Measuring of Calcium In Milk - Principle, Procedure, Result

Calcium in milk is measured by first releasing the calcium from milk matrix and then it is quantified by titration or instrumental method. In milk calcium are present in soluble form and also it is bound with casein complex so direct estimation without treatment is not accurate. The sample is prepared by mineralization in which organic matter is destroyed and calcium is brought into ionic form in clear solution.

For mineralization dry ashing method can be used in which milk sample is heated in furnace at high temperature and organic matter is burnt and ash is obtained and it is dissolved in acid. Wet digestion method is also used in which strong acid like nitric acid is used with heat or microwave and proteins and lipids are broken down rapidly. For routine analysis deproteinization is done by trichloroacetic acid and proteins are precipitated and calcium ions remain in filtrate.

After sample preparation complexometric titration is used as basic method. In this method standard EDTA solution is added slowly and EDTA forms stable complex with calcium ions. Because magnesium can interfere the titration is done in highly alkaline condition (pH 12–14) so magnesium is precipitated out. Indicator like Patton–Reeder indicator or murexide is added and end point is detected by sharp permanent colour change (wine red to pure blue) and calcium amount is calculated from EDTA volume used.

For more sensitive measurement Flame Atomic Absorption Spectrometry (FAAS) is used in which diluted or digested sample is atomized in flame and absorption of specific light by calcium atoms is measured. Milk contain phosphate and sulfate which can interfere so releasing agent like lanthanum chloride is added and phosphate is bound and calcium signal is detected properly.

For advanced analysis ICP-OES and ICP-MS are used. The digested milk sample is introduced as aerosol into hot argon plasma. ICP-OES measures emitted light from excited calcium atom and ICP-MS measures calcium ions by mass to charge ratio. These methods are used for high sensitivity and it can measure calcium along with other macro and trace elements in same sample.

Principle for Measuring of Calcium In Milk

Principle for measuring calcium in milk is based on releasing calcium from milk matrix and then it is measured by titration or instrumental method. Milk contains calcium in free ionic form and also it is bound with casein complex so sample digestion or deconstruction is required. Wet digestion or dry ashing are used and organic matter is destroyed and calcium is converted into measurable ionic form in clear solution.

In complexometric titration method the principle is formation of stable chelate between calcium ions and EDTA. The titration is carried out in highly alkaline condition (pH 12–14) so that interfering magnesium is precipitated as magnesium hydroxide and it do not react in titration. Metallochromic indicator like Patton–Reeder forms pinkish red complex with calcium first and when EDTA is added it displaces the indicator and sharp colour change to pure blue is obtained at end point. The volume of EDTA used is proportional to calcium present in sample.

In Flame Atomic Absorption Spectrometry (FAAS) the principle is based on absorption of light by ground state calcium atoms after atomization of sample in air–acetylene flame. Calcium absorbs at specific wavelength (422.7 nm) and absorbance is proportional to calcium concentration. Milk phosphate and sulfate can cause chemical interference so releasing agent like lanthanum chloride is added and calcium is freed for accurate measurement.

In ICP techniques the principle is based on high temperature argon plasma which decomposes atomizes and ionizes the digested sample. In ICP-OES excited calcium atom emits characteristic light and emission intensity is measured for calcium quantification. In ICP-MS calcium is converted into ions and it is separated and quantified by mass to charge ratio (m/z) in mass spectrometer and accurate result is obtained due to high sensitivity.

Requirements for Determination of Calcium In Milk

Requirements are as follows–

General requirement and quality control–

Milk sample is homogenized properly for getting uniform sample before analysis.
Quality control is followed by using blank and spike recovery test and Certified Reference Material (CRM) like NIST SRM 1849a for checking accuracy.

Sample preparation (mineralization)–

Milk matrix is deconstructed so that protein bound calcium is released into measurable ionic form.
Microwave assisted acid digestion is used in closed vessel system and strong acids like nitric acid with hydrogen peroxide are used.
Dry ashing is done in muffle furnace at 450–600°C for removing organic matter and ash is obtained.

Wet ashing is done by strong oxidizing acids like nitric acid perchloric acid or sulfuric acid with heating on hot plate or digester block.
Deproteinization is done by trichloroacetic acid (TCA) for precipitating protein and it is used for rapid routine volumetric analysis.

Complexometric titration requirement–

Standard EDTA solution is required as chelating titrant.
Sodium hydroxide (NaOH) or potassium hydroxide (KOH) is required for raising pH to 12–14 and magnesium interference is removed by precipitating as magnesium hydroxide.

Indicator is required like Patton–Reeder (PR) or murexide for detecting colour change at end point.

FAAS requirement–

Calcium hollow cathode lamp is required and wavelength 422.7 nm is used for maximum sensitivity.
Air–acetylene flame is used in slightly reducing condition so that stable calcium oxide is not formed.

Lanthanum chloride is added in sample and standard as releasing agent to mask phosphate and sulfate interference.
Sample dilution is required (1:250 or 1:500) because calcium concentration in milk is high and it exceed linear range.

ICP-OES / ICP-MS requirement–

High purity argon gas is required for generating plasma and for atomization and ionization of sample.

Internal standard like Yttrium Scandium or Germanium is added for correcting matrix effect and instrumental drift.
In ICP-MS collision/reaction cell is used (helium or hydrogen) or cold plasma condition is used for removing polyatomic and isobaric interference.

Procedure for Measuring of Calcium In Milk

Procedure are as follows–

Phase 1 (sample preparation)–

Milk sample is mixed properly and it is taken for analysis and calcium is released from milk matrix before measurement.

In dry ashing method milk sample is heated in muffle furnace (450°C–600°C) for 8–18 hours and organic matter is burnt completely and inorganic ash is obtained. The ash is dissolved in acid like nitric acid or hydrochloric acid and clear solution is prepared.
In wet digestion method milk is mixed with strong oxidizing agents like nitric acid and hydrogen peroxide and it is heated in open tube digester block (about 120°C for 4 hours) or it is digested in closed vessel microwave system and proteins and lipids are broken down and digested solution is obtained.
In deproteinization method (for routine titration) trichloroacetic acid (TCA) is added in milk and proteins are precipitated. The mixture is centrifuged and clear liquid is collected and it is neutralized and calcium ions remains in solution.

Phase 2 (measurement technique)–

Complexometric titration (EDTA method)–

The sample pH is adjusted to 12–14 by sodium hydroxide or potassium hydroxide and interfering magnesium is precipitated as magnesium hydroxide.
Indicator like Patton–Reeder or murexide is added and solution becomes pink or wine red.
Standard EDTA is added slowly and calcium is bound with EDTA and sharp colour change to pure blue or violet is obtained and end point is noted.

Flame Atomic Absorption Spectrometry (FAAS)–

The digested milk sample is diluted because calcium concentration in milk is high and it exceeds instrument linear range.
Lanthanum chloride is added as releasing agent for masking phosphate and sulfate interference.
The sample is aspirated into air–acetylene flame and absorption of calcium atoms is measured at 422.7 nm.

ICP-OES / ICP-MS–

Internal standard like Yttrium Scandium or Germanium is added in digested sample for correction of matrix effect and instrumental drift.
The sample is introduced as aerosol into argon plasma and calcium is atomized and ionized.
Calcium is quantified by measuring emitted light (ICP-OES) or by mass to charge ratio measurement in mass spectrometer (ICP-MS).

Results and Calculation for Determination of Calcium In Milk

Calculation (complexometric EDTA titration)–

In EDTA method calcium reacts with EDTA in 1:1 ratio and the calculation is based on EDTA molarity and EDTA volume used.

Moles of Ca2+ = Molarity of EDTA × Volume of EDTA (in litre).
Mass of Ca (g) = Moles of Ca2+ × 40.078 g/mol.

Final concentration is calculated by converting this mass to the total volume or mass of original milk sample (dilution factor is applied).

Calculation (potassium permanganate titration method)–

If precipitation and permanganate titration method is followed then calcium percentage is calculated by formula.

Formula–

Calcium (%) = 1.2 × (V / W)
Where V = Volume (ml) of standard KMnO4 used in titration.
W = Weight (g) of milk sample taken for test.

Calculation (instrumental method AAS / ICP-OES / ICP-MS)–

In instrumental method the instrument reading is corrected by dilution factor because sample is diluted during preparation.

Formula–

Original concentration (mg/kg) = Instrument reading (mg/L) × [Final diluted volume (ml) / Initial sample mass (g)]

If result is required in mg/100 ml then milk density factor is used (milk density is about 1.03 g/ml) and unit conversion is done.

Typical result (calcium content in milk)–

Whole cow milk shows calcium about 118–120 mg/100 ml.
Skimmed milk shows calcium about 113–132 mg/100 ml.
Semi skimmed milk shows average calcium about 128 mg/100 ml.
Raw cow milk shows average value about 107.2 mg/100 g (reported in Australia study).
Australian retail pasteurised milk shows average calcium about 1171–1174 µg/g (about 117 mg/100 g).

Infant formula (ready to feed) shows calcium about 53 mg/100 ml.

Importance of Measuring Calcium In Milk

Uses/importance are as follows–

It is used for evaluating nutritional value of milk because calcium is essential for formation and maintenance of bones and teeth. It is also required for muscle control blood circulation and enzyme reaction so calcium measurement helps in checking daily nutritional requirement specially in newborn and growing children.
It is used for regulatory compliance and accurate labeling and dairies monitor calcium level to meet government regulation and international food standard (Codex Alimentarius). The calcium quantification is required for verifying composition and correct nutritional labeling.

It is used in quality control during dairy processing because calcium helps in structural integrity of milk and dairy products. Monitoring calcium concentration helps in maintaining consistency in production and processing.
It is used for detecting illegal adulteration and abnormal high calcium level can indicate addition of chemical neutralizer like calcium hydroxide for masking acidity increase due to bacterial growth in poorly refrigerated milk.

Limitations

Limitations are as follows–

In complexometric titration milk turbidity and pale colour can make end point colour change difficult to observe properly.

Magnesium interference occur and pH has to be kept highly alkaline (pH 12–14) for precipitating magnesium before titration.
Milk proteins and other ions can cause matrix masking and slow reaction and back titration may be needed for accurate result.
It is single element method and large sample volume is required and only one element is analysed at a time.

In FAAS phosphate sulfate and aluminium present in milk can form heat resistant compound (calcium orthophosphate) and reading becomes low and lanthanum chloride masking agent is required.
FAAS is single element technique and separate hollow cathode lamp is required for each element so throughput become slow.
Sample preparation is lengthy because ashing and acid dissolution are needed before analysis and total time is increased.

ICP-OES and ICP-MS instruments are costly and operational cost is high due to continuous requirement of high purity argon gas.
Operation is complex and skilled analyst is required for handling instrument and software.
In ICP-MS spectral and polyatomic interference occur and argon ions can overlap with calcium isotope (40Ca) and error can occur if correction is not done.

Milk has high total dissolved solid and organic component and matrix effect occur and signal suppression and mass bias can occur so internal standard correction or gas dilution system is required.
Dry ashing is time consuming (8–18 hours) and loss of volatile element can occur and insoluble metal oxides can be formed and it is difficult to redissolve.
Open vessel wet digestion needs constant monitoring and large volume corrosive acids are used and safety hazard is present (perchloric acid can form explosive by product).

Microwave digestion is faster but equipment is expensive and maximum sample mass is limited for safe digestion.

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Measuring of Calcium In Milk – Principle, Procedure, Result