PHOSPHATE CHEMISTRY RELEVANT TO FOODS

I. PHOSPHATE CHEMISTRY RELEVANT TO FOODS

The goal of this section is to offer an overview of the chemistry that is relevant to the use of phosphates in food systems. Those seeking to develop new uses for phosphates should find some useful information in the references. This discussion does not examine phosphorus chemistry in general since it has been covered in excellent works by Van Wazer (1958) and Corbridge (1986). Reviews covering food phosphate chemistry can also be found in Molins (1991), Ellinger (1972a,b) and DeMan and Mehnychyn (1971). Nomenclature is covered since it is often archaic with many names referring to the same chemical. Phosphates are almost exclusively used in aqueous media and for this reason, the rate of dissolution and the hydrolysis of phosphates are discussed. Sequestration of metal ions by condensed phosphates is described. Methods of analysis are also re-

A. Nomenclature

Phosphates are defined and distinguished from other phosphorus-containing molecules as those in which the central phosphorus atom is surrounded by four oxygen atoms. The oxygen atoms spatially occupy a structure resembling a tetrahedron with the oxygen atoms at the corners. Sharing of oxygen atoms between two phosphorus atoms gives rise to a great number of different phosphate types. For mainly historical reasons, different names have often been given to the same chemicals. Attempts to improve the naming systems have not always met with universal acceptance but are still used. For these reasons, Table

1 is offered indicating the different names and which compounds they chemically refer to. Ideally, phosphate nomenclature provides information on (1) the structure of the phos- phate and (2) the type and number of charge balancing cations.

The structural arrangement of the phosphate tetrahedron forms the basis of its no- menclature. Phosphates can form structures containing from one to hundreds or even thou- sands of phosphate tetrahedra. Structurally, the simplest is orthophosphate containing one tetrahedron. Joining two orthophosphates together produces a diphosphate. Diphosphates of this type are also commonly known as pyrophosphates. Continuing in the same vein leads to, sequentially, triphosphate, tetraphosphate, etc. Triphosphates, or tripolyphos- phates as they are also known, are the longest ‘‘pure’’ phosphate oligomer commercially available. Higher polymers containing the phosphate tetrahedron are commercially sold as mixtures. For example, sodium hexametaphosphate is actually a mixture of polyphos- phates containing from about 13 up to 35 phosphate tetrahedra linked in a linear fashion. If a polyphosphate is joined back on itself to form a ring, it is referred to as a true metaphos- phate (in contrast to hexametaphosphate which retains this name today for historical rea- sons). Trimetaphosphates are available commercially. One other phosphate structural type is also known, the ultraphosphates. These are phosphate anions which contain a structural unit in which a phosphate tetrahedron is linked to three other phosphate tetrahedra. It is

a branched phosphate structure. There are then structurally only four phosphate types: (1) orthophosphates which are not attached to any other phosphate tetrahedron; (2) polyphosphates (including pyro- phosphates) in which a phosphate tetrahedron is linked to at most two other phosphate tetrahedra; (3) true metaphosphates in which the polyphosphate joins back upon itself to form a ring; and (4) ultraphosphates which contain at least one phosphate tetrahedron joined to three others. Polyphosphates, metaphosphates, and ultraphosphates are all pre- pared from orthophosphates by heating to drive off water. For this reason, they are called, as a group, condensed phosphates.

Composed of (formally) pentavalent phosphorus (P ⫹5 ) and divalent oxygen (O ⫺2 ) the phosphates are negatively charged and require charge-balancing cations. These can

be protons (H ⫹ ), metal ions, or molecular ions like ammonium. Phosphate nomenclature then includes the type of cation and a code describing the type of phosphate structure. For example, potassium tripolyphosphate is a polyphosphate containing three phosphate tetrahedra and the phosphate anion is charge balanced by potassium ions. Other common terms are shown in Table 1.

Calcium phosphates are a bit confusing since the calcium ion is doubly positive. The naming for this orthophosphate is a carryover from the past when phosphates were expressed as an oxide and related to its P 2 O 5 content. Monocalcium phosphate contains one calcium oxide molecule for each P 2 O 5 moiety; it is CaO : P 2 O 5 : 2H 2 O or Ca(H 2 PO 4 ) 2 .

Dicalcium phosphate is 2CaO : P 2 O 5 :H 2 O or CaHPO 4 , containing two molecules of cal-

cium oxide for each of P 2 O 5 .

B. Production Methods

The production of phosphates begins with phosphoric acid (Ullmann’s, 1991; Kirk- Othmer, 1982). The acid itself is made by one of two routes. ‘‘Thermal’’ phosphoric acid

is made by burning phosphorus (P 4 ), which is itself prepared by smelting phosphate ore (shown as Ca x [PO 4 ] y in Reaction [1]) in an electric furnace using coke (C in Reaction [1]) as a reducing agent. Silica (SiO 2 ) is added as a flux to lower the melting point of the mixture.

[1] The phosphorus is then burned in specially designed torches in an excess of air and

Ca x [PO 4 ] y ⫹ C ⫹ SiO 2 → CaSiO 3 ⫹P 4 ⫹ CO

quenched with water, yielding phosphoric acid: P 4 ⫹O 2 → ‘‘P 2 O 5 ’’

[3] Since phosphate rock naturally contains arsenic and the chemistry of arsenic compounds

‘‘P 2 O 5 ’’ ⫹ H 2 O →H 3 PO 4

are similar in many respects to phosphorus the arsenic is carried over into the acid. To bring it up to food grade quality, the arsenic can be removed using various methods.

‘‘Purified wet’’ phosphoric acid starts from fertilizer grade phosphoric acid made by reacting phosphate rock with sulphuric acid. This is purified using solvent extraction methods in which essentially pure phosphoric acid is extracted into an organic solvent and then in a subsequent step released into an aqueous phase. The impurities remain in the fertilizer grade acid. Both the thermal process and purified wet process result in the production of food grade phosphoric acid.

To produce phosphates from phosphoric acid, reaction with an alkali is required. The alkali can be any of a large number of different reagents. The following list gives an idea of the variety of alkali sources that can be used: NaOH, Na 2 CO 3 , NaCl, KOH, K 2 CO 3 , KCl, CaO, Ca(OH) 2 , CaCO 3 , and NH 3 . Depending on the source of the alkali raw material, impurities common to its origin can be carried through into the phosphate. Food grade alkali sources are used to make food grade products.

The product from the neutralization of phosphoric acid with alkali is then usually dried to give an orthophosphate. While the orthophosphates are themselves available com- mercially, they are also the raw materials for subsequent processing to give condensed phosphates. The mechanisms by which these reactions occur can be complex (e.g., in the case of STPP), but all involve the removal of the constituents of water from the orthophos- phates; for example, the synthesis of tetrapotassium pyro-(or di-) phosphate from dipotas- sium phosphate is

[4] The fact that the synthesis of condensed phosphates requires significant amounts of

2K 2 HPO 4 (s) ⫹ Heat ↔ H 2 O(g) ⫹ K 4 P 2 O 7 (s)

heat to effect the transformation immediately alerts one to the fact that they are thermody- namically unstable to decomposition by water. This will be discussed in the section on hydrolysis.

Table 1 Nomenclature, Acronyms, and Chemical Formulae of Phosphates Used in Food Applications Common names

Solubility Phosphoric acid

High Orthophosphoric acid Monoammonium phosphate

PA

H 3 PO 4 Very low

NH 4 H 2 PO 4 4.5 38 (20 °) Ammonium phosphate monobasic

MAP

120 (80 °) Monoammonium dihydrogen phosphate Primary ammonium phosphate Diammonium phosphate

(NH 4 ) 2 HPO 4 8.0 69 (20 °) Ammonium phosphate dibasic

DAP

97 (60 °) Diammonium hydrogen phosphate Secondary ammonium phosphate Monocalcium phosphate monohydrate

3.4 Disproportionates Calcium phosphate monobasic Acid calcium phosphate Monocalcium phosphate anhydrous

MCP-1

Ca(H 2 PO 4 ) 2 ⋅H 2 O

Ca(H 2 PO 4 ) 2 3.4 Disproportionates Coated monocalcium phosphate

MCP-O

cAMCP

Dicalcium phosphate anhydrous

CaHPO 4 7.5 Insoluble Calcium phosphate dibasic

DCP-O

DCPA

Calcium hydrogen orthophosphate Dicalcium phosphate duohydrate

7.5 Insoluble Tricalcium phosphate

DCP-2

Ca(HPO 4 ) ⋅ 2H 2 O

Ca 5 (PO 4 ) 3 (OH) a 7.2 Insoluble Calcium phosphate tribasic Precipitated calcium phosphate Synthetic hydroxy apatite Ferric orthophosphate

TCP

Insoluble Monopotassium phosphate

FePO 4 a 3.8–4.4

KH 2 PO 4 4.4 20 (20 °) Potassium phosphate monobasic Monopotassium dihydrogen phosphate Potassium acid phosphate Potassium biphosphate Dipotassium phosphate

MKP

K 2 HPO 4 9.5 120 (10 °) Potassium phosphate dibasic

DKP

260 (50 °) Dipotassium hydrogen phosphate Dipotassium acid phosphate Secondary potassium phosphate Tripotassium phosphate

TKP

K 3 PO 4 12 51 (20 °)

Potassium phosphate tribasic Hemisodium phosphate

NaH 2 PO 4 ⋅H 3 PO 4 2.2 High Monosodium phosphate

HSP

NaH 2 PO 4 4.4 85 (20 °) Sodium phosphate monobasic

MSP

212 (80 °) Monosodium dihydrogen phosphate Disodium phosphate

Na 2 HPO 4 8.8 7.7 (20 °) Sodium phosphate dibasic

DSP, DSP-2,

93 (80 °) Disodium hydrogen phosphate Trisodium phosphate dodecahydrate

DSP-7

12 13 (20 °) Sodium phosphate tribasic Trisodium phosphate anhydrous

TSP-12

Na 3 PO 4 ⋅ 1/4NaOH ⋅ 12H 2 O

TSP-0

Sodium aluminum phosphate, acidic

SALP

NaAl 3 H 14 (PO4) 8 ⋅ 4H 2 O

Calcium acid pyrophosphate

CaH 2 P 2 O 7 3 Slight Calcium pyrophosphate

CAPP

Ca 2 P 2 O 7 6.0 Insoluble Calcium diphosphate Dimagnesium phosphate

6.5 Slight Tetrapotassium pyrophosphate

DMP

MgHPO 4 ⋅ 3H 2 O

K 4 P 2 O 7 10.4 180 (20 °) Potassium diphosphate

TKPP

260 (50 °) Sodium acid pyrophosphate

Na 2 H 2 P 2 O 7 4.2 12 (20 °) Acid sodium pyrophosphate

SAPP

22 (60 °) Disodium dihydrogen diphosphate Disodium pryophosphate Tetrasodium pyrophosphate

ASPP

Na 4 P 2 O 7 10.2 6 (20 °) Sodium diphosphate

TSPP

50 (80 °) Potassium tripolyphosphate

K 5 P 3 O 10 9.6 178 (20 °) Pentapotassium triphosphate

KTPP

216 (80 °) Pentapotassium tripolyphosphate Sodium tripolyphosphate

Na 5 P 3 O 10 9.8 15 (20 °) Pentasodium triphosphate

STPP

17 (80 °) Pentasodium tripolyphosphate Potassium metaphosphates (Kurrol’s salt)

Insoluble Sodium hexametaphosphate (Graham’s salt)

(KPO 3 ) n

7.0 High Sodium trimetaphosphate

SHMP

(NaPO 3 ) n

Na 3 (PO 3 ) 3 6.7 30 (35 °) Note : The pH values are for a 1% solution or, for insoluble materials, a 10% slurry. The solubility data are in grams of phosphate per 100 g of water. MCP reacts with water

STMP

leading to phosphoric acid and DCP. a Approximate chemical composition.

C. Solubility in Water

The generally accepted values for the solubility of the different phosphates are collected in Table 1 . Some condensed phosphates are difficult to obtain in solution at the maximum solubility level due to both slow solubility and hydrolysis, which can give the appearance of an insoluble phosphate due to precipitation of orthophosphates. Since phosphates are seldom used by themselves in pure water, the values shown in Table 1 are acceptable for most applications.

The rate at which a phosphate dissolves in water is important for practical, commer- cial reasons; food applications generally occur in aqueous solutions, while phosphates are almost always sold as solids. Two examples will serve to illustrate this concept. First, control over their rate of dissolution is a useful feature of the use of phosphate-based acids for chemically leavened doughs. This is accomplished either by barrier methods operating on quickly dissolving leavening acids (MCP, SAPP) or by producing poorly soluble phos- phate salts (SALP, DMP). Second, the method of preparation of a phosphate solution is of great importance in preparing brines since the phosphate is usually the most difficult compound to dissolve. It is usually recommended that the phosphate be the first thing dissolved in water before any of the other ingredients. Due to solubilization difficulties,

a number of phosphate manufacturers have focused on producing rapidly dissolving phos- phates and phosphate blends for food uses. This is accomplished by either increasing the surface area of the phosphate that is available to dissolve in water or by increasing the thermodynamic driving force to dissolve, or both. Both these aspects are discussed in more detail in the next section.

1. STPP Dissolution Improving the dissolution rate of STPP has been an important advance in the use of STPP.

This is the most poorly soluble sodium phosphate and among the most important in food uses. STPP can be the slowest useful food phosphate to dissolve, but by increasing the thermodynamic driving force to dissolution, this situation can be improved. Two anhy- drous crystalline phases of STPP exist with differing heats of solution (the amount of energy released when dissolved), a high temperature crystalline phase, denoted I, and a lower temperature phase, II (see Fig. 1 ). In commercially available STPP for food use, there is generally a mixture of the two different crystalline phases. Phase I releases a greater amount of heat during its reaction and dissolution in water than does phase II. It also dissolves more rapidly, which is thought to be a direct consequence of the environ- ment of some of the sodium ions in the phase I crystal. By adjusting the production condi- tions, the proportion of the different phases can be controlled (see Fig. 2 ).

The proportion of phase I crystals in STPP can be determined by at least a few methods. The most accurate, but most resource intensive, is via X-ray diffractive methods. Rapid estimations of the phase I content can be achieved by either an infrared spectro- scopic method (AWA, 1997) or by measurement of the amount of heat released by the phase I fraction (McGilvery, 1953).

2. Dissolution of Other Phosphates While they have very high maximum solubility, the rate of dissolution of long chain

polyphosphates is generally slow. High shear mixers can rapidly dissolve SHMP solutions with minimal heating of the solution, which leads to hydrolysis. The rate of dissolution

Figure 1

A comparison of the rates of dissolution of typical food grade STPP to that of a rapidly dissolving STPP at 5 °C. The difference in the initial rate of dissolution can make a large difference in commercial applications.

of SAPP and MCP-1 are important in their application to chemically leavened doughs and the amount of ‘‘bench action’’ carbon dioxide that is generated. Doping of the ortho- phosphate in the preparation of SAPP can change the rate of dissolution either by introduc- ing poorly soluble impurities or more rapidly dissolving ones. This is accomplished by doping with K ⫹ , Ca ⫹2 , or Al ⫹3 . Coated MCP-0 has a polyphosphate coating which retards the rate of reaction of the MCP-0 with bicarbonate. The history behind the development of this coating makes interesting reading (Toy, 1987).

Figure 2

A comparison of the rate of dissolution of a physical blend (simple mixing of powders) of STPP and SHMP with that of an instantized blend with the same composition.

3. Measurement of the Rate of Dissolution The rate at which phosphates dissolve can be readily measured indirectly using a conduc-

tivity probe (AWA, 1994). This is a useful tool that can be used to predict the solubility characteristics of various phosphates alone or in combination under a wide variety of conditions like water hardness, temperature, pH, previously dissolved additives, etc.

D. Phosphate Hydrolysis

The hydrolysis of complex phosphates is of interest in two areas: in solution and in use. The major difference between the two being that in food there are more avenues for hydrolysis and thus the rates are faster. The formation of orthophosphates from polyphos- phates is of concern to meat workers since sodium orthophosphates effloresce on the sur- face of meat in a phenomenon known as ‘‘snow formation,’’ or ‘‘whiskering.’’ The reduc- tion in favorable properties such as microbial inhibition is also of importance. To gain an understanding, it is useful to examine the thermodynamic and kinetic aspects of hydrolysis (Osterheld, 1972).

1. Thermodynamics of Hydrolysis As noted, condensed phosphates are thermodynamically unstable with respect to hydroly-

sis over all pH and temperature ranges. An examination of the free energy changes (Table

2) shows that hydrolysis is spontaneous for all condensed phosphates. Since the mecha- nism of hydrolysis is molecularly similar for all polyphosphate chain lengths, it is not necessary to examine all the different polyphosphates separately, and only the energetics of pyrophosphate hydrolysis are shown in the table.

To penetrate further into the thermodynamics of polyphosphate hydrolysis, investi- gators have resorted to complex calculations of the energy changes and modeling of the reaction (Saint-Martin et al., 1991; Romero and DeMeis, 1989; Dupont and Pugeois, 1983). The interest in polyphosphate hydrolysis is driven by a desire to more fundamen- tally understand the driving force behind many biochemical reactions. This began in about 1940 with the discovery of ATP and its involvement in energy storage and release in living tissues. According to the current understanding (Saint-Martin et al., 1994) about half of the energy that is released in the hydrolysis comes from a rearrangement of chemi- cal bonds, and the other half comes from changes in the hydration of the products and reactants. The interactions of the products and reactants with water are quite different. The contribution of hydration energy to the enthalpy of the pyrophosphate hydrolysis accounts for about half of the energy released during hydrolysis.

Table 2 The Equilibrium Constant (K), Enthalpy ( ∆H°), and Entropy ( ∆S°) for the Hydrolysis of Pyrophosphate

Reaction

∆H° (kcal/mole)

∆S° (eu)

⫺12 P 2 O 7 ⫺4 ⫹H 2 O → 2HPO 4 ⫺2

H 2 P 2 O 7 ⫺2 ⫹H 2 O → 2 H2PO 4 ⫺

2. Kinetics It is interesting to note that the mechanisms of energy transfer in living organisms are

possible because at in vivo temperatures and pH values, the hydrolysis of condensed phos- phates is slow. The rate is dependent on a number of factors, the main ones are described herein.

Hydrolysis of condensed phosphates can occur in two ways. The majority of the hydrolysis occurs via end group ‘‘clipping’’ in which a terminal phosphate tetrahedron is cleaved from the chain. Much less probable is ‘‘random cleavage’’ of the middle of the chain, but it has been established that this does occur with long chain phosphates (McCullough et al., 1956).

End group clipping:

Random cleavage:

The rate of this type of hydrolysis is negligibly slow for polyphosphates up to about hexaphosphate. Whether the loss of orthophosphate from the end of a polyphosphate oc- curs via a concerted mechanism in which water is split apart at the same time the P–O– P bonds are broken or by a stepwise mechanism has been debated. Most of the work has been done on organic substituted phosphates. The precise details of the mechanism has been extensively studied (Jencks, 1992). It is believed that hydrolysis occurs because of an increase in the positive character of the central P atom in the (soon to be) orthophos- phate leaving group. Either via a concerted or stepwise loss mechanism, the chain is short- ened by one phosphate tetrahedron.

The rates of hydrolysis are strongly affected by temperature and pH. They are slower The rates of hydrolysis are strongly affected by temperature and pH. They are slower

The variation in hydrolysis rates with pH is not actually a linear relationship. Pla- teaus occur at different pH values indicating that certain protonated or unprotonated spe- cies are more resistant to the type of polarization that is necessary to occur before the orthophosphate leaving group is cleaved from the chain. A useful nomograph relating the half-life of a phosphate under different conditions of pH and temperature has been pub- lished (Griffith, 1959). This is useful for estimating the amount of hydrolysis under differ- ent pH and temperature conditions.

Metal ions in solution also have an effect on the hydrolysis rates. The effect is not completely understood, but studies have found that hydrolysis is often accelerated by the presence of some metal ions like calcium, and inhibited slightly by the presence of magne- sium. The influence the metal ion has on a complex phosphate is thought to involve polar- ization of the P–O bonds and thus affects the electropositive character of the central phos- phorus atom (Osterheld, 1972).

3. Trimetaphosphate Formation While not truly a hydrolysis, the formation of trimetaphosphates in solution from long

chain phosphates is a chemically intriguing reaction. It is mentioned here since the seques- tration ability of trimetaphosphate is essentially nonexistent. As well, the polyanionic char- acter of trimetaphosphates is distinctly different from that of polyphosphates, and therefore

a solution which starts out as polyphosphate can rapidly change its sequestering power even though condensed phosphates remain in solution. The reaction is simply

[7] Metaphosphate abstraction requires the presence of sodium ions. This was demon-

[NaPO 3 ] n → Na 3 P 3 O 9 ⫹ [NaPO 3 ] n⫺3

strated in studies using tetramethyl ammonium salts of oligophosphates which showed essentially no metaphosphate abstraction products (Iler, 1952). It is thought that the meta- phosphate abstraction reactions occurs via a coiling of the phosphate chain around a so- dium ion, followed by an intramolecular scission to yield the trimetaphosphate ion and a polyphosphate chain shorter by three units (Thilo, 1962).

Table 3 The Relative Effects of Varying Certain Parameters on the Rate of Hydrolysis of Condensed Phosphates

Factor

Approximate effect on rate

Temperature

10 5 –10 6 faster from freezing to boiling

pH

10 3 –10 4 slower from strong acid to base

Enzymes

At most 10 5 –10 6 faster

Cations Manyfold increase in some cases Concentration

Roughly proportional Ionic environment

Severalfold change Colloidal gels

As much as 10 4 –10 5 faster

4. Hydrolysis of Complex Phosphates in Meats The hydrolysis chemistry in meats is similar to that occurring in solution, but the rates

are faster (Sutton, 1973). This is due to the presence of phosphatase enzymes, which continue to be active for a period of time after death. The phosphatase activity decreases with time. After rigor mortis, little change in the phosphatase activity is observed. For example, the hydrolysis of STPP in cod muscle proceeds even when stored at 0 °C (Sutton, 1973). Large deviations in the rates of hydrolysis have been observed depending on the type of animal and on the animal themselves (Neraal and Hamm, 1977a,b). Interestingly, although the condensed phosphates are degraded in the tissue, their effects on the food often continue. This is best demonstrated by the fact that the increase in the water holding capacity of meat due to the presence of pyrophosphates is maintained after hydrolysis to orthophosphate (Hamm and Neraal, 1977c). This is suggested to be due to irreversible changes induced in the structures of the muscle (Sutton, 1973).

E. Sequestration

Sequestration of metal ions by condensed phosphates is an important function of complex phosphates in food applications. In its most general definition sequestration is the elimina- tion of chemical effects of metal ions initially dissolved in aqueous media. Sequestration generally refers to the formation of a stable, water-soluble complex with a metal ion that prevents the ion from participating in reactions. Orthophosphates can be said to reduce the effect of hardness ions (e.g., Ca ⫹2 , Mg ⫹2 , Fe ⫹2/⫹3 , etc.) by precipitation, but this is not strictly sequestration. Sequestration requires a complex phosphate since it needs at least two separate sites of attachment on the same molecule.

The ability of a particular polyphosphate to sequester a particular metal ion is a function of pH, temperature, and the other species that are present. The sensitivity of the sequestration capacity to pH value of a polyphosphate decreases with increasing chain length. Sequestration is best understood with respect to water softening and reducing scale. Reference to the original papers is recommended for those interested in the subject (Van Wazer and Campanella, 1950; Van Wazer and Callis, 1958; Thilo, 1955; Irani and Callis, 1962; Irani and Morgenthaler, 1963). The sequestration of cations in foods is strongly associated with the inhibition of certain reactions involving metal ions like catalysis of lipid oxidation or enzymatic browning. Because of the flexible polyphosphate backbone and the presence of many anionic ‘‘sites,’’ polyphosphates can accommodate the ‘‘needs’’ of many different cations in forming complexes with them (Table 4).

Table 4 Sequestering Power of Selected Sodium Phosphates

STPP TSPP

Note : The table illustrates the number of grams of the metal ion that can be seques- tered by 100 g of the indicated phosphate. Source : Albright & Wilson Americas, Inc.,

F. Buffering of Solutions

Mixtures of orthophosphates are excellent buffers. Condensed phosphates are less useful at buffering. Diphosphates (pyrophosphates) are of some utility, but chains longer than 2 are not good buffers at all. The ranges of pH from 2 to 3.5, 5.5 to 7.5, and 10 to 12 can all be buffered by the ortho- and pyrophosphate anions. Long phosphates can perform some buffering in the range of 5.5 to 7.5, but it is not very cost effective compared to orthophosphates. The problem with long chain phosphates is that except for the end groups there are no weak acid titratable protons.

G. Analytical Methods

Analytical methods for phosphates can be broadly separated into two groups, those for total phosphate and those sensitive to the different phosphate species present (that is ortho-, pyro-, triphosphate, etc.). The difficulty with the analysis of phosphates in foods is the presence of compounds other than phosphate. Chemical methods of analyzing phosphate species usually suffer from interferences. It is necessary to somehow remove these, leaving behind the phosphate. There are many methods found in the literature. If the total amount of phosphate is all that is of concern, then exhaustive alkali or acid digestion techniques, either with an oxidizer or by charring, will eliminate the interferences caused by the food. Physical methods (like NMR spectroscopy) will in many cases succeed since it is sensitive only to the presence of a certain nuclei with specific chemical shift ranges specific to certain phosphates, and J–J coupling indicating the types and relative amounts of the phosphate species.

1. Analytical Methods for Total Phosphate The best methods for total phosphate are colorimetric. Both vanado-molybdate and ammo-

nium molybdate (Vogel, 1989) methods are excellent for determining the amount of penta- valent phosphate present as orthophosphate. The vanado-molybdate method is best suited for when phosphate is the major component in solution. The molybdenum blue method is more suited for solutions when phosphate is a minor component. All phosphate species present in a sample can be confidently converted to orthophosphates by refluxing with nitric or hydrochloric acids. The ashing of all organic components of the food is required to prevent the development of turbidity in the colored solutions created by addition of the molybdo reagents. This can be time consuming. Less sensitive to the presence of impurities are inductively coupled plasma–atomic emissions spectroscopy (ICP-AES) methods. While the equipment is more expensive than other analytical laboratory equipment, it does allow for the determination of many elemental components from one sample. Gravimetric methods of P determination may suffer from interferences from insoluble monophosphate sources.

2. Analytical Methods for Different Phosphate Species Ion chromatography is the method of choice for speciation of phosphates. The method is

very cost effective, accurate, and straightforward. This method relies on the fewest as- sumptions of any analytical method aimed at quantifying the different phosphate species present. Like all other chemical methods, it requires careful pretreatment of the sample to remove other components in the food. Once this is achieved, quantitative analysis of the phosphate species is readily achieved. Depending on the chromatography column and

The application of 31 P NMR spectroscopy to the quantitative analysis of condensed phosphates has been recently reviewed in which it is compared to other methods of analy- sis. It is a precise and accurate method for the quantification of phosphate species with the potential for automation allowing for the analysis of a large number of samples. Two- dimensional J resolved spectroscopy can be used to semiquantitatively determine the mix-

tures of phosphate chains that exist in phosphate melts (Gard et al., 1992). The use of 31 P NMR in the study of whole cells and in particular polyphosphates has been achieved. Addition of chelating agents, like EDTA, to reduce signal broadening by metal ions attached to the polyphosphates is required. The potential for this technique in terms of the reduced need for extensive sample preparation is great (Roberts, 1987).