced to a minimum by the sequential suppression (chemical suppressor and CO₂-suppressor), that means that especially anions can be detected better. Currently, the instrument has two columns Metrosep C4 - 150 (Metrohm 2015, p. 154) and Metrosep A Supp 5 - 150 (Metrohm 2015, p. 64), both including the precolumns. The Metrosep C4 – 150 is a universal standard column for analysis of cations of alkaline and alkaline earth metals (Li⁺, Na⁺, K⁺, Rb⁺, Cs⁺, Mg²⁺, Ca²⁺, Sr²⁺, Ba²⁺, NH⁴⁺) in aqueous solutions. The Metrosep A Supp 5 – 150 is an anions column, which can separate F^–, Cl^–, Br^–, I^–, ClO₂^-, ClO₃^-, ClO₄^-, BrO₃^-, und CrO₄^2– and phosphate ions (PO₄^3-) (Fig. 5.6-4). The instrument contains conductivity detectors for anions and cations and an UV/VIS detector for anions (to suppress high Cl concentrations). Additionally, for high ion concentrations an inline-dilution and inline-dialysis for anions is available.

In the working group Soil Physics mainly water samples from a drain of an arable field are analysed currently. The following sample matrices can be analysed by this IC: fresh water, seawater, aqueous solutions of extracts from solid samples (has not been tested so far). Currently, samples of moor water of the projects WETSCAPES und BALTIC TRANSCOAST are analysed.

► Sample preparation:

► Filtration of water samples with folded filters is only necessary if too much solids or larger particles are in the sample
► Setting of pH of the sample is only necessary if the pH value is outside the pH range of the column (A Supp 5-150: 3 bis 12); for this column that means pH values < 3
► Conductivity of the sample should be measured before to get an estimation of the concentration of anions in the sample
► If enough sample is available, a minimum of 8 ml should be filled in the IC vials and placed in the fridge (if necessary) until measurement
► If there is too little sample material in the vial, it is diluted automatically in the instrument.

► Measurement:

► It is calibrated once for a lot of measurements. The calibration is valid as longs as control standards do not exceed defined deviation. In this case the calibration has to be done again.
► Currently, the calibration range is between 0.2 bis 200 mg phosphate l-1 in 2 measurement ranges (0.2…20 mg l-1 and 20…200 mg l-1). The samples are aromatically assigned to the range.

The limit of detection is in the µg per litre range and could be lowered to ng per litre by previous enrichment process (Jensen 2013, p. 113). Until now, it has been calibrated to 0.2 mg phosphate per litre in the working group Soil Physics. Since the instrument is new, limits of detection and quantification are still in work for specific matrices. According to preliminary results, the limit of detection seems to be in the range of 5 µg phosphate per litre.