- Email: [email protected]
ION EXCHANGE | Chelation Ion Chromatography
Response (AU)
ION EXCHANGE / Chelation Ion Chromatography 0.2 0.18 0.16 0.14 0.12 0.1 0.08 0.06 0.04 0.02 0
467
See also: Ion Exchange: Overview; Principles; Ion Chromatography Instrumentation; Chelation Ion Chromatography; Isolation of Biopolymers; Isotope Separation. Thiocyanate
Further Reading
0
1
2
3
4
5
6
7
8
9
10
Time (min) Figure 4 Analysis of urine for thiocyanate using ion interaction liquid chromatography. Lower trace: sample (heavy smoker) diluted 1:20. Upper trace (heavy smoker) diluted 1:20 and spiked with thiocyanate.
alkaline earth cations in saliva. The sample matrix is relatively simple compared to other biological fluids and can be analyzed directly or simply diluted prior to injection. IC methods have also been developed for the determination of sweat samples for ionic analytes. Methods looking at sulfate levels and also concentrations of sodium and potassium in sweat samples have been developed, with relative levels of the latter metal ions being useful indicators of several important diseases, one of which is cystic fibrosis.
Betti M (1997) Use of ion chromatography for the determination of fission products and actinides in nuclear applications. Journal of Chromatography A 789: 369–379. Buldini PL, Cavalli S, and Trifiro A (1997) State-ofthe-art ion chromatographic determination of inorganic ions in food. Journal of Chromatography A 789: 529–548. Haddad PR and Jackson PE (1990) Ion Chromatography. Principles and Applications. Amsterdam: Elsevier. Lopez-Ruiz B (2000) Advances in the determination of inorganic anions by ion chromatography. Journal of Chromatography A 881: 607–627. Singh RP, Smesko SA, and Abbas NM (1997) Ion chromatographic characterisation of toxic solutions: analysis and ion chemistry of biological liquids. Journal of Chromatography A 774: 21–35. Small H (1990) Ion Chromatography. New York: Plenum. Smith RE (1987) Ion Chromatography Applications. Boca Raton, FL: CRC Press. Vanatta LE (2001) Application of ion chromatography in the semiconductor industry. Trends in Analytical Chemistry 20: 336–345.
Chelation Ion Chromatography P Jones, University of Plymouth, Devon, UK P N Nesterenko, Lomonosov Moscow State University, Moscow, Russia & 2005, Elsevier Ltd. All Rights Reserved.
Introduction It is now over 60 years since the development of column ion-exchange chromatography using polymeric resins. Interestingly, little has changed since then in terms of the basic separation processes for inorganic anions and cations. The renaissance in the 1970s, where ion-exchange was the principal process in a group of techniques now known as ion chromatography (IC), was mainly associated with the improvement in efficiency of stationary phases and detection systems rather than new types of ionexchange groups or elution systems. In essence, the IC separation of metal ions principally involves the use of eluents containing complexing organic acids,
such as tartaric, citric, oxalic, combined with high efficiency stationary phases. The complexing strength of acid and concentration chosen depended on whether the ion-exchange substrate was cationic, anionic, or mixed. Although poly(styrene–divinylbenzene) (PS–DVB) based resins are still the most common substrate, silica-based materials are increasingly being used. There are a number of problems associated with present IC methods involving high efficiency ion-exchange separations of metal ions, the two principal ones being sensitivity to ionic strength and limited selectivity. The influence of ionic strength is particularly serious as a relatively large salt concentration in the sample can drastically affect the chromatography, in many cases making it impossible to resolve the analytes. Limited selectivity is also a restriction, as once the substrate is chosen for conventional ion-exchange separations there are only a small number of cases where changes in eluent composition can significantly alter separation order.
468 ION EXCHANGE / Chelation Ion Chromatography
There is an alternative to ion-exchange, known generally as chelation ion-exchange, giving greater control over selectivity and relatively unaffected by changes in ionic strength. Although related, chelation ion-exchange differs fundamentally from ionexchange in that it involves the formation of coordinate bonds between metal ions and a complexing ligand immobilized on the surface of the substrate. Thus, the sorption process in chelation ion-exchange depends mainly upon the values of the stability constants of the metal complexes formed on the stationary phase. Inevitably, because ionexchange and chelation ion-exchange arise from the same functional group, they can occur together. Nevertheless, conditions can be chosen where chelation ion-exchange is the dominant mechanism controlling separation, as explained in the next section. It can be seen that the greater versatility of chelation ion-exchange arises from the wide range of complexing ligands available, where selectivity can be altered to suit the trace metal composition of a particular interest and sample type. Chelation ionexchange is not a recently exploited technique, being used for nearly as long as ion-exchange itself. However, until recently, chelation ion-exchange columns have principally been used for matrix isolation and preconcentration of suites of metals rather than chromatographic separation. The isolation of trace metals from seawater using Chelex 100 (iminodiacetic acid functionalized PS-DVB) is a classic example. Later, chelating ion-exchangers such as 8-hydroxyquinolinol attached to silica (Table 1) have been extensively used in flow injection analysis for determination of metals. In the last 10 years or so, chelation ion-exchange columns have become available for use as precolumns in IC systems where matrix isolation and preconcentration occur online before the suite of metals is swept onto an ion-exchange column for separation (see later). This approach became known as chelation ion chromatography (CIC). Another use of chelation ion-exchange columns, the main focus of this article, is for the high-efficiency separation of metal ions on small particle size substrates. It is a relatively new approach and shows great potential for the IC determination of metals in a wide variety of complex samples, including those with very high ionic strengths. Although the term CIC can be used to describe both uses of chelation ion-exchange substrates, it should be noted that some workers have preferred to use the term high-performance chelation ion chromatography (HPCIC) to differentiate the high-efficiency separation technique from CIC methods where chelation ion-exchange is only
used for preconcentration or matrix isolation purposes. The principal purpose of this article is to describe the latest work involving high-efficiency chelation ion-exchange columns for determining trace metals in a wide variety of complex matrices. The basic principles relating to the sorption of metal ions will be described first, followed by illustrations of the different separation modes available, and, finally, key examples will be given of the wide range of samples analyzed by the technique.
Chelation Ion-Exchange Equilibria The efficient separation of metal cations can be achieved with three main methods: 1. using conventional cation-exchange based on electrostatic interactions; 2. by preconcentration of metal cations on a short column packed with chelating substrate with further separation by cation-exchange ion chromatography; 3. using the ability of chelating ion-exchangers to achieve analytical separations of metal ions with a single column. The last two methods can both be described as CIC as discussed earlier. However, it is only with approach (3), the principal concern of this article, is it important to fully understand the factors affecting efficiency and selectivity to obtain good chromatography. Therefore, the basic principles described below will focus on these aspects. Distribution Ratio
Pure sorption mechanisms are very rare in chromatography and chelation ion-exchange is no exception. As the chelating groups are usually charged, the chromatographic retention of alkaline-earth, transition, and heavy metal ions on such a column, in a noncomplexing eluent, occurs through the combination of electrostatic and coordination interactions between cations and chelating functional groups on the substrate. The distribution ratio DM of metal cation My þ between chelating ion-exchanger and the contacting mobile phase can be expressed by the following equation: yþ C I yþ DM ¼ ð½Myþ R þ ½MR Þ=½Maq
yþ
yþ
½1
where ½MR I and ½MR C are equilibrium concentrations of cation retained by the stationary phase due to electrostatic interactions (conventional ion-exchange) and chelation ion-exchange, respectively.
ION EXCHANGE / Chelation Ion Chromatography
469
Table 1 Selectivity of chelating ion-exchangers for HPCIC Functional groups
Selectivity
(A) Chemically bonded 8-Hydroxyquinolinol
O
Ni2 þ 4Co2 þ 4Zn2 þ 4Pb2 þ 4Cd2 þ 4Mn2 þ Yb3 þ 4Gd3 þ 4La3 þ
(CH2)3NHC(O)
Si
N
N
OH N
(a) Mg2 þ 4Ca2 þ 4Sr2 þ 4Ba2 þ (b) Fe3 þ 4Cu2 þ 4In3 þ 4UO22 þ 4Pb2 þ 4Ni2 þ 4Zn2 þ 4 Cd2 þ 4Co2 þ 4Be2 þ 4Fe2 þ 4Mn2 þ (c) Lu3 þ 4Yb3 þ 4Tm3 þ 4Er3 þ 4Ho3 þ 4Tb3 þ 4Eu3 þ 4 Gd3 þ 4Dy3 þ 4Sm3 þ 4Y3 þ 4Nd3 þ 4Pr3 þ 4Ce3 þ 4La3 þ
Iminodiacetic acid
CH2COOH O
Si
(CH2)3OCH2 CH(OH)CH2 N CH2 COOH
(a) Mg2 þ 4Ca2 þ 4Sr2 þ 4Ba2 þ
Aminophosphonic acid
(b) Mn2 þ 4Cd2 þ 4Pb2 þ 4Cu2 þ 4Zn2 þ 4Co2 þ 4Ni2 þ
O O
Si
(c) Lu3 þ 4La3 þ 4Be2 þ 4Al3 þ
(CH2)3NHCH2 −P−OH O− UO22 þ 4Cu2 þ 4Cd2 þ 4Co2 þ
Amidoxime
NOH O
Si
(CH2)3C NH2 Fe3 þ 4Cu2 þ 4Zn2 þ
p-Aminosulfosalycilic acid
OH O
Si
(CH2)3OCH2 CH(OH)CH2NH
COOH
K þ 4Rb þ 4Cs þ 4Na þ 4Li þ (as Cl salts)
Bis(benzo-15-crown-5)
Ba2 þ 4Sr2 þ 4Ca2 þ 4Mg2 þ (as Cl salts) I 4Br 4Cl (as K þ salts) O
Si
(CH2)2NH
(CH2)3N C
C
O
O
O
O O
O
O
O
O
O
O
O
Cu2 þ 4Co2 þ 4Zn2 þ 4Cd2 þ Fe2 þ 4Mg2 þ
Poly(amines)
O
Si
(CH2)3OCH2CH(OH)CH2N(CH2CH2NH)nH n = 0−4 Continued
470 ION EXCHANGE / Chelation Ion Chromatography Table 1 Continued Functional groups
Selectivity
Poly(butadienemaleic acid) PBDMA
Cu2 þ 4In3 þ 4UO22 þ 4Pb2 þ 4Ni2 þ 4Co2 þ 4Zn2 þ 4 Cd2 þ 4Mn2 þ 4Mg2 þ
COOH [
]
n
COOH Cu2 þ 4Pb2 þ 4Zn2 þ 4Ni2 þ 4Mn2 þ 4Cd2 þ 4Co2 þ 4 Mg2 þ 4Ca2 þ 4Ba2 þ
Aspartic acid
COOH O
(CH2)3OCH2CH(OH)CH2NHCH
Si
CH2CH2COOH (B) Precoated or impregnated substrates (a) Mg2 þ 4Ca2 þ 4Sr2 þ 4Ba2 þ
Xylenol Orange
(b) Cu2 þ 4Ni2 þ 4Zn2 þ 4Cd2 þ 4Mn2 þ CH2COOH
O
CH2NCH2COOH
CH3
CH3
C
OH
CH2NCH2COOH SO3H
CH2COOH
(a) In3 þ 4Ga3 þ 4Al3 þ
Aurin tricarboxylic acid
(b) Cu2 þ 4Pb2 þ 4Cd2 þ 4Ni2 þ 4Zn2 þ 4Co2 þ 4Mn2 þ O COOH
C
HOOC
COOH
HO
OH
Ca2 þ 4Mg2 þ 4Sr2 þ 4Ba2 þ
o-Cresolphthalein complexone O
CH2COOH CH2NCH2COOH
CH3
CH3
C
OH
CH2NCH2COOH COOH
CH2COOH
ION EXCHANGE / Chelation Ion Chromatography
471
Table 1 Continued Functional groups
Selectivity
Methyl thymol blue
Ca2 þ 4Mg2 þ 4Sr2 þ 4Ba2 þ CH2COOH
O
CH2NCH2COOH
(CH3)2CH
CH(CH3)2 CH3 C
OH
CH2NCH2COOH
CH3 SO3H
CH2COOH
Fe3 þ 4Al3 þ
Chrome Azurol S O COOH
CH3 Cl
SO3H
C HOOC Cl HO CH3
(C) Dynamically coated Methyl thymol blue (see structure in section 1b)
(a) Cu2 þ 4UO22 þ 4Mg2 þ 4Ca2 þ (b) Lu3 þ 4Tm3 þ 4Tb3 þ 4Sm3 þ 4Nd3 þ 4Ce3 þ 4La3 þ 4 Cu2 þ 4Pb2 þ 4Cd2 þ 4Zn2 þ 4Co2 þ 4Mn2 þ 4Mg2 þ (a) Cd2 þ 4Pb2 þ 4Zn2 þ 4Ni2 þ 4Cu2 þ 4Mn2 þ (b) Zr(IV)4Hf(IV)4U(VI)4Bi(III)4V(V)4Th(IV)4Fe(III)
Dipicolinic acid
N
HOOC
COOH (a) Cd2 þ 4Pb2 þ 4Cu2 þ 4Zn2 þ 4Ni2 þ 4Co2 þ 4Mn2 þ (b) UO22 þ 4Fe3 þ 4Lu3 þ 4La3 þ 4Al3 þ
4-Chlorodipicolinic acid
Cl
N
HOOC
COOH (a) Cu2 þ 4Ni2 þ 4Co2 þ 4Zn2 þ 4Cd2 þ 4Pb2 þ 4Mn2 þ (b) Lu3 þ 4La3 þ
Quinaldic acid
N
COOH
472 ION EXCHANGE / Chelation Ion Chromatography
½Myþ aq is the concentration of cation in the mobile phase. The retention factor k can be expressed as k ¼ ðt t0 Þ=t0 ¼ DM j
½2
where t is retention time, t0 the void time, and j is a characteristic constant for a given chromatographic column expressed as the ratio of the volumes of mobile Vaq and stationary VR phases: j ¼ Vaq =VR
½3
As suitable chromatographic separation can be achieved at values of ko10–15 and the formation of complexes at the surface with more than one ligand is for simplification assumed not to take place due to thermodynamic and steric restrictions; then, x Myþ ¼ MLðyxÞþ aq þ L
½4
ðyxÞþ C
Thus, ½MR
can be expressed as follows: ðyxÞþ C
n ¼ b1 ½Myþ aq ½L
½MR
½5
where [Ln ] is the concentration of functional groups and b1 is the stability constant of complex ML(y–x) þ formed at the surface in accordance with (4). Selectivity Ratio
The ion-exchange process of cation My þ and alkali metal cation B þ as competing cation at constant pH of the eluent on the chelating ion-exchanger can be expressed as follows: yþ yþ þ yBþ R þ Maq ¼ yBaq þ MR
½6
where the subscript ‘aq’ denotes the mobile phase and ‘R’ denotes the stationary phase. The following selectivity ratio is given by KBM ¼
þ y ½Myþ R ½Baq þ y ½Myþ aq ½BR
½7
Taking into account the two possible types of interactions in accordance with [1], the retention factor k of metal cation My þ can be expressed as k¼
KBM
! y ½Bþ n R y þ b1 ½L j ½Bþ aq
½8
The first member of the sum in brackets expresses the impact of conventional ion-exchange interactions on retention, and the second member expresses the impact of chelation on retention. Several conclusions can be formulated from eqn [8]. Assuming little change in b1 with high ionic strength (40.1), the retention of a metal ion due to
chelation should not depend upon the concentration þ ], but solely on the conof alkali metal cations [Baq centration of functional groups in the chelating ion-exchanger. In practice, it means electrostatic interactions need to be suppressed for chelation to be the dominant sorption mechanism. The retention of M due to conventional ion-exþ ]. change can be suppressed by increase of [Baq Thus, in the case of domination by chelation y þ y (b1 ½LcKBM ð½Bþ R =½Baq Þ at high concentration level þ of Baq in the eluent) the separation selectivity a is defined by ratio of corresponding stability constants of metal chelates formed at the surface: M1 a ¼ k2 =k1 ¼ bM2 1 =b1
½9
Strictly, it should be the ratio of conditional stability constants, but for a given ligand with no hydrolysis of the metal ions, the ratio is the same as the thermodynamic stability constants. Temperature Effects
As well as varying the ionic strength of the eluent, the separation selectivity KBM in CIC can be changed by variation of column temperature. Sorption heat relates the retention factor k with the column temperature by the van’t Hoff equation: ln k ¼ DH=RT þ DS=R þ ln j
½10
where DH and DS are sorption enthalpy and entropy, respectively, and j is the phase volume ratio. The temperature effects are exothermic (negative values of DH) in the case of conventional ion-exchange. However, the heat values do not exceed 8–13 kJ mol 1 and therefore have little influence on retention. In contrast, for stationary phases where metal ion complexation is present, the sorption process will normally exhibit much higher values of DH in both exothermic and endothermic conditions. Thus, temperature effects will be significant and some regulation of selectivity is possible.
Factors Affecting the Efficiency of Chelation Ion-Exchange It is very important to understand the factors controlling the kinetics of the sorption process as they will have a major bearing on separation efficiency. There are three factors affecting the kinetics of chelating ion-exchange: 1. transport of metal ion from the mobile phase to the surface of chelating ion-exchanger;
ION EXCHANGE / Chelation Ion Chromatography
2. diffusion of the ions inside of the stationary phase; and 3. kinetics of chelation of metal ion with functional groups. The first two factors are similar to those occurring in simple conventional ion-exchange so they will not be discussed further. The resulting kinetics of chelation depends upon the chemical nature of the functional groups and the structure of the chelating ion-exchanger, with the former being the most crucial for good separations. Chemical Nature of Functional Groups
Charge on the functional groups It was empirically established that better kinetics of CIC arise when negatively charged functional groups are present. Obviously, negatively charged groups at the surface, attracting oppositely charged cations from the mobile phase, will provide better transport of metal ions from the mobile phase than neutral or positively charged chelating surfaces. Dentation of the ligand It would be expected that the higher the denticity of the ligand, the slower the kinetics as more groups need to be dissociated and more coordinate bonds formed for each metal exchange reaction. In practice, polydentate ligands are usually necessary to achieve adequate retention and selectivity. Fortunately, for most ligands with a denticity not greater than 3, the slower kinetic effect in most cases appears to be relatively minor, as long as the eluent conditions are chosen to ensure small conditional constants. Magnitude of the conditional constants Of all the ligand properties affecting chelation ion-exchange kinetics, the value of the metal/ligand conditional constant has the largest influence. In simple terms, the magnitude of a stability or conditional constant is governed by the ratio of the velocity of the forward reaction (association) to the velocity of the back reaction (dissociation). Except for a small number of metal ion/ligating atom combinations (see later), the velocity of the forward reaction is very fast. Thus, the kinetics of chelation ion-exchange will be controlled by the value of the velocity of the back reaction (dissociation), which will increase as the conditional constant decreases. However, the retention factor k is directly proportional to the value of the conditional constant (eqn [8]). Therefore, to achieve optimum efficiency of separation, the conditional constants should be as low as possible, but not so low as to cause serious overlap of peaks close to
473
the solvent front. As most ligands arise from dissociation of an acid group, change in pH is the simplest way of changing the conditional constant and hence k. It should be noted that this property is very important for the chromatographic separation of metal cations, but not so critical when used for preconcentration or matrix isolation of groups of metal ions. Type of ligating atom Nitrogen and oxygen are the most common ligating atoms in the chelating groups used in CIC. Sulfur-containing ones have been investigated, but are inherently unstable due to air oxidation. Nitrogen-ligating atoms can give slower kinetics than oxygen atoms with certain common metals, nickel being most notable. Even N, O, O chelaters like iminodiacetic acid (IDA) give broad nickel peaks unless the conditional constant is very low and the nickel is close to the solvent front. Structural Properties of the Chelating Exchanger
Conformational mobility of the bonded ligand Chelating ion-exchangers having functional groups chemically attached as a monolayer to the surface through flexible long linkers will provide better selectivity and separation efficiency as they are more easily accessible to metal ions. On the other hand, chelating substrates formed by immobilizing ligands in micropores will be more sterically hidden, subsequently decreasing the kinetics of metal ion association. The distribution density of functional groups at the surface A very high concentration (capacity) of functional groups can lead to nonuniform complexation at the surface of the chelating ion-exchanger. Perhaps a more important consideration is that close proximity of ligands allows the theoretical possibility of formation of ML2 and even ML3 complexes with much less favorable kinetics of dissociation, causing poor (broad and tailing) peak shapes in CIC. It should be noted that for some chelating substrates such as silica bonded IDA, due to thermodynamic considerations, ML2 and ML3 complexes are not formed at the typical low pHs used for separations. It is interesting to note that, depending on the distribution and density of the functional groups, weak cation-exchangers with carboxylic and phosphonic acid functional groups could serve as chelating ionexchangers as two or more groups in close proximity may be able to form chelate-related structures with metal ions, though this may have a bearing on peak broadening as discussed above.
474 ION EXCHANGE / Chelation Ion Chromatography
Pore size The structure of the substrate can also affect the stoichiometry of complexes at the surface. In narrow pores the coordination of two attached ligands is much more probable.
Main Types of Chelation Ion-Exchangers Rigidity and mechanical stability of the substrate under conditions of high-performance liquid chromatography are important and microspherical particles of macroporous highly crosslinked PS-DVB or poly(metacrylate) and silica are well suited for CIC. There are three main methods of producing a chelating ion-exchange surface. Chemically Bonded
This is the most common approach to producing a chelating surface as can be seen from the selection shown in Table 1A. Although they are all capable of producing separations, only a small number appear to have sufficiently good kinetics for chromatographic separations, the most popular being IDA and aminophosphonic acid. Impregnation
Relatively large chelating dyestuff molecules can form an essentially permanent coating by becoming ‘trapped’ in the pores of the substrate. PS-DVB resins are the most successful for this mode as p–p interactions between the aromatic rings of the dye and the resin help to hold the chelating molecule in place. Table 1B shows some representative examples of those used for chromatographic separations. Those containing the IDA group appear to give the best efficiency, though not as good as the chemically bonded type, presumably due to the chelating groups being more sterically hindered in the pore structure. Dynamic Coating
Dynamic coating or modification involves creating a chelating surface by having a ligand continuously present in the eluent. Conditions are chosen so that no permanent impregnation occurs, but rather a dynamic equilibrium is established between a sorbed layer of ligand on the surface of the substrate and the concentration of the ligand in the mobile phase. Due to the preferred adsorption of the ligand, its concentration at the surface is much higher than in the eluent, thus producing a chelation ion-exchange mechanism. Altering such parameters as the concentration of ligand in the eluent, mobile phase pH, and the nature of the stationary phase allows the dynamic
equilibrium to change, thus affecting metal selectivity. This is a relatively new approach and some representative examples are given in Table 1C. Although large molecules such as chelating dyestuffs have been successfully used in the dynamic mode for chromatographic separations, most work has concentrated on small molecules with aromatic groups such as dipicolinic acid.
Practical Aspects of Chelation Ion-Chromatography Precolumn Systems with Common Chromatographic Equipment
The original and most widespread application of chelating ion-exchangers is in the selective adsorption of one or more metal ions of interest from diluted solutions followed by sensitive determination. The short columns or cartridges are not designed for the efficient chromatographic separation of metal ions, but rather for collection on the column before being swept off as a concentrated ‘plug’. By this means matrix isolation and/or preconcentration can be carried out before further analysis online or offline. For CIC, one approach, as explained previously, is to use a chelating precolumn as part of an online IC system and can be considered as one constituent of the overall separation/determination process. Conventional ion-exchange columns, connected in series with the chelating column, are used for the analytical separation of metal ions before postcolumn reaction detection. The most common chelating columns are based on IDA or 8-hydroxyquinoline, bonded to controlled pore glass, silica, or PS-DVB resins. Some are available commercially, though many ‘home made’ columns are described in the literature. These systems have been used successfully to determine trace metals in a number of complex samples, though of necessity the set-up can be rather complex. One commercially available system, for example, involves three columns (the first one being the chelating ion-exchange column) several pumps, eluents, and switching valves. High-Performance Chelation Ion Chromatography
A relatively recent approach in CIC involves more efficient, small particle size substrates as the chelating surface. The use of these high-performance stationary phases means that reasonably fast analytical separations of groups of metal ions can be obtained similar to conventional high-performance ion-exchange. Matrix isolation is not normally required as the chelating column is relatively insensitive to concentrated salt solutions. Preconcentration, if necessary, can also
ION EXCHANGE / Chelation Ion Chromatography
be carried out using a step pH gradient. Thus, matrix isolation, preconcentration, and analytical separation can be achieved using a single high-efficiency chelating column, removing the need for a complex multicolumn system as described in the previous section. The sorption process controlling separation can be almost pure chelation or a combination of chelation and simple ion-exchange as discussed in detail in the section on chelation ion-exchange equilibria. A straightforward way of controlling the relative contribution of chelation and simple ion-exchange is
475
through the ionic strength of the eluent. Increasing the concentration of an alkali metal salt such as potassium nitrate in the eluent will suppress simple ionexchange interactions. Concentrations as high as one molar have been used to achieve almost complete suppression of ion-exchange for many metal ions. The chromatograms in Figures 1–6 were selected to show the kind of analytical separations and selectivities available for groups of metal ions for the three types of chelating surface: bonded,
La
658 nm 0.002 AU
0.10 Mn Y Nd Pr
Absorbance
0.08 Cd
0.06
Ce
Zn
Sm Eu Gd Tb
Co
Dy Ho Er
0.04
Tm
Yb
Lu
0.02 0
0 0
4
2
(A) 0.35
6 8 Time (min)
10
12
Mn
Absorbance
0.25 Co
0.20 0.15 Zn
0.10
40
50
60
70
Time (min)
:——————————————————————
0.00 0
2
4
6
8 10 12 14 Time (min)
(B)
16
Co Mn
1050 Absorbance (mAU)
30
Pb
0.05
650 Cd
250
Zn
Pb
−150 0 (C)
20
Figure 2 Isocratic separation of standard mixture of 14 lanthanides and yttrium on a 250 mm 4 mm column, packed with 5 mm silica IDA. Eluent: 1.6 10 2 mol l 1 HNO3 with 0.5 mol l 1 KNO3; flow rate 1.0 ml min 1; column temperature 651C, sample volume 20 ml, sample concentration of each metal was 4 ppm in 0.2% HNO3. Detection, Arsenazo III postcolumn reaction at 658 nm. (Reprinted with permission from Nesterenko PN and Jones P (1998) Isocratic separation of lanthanides and yttrium by high performance chelation ion chromatography. Journal of Chromatography A 804: 223–231; & Elsevier.)
Cu
0.30
10
2
4 6 Time (min)
8
10
18 20
Figure 1 Separations on silica IDA columns with different elution protocols. (A) Isocratic separation of four metal ions on a 100 mm 4 mm column packed with 5 mm IDA silica. Eluent, 10 mmol l 1 nitric acid. Detection, PAR postcolumn reaction at 510 nm. (Unpublished work, Nesterenko PN and Jones P.) (B) Isocratic separation of five metal ions on a 250 mm 4 mm column packed with 5 mm IDA silica. Eluent, 0.5 mol l 1 KCl, 20 mmol l 1 picolinic acid, and 12.5 mmol l 1 nitric acid. Detection, PAR postcolumn reaction at 510 nm. (Unpublished work, Nesterenko PN and Jones P.) (C) Step gradient separation of Mn(II), Cd(II), Co(II), Zn(II), and Pb(II). Eluent conditions: 0.1 mol l 1 NaCl (pH 2.6) switched to 0.1 mol l 1 NaCl (pH 1.6) at time ¼ 3 min prior to standard injection. Column, 250 mm 4 mm, packed with 8 mm silica IDA. Detection, PAR postcolumn reaction at 495 nm. (Reprinted with permission from Bashir W and Paull B (2002) Ionic strength, pH and temperature effects upon selectivity for transition and heavy metal ions when using chelation ion chromatography with an iminodiacetic acid bonded silica get column and simple eluents. Journal of Chromatography 942: 73–82; & Elsevier.)
476 ION EXCHANGE / Chelation Ion Chromatography 0.05
Absorbance
0.04 Ba
0.03
Mn
Zn
Pb Cd
Mn Cu
0.02 0.01
0.04
−0.01
0
2
4
6
8
10
12
14
16
18
20
Time (min) Figure 3 The separation of Ba(II) 1.5 ppm, Ni(II) 1 ppm, Co(II) 2 ppm, Zn(II) 1 ppm, Pb(II) 5 ppm, Cd(II) 2.5 ppm, Mn(II) 1.5 ppm, and Cu(II) 5 ppm on the aminophosphonic acid functionalized silica column, 250 mm 4.6 mm. Eluent 1 mol l 1 KNO3, 5 mmol l 1 HNO3. Detection, PAR/ZnEDTA postcolumn reaction at 495 nm. (Reprinted with permission from Nesterenko PN, Shaw MJ, Hill S, and Jones P (1999) Aminophosphonatefunctionalised silica: A versatile chromatographic stationary phase for high performance chelation ion chromatography. Microchemical Journal 62: 58–69; & Elsevier.)
Cu Zn Cd
Co 0.02
0
0.28
0
Zn(II)
0.18 Pb(II)
0.13
Cu(II)
Mn(II) Cd(II)
0.08
0.03
0
2
4
6 Time (min)
5
10 15 Retention time (min)
20
Figure 5 The separation of Mn(II) 0.5 mg l 1, Co(II) 0.5 mg l 1, Ni(II) 0.5 mg l 1, Zn(II) 2 mg l 1, Cu(II) 1 mg l 1, Pb(II) 10 mg l 1, and Cd(II) 20 mg l 1 on a 300 4.6 mm PRP-1 7 mm PS-DVB dynamically coated column. Eluent: 1 mol l 1 potassium nitrate, 0.25 mmol l 1 chlorodipicolinic acid, and 6.25 mmol l 1 nitric acid (pH 2.2). Detection: PAR at 520 nm. (Reprinted with permission from Shaw MJ, Jones P, and Nesterenko PN (2002) Dynamic chelation ion chromatography of transition and heavy metals using a mobile phase containing 4-chlorodipicolinic acid. Journal of Chromatography A 953: 141–150; & Elsevier.)
0.23
−0.02
Ni
Absorbance
0.00
Absorbance (AU)
Pb
0.06
Ni Co
8
10
12
Figure 4 Gradient elution of a 0.5 mg l 1 Mn(II), 20 mg l 1 Cd(II), 10 mg l 1 Zn(II), 10 mg l 1 Pb(II), and 1 mg l 1 Cu(II) mixture on the 100 mm 4.6 mm Xylenol Orange impregnated Hamilton resin column. Injection volume used was 100 ml with detection at 520 nm with PAR postcolumn reaction. (Reprinted with permission from the Ph.D. dissertation of James Cowan (2002) The development and study of chelating substrates for the separation of metal ions in complex matrices. University of Plymouth, Figure 2.9, p. 84.)
impregnated, and dynamically loaded. For many chelating substrates the selectivity factors for metal ions in simple eluents can vary over a large range. This means that to separate four or more metal ions isocratically in a reasonable time can be a problem. Figure 1 illustrates this situation quite clearly for silica IDA columns, where a simple dilute nitric acid eluent can separate four relatively weakly retained metal ions, but the more strongly held metal ions such as copper or lead require complexing eluents and/or gradients to elute them more quickly. Although the application of gradients is more complex, gradients have the advantage of sharpening later eluting peaks and allowing on-column preconcentration if required. Two exceptions to this general situation have been found so far. The first involves aminophosphonate bonded silica (Figure 3) where eight common metal ions can be separated isocratically in
ION EXCHANGE / Chelation Ion Chromatography 0.006
477
Th(IV) Sr
Ca+Mg
Absorbance
0.005 0.004 U(VI)
Bi(III)
0.003
Zr(IV) Hf(IV) Absorbance
0.002 Fe(III)
0.001 0 0
5
10 Time (min)
15
20
Figure 6 Chromatogram showing the separation of six acid hydrolyzing metal ions on a dynamically coated column (100 4.6 mm ID), packed with 5 mm PLRPS polystyrene divinylbenzene resin. Eluent, 1 mol l 1 potassium nitrate and 0.1 mmol l 1 dipicolinic acid in 0.5 mol l 1 nitric acid. Detection, Arsenazo III postcolumn reaction monitored at 654 nm. Sample injection, 100 ml of a mixture of 20 ppm Fe(III), 0.5 ppm Th(IV), 20 ppm Bi(III), 0.5 ppm U(VI), 25 ppm Hf(IV), and 5 ppm Zr(IV). (The ion chromatographic separation of high valence metal cations using a neutral polystyrene resin dynamically modified with dipicolinic acid. Cowan J, Shaw MJ, Achterberg EP, Nesterenko PN, and Jones P (2000) The analyst communication. Analyst 125: 2157–2159; reproduced by permission of The Royal Society of Chemistry.)
Cu(II)
Zn(II) Cd(II) Co(II)
Ni(II)
Absorbance, 490 nm
0.1 AU
15 ppb Co(II) 60 ppb Ni(II) 15 ppb Cd(II)
20 ppb Co(II) 40 ppb Ni(II) 10 ppb Cd(II)
Sample
Ba 60
0 Time (min)
Figure 8 Chromatogram showing the separation of barium and strontium from magnesium and calcium in an oil-well brine (sample 1, diluted 1:1 v/v with double distilled water) using a Methylthymol Blue impregnated column (pH 9.2). (Determination of alkaline earth metals in offshore oil-well brines using high performance chelation ion chromatography. Paull B, Foulkes M, and Jones P (1994) Analytical Proceedings 31: 209–211; reproduced by permission of The Royal Society of Chemistry.)
16 min using dilute nitric acid eluent, though iron(III) is strongly retained under these conditions. The second exception concerns the separation of the lanthanides and yttrium on IDA bonded silica (Figure 2). The separation of such a large number of metals isocratically is possible because of the relatively small differences in stability constants and hence k values between the individual lanthanides. In contrast, this very difficult lanthanide separation is impossible to achieve isocratically by conventional ion-exchange in a realistic time frame and all published methods use gradients with complexing eluents. Detection in Chelation Ion Chromatography
Blank run 0
20
40
60
Time (min) Figure 7 Determination of trace metals in seawater showing two standard additions. Column, 250 4.0 mm, 6.5 mm silica IDA. Step gradient: 0–10 min, 0.5 mmol l 1 nitric acid–0.5 mol l 1 KCl; 10–30 min, 80 mmol l 1 tartaric acid; 30–50 min, 10 mmol l 1 picolinic acid. Flow rate, 0.8 ml min 1. Detection, 490 nm, postcolumn reaction with PAR/NH3/HNO3 reagent. Sample, 6 ml Carnon estuary water. (From Nesterenko PN and Jones P, unpublished data.)
The main mode of detection of eluted metal species is the same as that used in conventional high-performance ion-exchange chromatography, so the reader is referred to the Further Reading section for more detailed information. Essentially, postcolumn reactions involving colorimetric reagents are employed to achieve the highest sensitivity detection. 4-(2pyridylazo)resorcinol (PAR) is the most commonly used colorimetric reagent, reacting sensitively with a wide range of metals. However, it should be pointed
Table 2 Practical applications of HPCIC for the determination of trace metals Functional groups
Sorbent (particle size, pore Column (mm) diameter, surface area, and capacity)
(A) Chemically bonded ligands Iminodiacetic acid 8 mm silica, 13 nm, 350 m2 g 1, 320 mmol g 1
Aminomethylphosphonic acid 2-Pyridinecarboxyaldehyde phenylhydrazone Amidoxime
10 mm polymer, 100 nm, 20 mmol ml 1
75 7.5
5 mm silica, 13 nm, 350 m2 g 1, 100 mmol g 1 5 mm silica, 12 nm, 400 m2 g 1, 18 mmol g 1 5 mm silica, 13 nm, 350 m2 g 1, 130 mmol g 1
50 4.6
(B) Precoated or impregnated substrates Xylenol Orange 8 mm PS-DVB, 10 nm, 414 m2 g 1, 31 mmol g 1
Methyl thymol Blue
250 4.0
8.8 mm PS-DVB, 12 nm, 470 m2 g 1, 48 mmol g 1 5 mm PS-DVB, 10 nm
250 4.6
Mobile phase
Metal ions separated (in order of retention)
Photometric detection
Application
Gradient elution 0.035 mol l 1 KCl– 0.065 mol l 1 KNO3, pH 2.5 1 mol l 1 KNO3, pH 4.9
Mg, Ca, Mn, Cd, Co, Zn, Ni, Pb, Cu Mg, Ca, Mn, Cd, Co, Zn, Pb, Ni, Cu Mg, Ba, Sr, Ca, Be
PAR, 550 nm PAR, 550 nm
0.4 mol l 1 KNO3, pH 2.5
Mg, Ba, Sr, Ca, Be
CAS, 590 nm
1 mmol l 1 dipicolinic acid, pH 3.0 0.2 mol l 1 KCl, 50 mmol l 1 phosphate buffer, 0.1 mmol l 1 CPC, pH 5.3 1 mol l 1 KNO3, 0.5 mol l 1 HNO3, 0.08 mol l 1 ascorbic acid Oxalate buffer, pH 4.5
Cu, Zn, Pb, Co, Cd
Direct UV, 290 nm Direct with buffering, 575 nm CAS, 560 nm
Mg, Ca, Mn, Cd, Co, Zn, Ni, Cu in seawater Mn, Cd, Co, Zn in freshwater Mg, Ca in NaCl and KCl brine solutions; eyewash saline solution Be in tap water, waste water, seawater Zn, Pb in waste waters of galvanic bath Mg, Ca in coastal seawater
Mg, Sr, Ca
Ni, Zn, Cu, Cd, Mn, Al, Be, La, Lu
o-CPC, 572 nm
Mn, Fe, Cd, Zn, Co, Pb, Cu
PAR, 550 nm
Be in stream sediment
Mn, Fe, Zn, Cu in tomato leaves and vitamin tablets Transition metals in seawater
50 3
5 mmol l 1 oxalic acid, pH 2.2
Cd, Pb, Co, Zn, Ni, Cu
PAR, 540 nm
100 4.6
1 mol l 1 KNO3, pH 7.7
Ba, Sr, Mg, Ca
PAR-ZnEDTA, 490 nm
Alkaline-earth metals in KCl or NaCl brines
1 mol l 1 KNO3, pH gradient
Ba, Sr, Mg, Ca, Mn, Cd, Zn, Ni, Cu, Fe
PAR-ZnEDTA, 490 nm
1 mol l 1 KNO3, 0.05 mol l 1 lactic acid, pH 8.5
Ba, Sr, Mg, Ca
PAR-ZnEDTA, 490 nm
Transition metals in coastal or estuarine seawater, inorganic chemicals Ba, Sr in mineral water
1 mol l 1 KNO3, pH 7.9
Ba, Sr, Mg, Ca
0.5 mol l 1 KNO3, pH 1.2
Ca, Mg, U, Cu
PAR-ZnEDTA, 490 nm Arsenazo III, 600 nm
100 4.6
150 4.1
Ba, Sr, Mg, Ca in oil-well brines U in saline lake water
100 4.6
1 mol l 1 KNO3, 0.05 mol l 1 lactic acid, pH 9.8
Ba, Sr, Mg, Ca
PAR-ZnEDTA, 490 nm
Sr in milk powder
100 4.6
1 mol l 1 KNO3, pH step gradient from 4.0 to 1.1
Al, Fe
PCV, 580 nm
Al in seawater
Aurin tricarboxylic acid (ATA)
8.8 mm PS-DVB, 12 nm, 470 m2 g 1, 48 mmol g 1 8.8 mm PS-DVB, 12 nm, 470 m2 g 1, 35 mmol g 1 7 mm PS-DVB, 10 nm, 150 mmol g 1
250 4.6
1 mol l 1 KNO3, 50 mmol l 1 acetic acid, pH step gradient from 4.5 to 1.0
Mn, Co, Ni, Cd, Pb, Cu, Ga, In
PAR, 520 nm
Pb, Cd, Cu in highly mineralized water
(C) Dynamically coated Dipicolinic acid
5 mm PS-DVB
100 4.6
Fe, Th, V, Bi, U, Hf, Zr
7 mm PS-DVB, 10 nm
300 4.6
Arsenazo III, 654 nm or PCV, 585 nm PAR, 520 nm
U, Bi, Zr in sediment. U in mineral water, seawater
4-Chlorodipicolinic acid
Pb, Cd, Cu in rice flour
o-Cresolphtalein complexone (CPC)
5 mm porous graphitic carbon, 25 nm, 120 m2 g 1 5 mm PS-DVB, 10 nm
100 4.6
1 mol l 1 KNO3, 0.5 mol l 1 HNO3, 0.1 mmol l 1 dipicolinic acid 1 mol l 1 KNO3, 0.25 mmol l 1 chlorodipicolinic acid, pH 1.5 45–58% MeOH, 0.4 mmol l 1 CPC, pH 10.0–10.5
Mg, Ca
Direct detection, 600 nm
Mg, Ca in seawater, saline lake water
Ba, Sr, Ca, Mg
CPC, 575 nm
Sr in Antarctic saline lake water
Methyl thymol Blue (MTB)
5 mm PS-DVB, 10 nm
150 4.1
0.5 mol l 1 KNO3, 0.2 mmol l 1 CPC, 20 mmol l 1 borate, pH 9.5 0.5 mol l 1 KNO3, 0.2 mmol l 1 MTB, pH 1.2
Mg, Mn, Zn, Cd, Pb
Zn in industrial gypsum
N-n-dodecyliminodiacetic acid
5 mm ODS-silica, 14 nm, 300 m2 g 1
150 4.0
75 mmol l 1 tartrate buffer, pH 5.5
Ca, Sr, Mg, Ba
Buffering at pH 5.9 with NH4OAc, 600 nm CPC, 575 nm or HQS-MgEDTA, fluorimetric 405/525 nm
o-Cresolphtalein complexone Chrome Azurol S
150 4.1
Mn, Co, Ni, Zn, Cu, Pb, Cd, Al, La, Lu, Fe, U
Mg, Ca in coastal seawater
Postcolumn reagents: PAR – 4-(2-pyridylazo)resorcinol; o-CPC – o-cresolphthalein complexone; CAS – chrome azurol S; PCV – pyrocatechol violet; HQS – 8-hydroxyquinoline-5-sulfonic acid.
480 ION EXCHANGE / Chelation Ion Chromatography 0.018
Mn
Cu 0.016 0.004
0.014
Cd
Absorbance
Absorbance
0.012 0.01 0.008
Zn Pb 0.006
Cu
0.004
Ni
0.002
Pb Cd
0 0
2
4
6
8
10
12
Retention time (min)
5
10
15
20 min
Figure 9 Chromatogram showing the preconcentration and separation of trace metals in 1 mol l 1 Na2SO4 sample spiked with 10 mg l 1 Mn2 þ , 25 mg l 1 Zn2 þ , and 0.1 mg l 1 Cd2 þ , Pb2 þ , Ni2 þ , and Cu2 þ . Sample volume was 10 ml adjusted to pH 6. Column, 100 mm 4.6 mm, packed with Xylenol Orange impregnated 8 mm PLRPS resin. (Reprinted with permission from Challenger OJ, Hill SJ, and Jones P (1993) Separation and determination of trace metals in concentrated salt solutions using chelation ion chromatography. Journal of Chromatography 639: 197–205; & Elsevier.)
out that lower limits of detection can be achieved using CIC compared to conventional IC separations. This is because many IC methods use strong complexing acids at relatively high concentrations in the eluents, which compete with colorimetric reagents such as PAR, reducing optimum sensitivity. In contrast, most CIC methods use either noncomplexing eluents or eluents containing relatively weak complexing acids. ——————————————————————9 Figure 11 Isocratic separation of Bi(III), U(VI), and Zr(IV) in GBW07311 sediment sample at pH 0 on the 15 cm PLRP-S column dynamically modified with 0.1 mmol l 1 dipicolinic acid. Injection volume used was 500 ml with detection at 654 nm with Arsenazo III postcolumn reaction. (Reproduced with permission from the Ph.D. dissertation of James Cowan (2002) The development and study of chelating substrates for the separation of metal ions in complex sample matrices. University of Plymouth, Figure 5.15, p. 218.)
0.001 9
0.001 4 Absorbance (AU)
0
Figure 10 The separation of Cd(II), Pb(II), and Cu(II) from matrix interferences in the certified rice flour GBW08502 on the 100 4.6 mm PRP-1 7 mm PS-DVB column. Eluent: 1 mol l 1 potassium nitrate, 30 mmol l 1 nitric acid, and 0.25 mmol l 1 chlorodipicolinic acid. Detection: PAR at 520 nm. (Reprinted with permission from Shaw MJ, Jones P, and Nesterenko PN (2002) Dynamic chelation ion chromatography of transition and heavy metals using a mobile phase containing 4-chlorodipicolinic acid. Journal of Chromatography A 953: 141–150; & Elsevier.)
0.000 9 U(VI)
Zr(IV)
0.000 4 Bi(III)
−0.000 1 0
5
10
15
Time (min)
20
25
ION EXCHANGE / Isolation of Biopolymers 481
Applications In this section, examples of applications involving high-efficiency CIC columns have been selected to demonstrate the wide range of complex samples analyzed using this technique (Table 2). The table is divided into three sections in terms of how the chelating substrate is formed, namely, bonded, impregnated, and dynamically coated. The insensitivity to ionic strength and special selectivity of these columns can be especially seen in the ability to analyze highly saline samples and digested sediment and mineral samples containing relatively enormous amounts of matrix metals. Figures 7–11 have been selected to illustrate some of the actual chromatographic separations obtained under these conditions. See also: Ion Exchange: Overview; Principles; Ion Chromatography Instrumentation; Ion Chromatography Applications; Isotope Separation. Liquid Chromatography: Ion Pair.
Further Reading Fritz JS and Gjerde DT (2000) Ion Chromatography, 3rd edn. Heidelberg and New York: Huethig.
Haddad PR and Jackson PE (1990) Ion Chromatography, Principles and Applications. Amsterdam: Elsevier. Jones P and Nesterenko PN (1997) High performance chelation ion chromatography: a new dimension in the separation and determination of trace metals. Journal of Chromatography 789: 413–435. Paull B and Haddad PR (1999) Chelation ion chromatography of trace metal ions using metallochromic ligands. Trends in Analytical Chemistry 18: 107–114. Paull B and Jones P (1996) A comparative study of the metal selective properties of chelating dye impregnated resins for the IC separation of trace metals. Chromatographia 42: 528–538. Sarzanini C (1999) Liquid chromatography: a tool for the analysis of metal species. Journal of Chromatography A 850: 213–228. Sarzanini C and Mentasi E (1997) Determination and speciation of metals by liquid chromatography. Journal of Chromatography A 789: 301–321. Timerbaev AR and Bonn GK (1997) Complexation ion chromatography – an overview of developments and trends in trace metal analysis. Journal of Chromatography 640: 195–206. Weiss J (1995) Ion Chromatography. Weinheim: VCH.
Isolation of Biopolymers P R Levison, Pall Europe Ltd., Portsmouth, UK & 2005, Elsevier Ltd. All Rights Reserved.
Introduction Biopolymers, the so-called ‘building blocks of nature’, are found in all living matter be it of animal, microbial, or vegetable origin. Biopolymers include proteins (polymers of amino acids), genetic material (polymers of nucleic acids), glycoforms (carbohydrates and glycosylated molecules), metabolites, and other structural molecules. By their very sequence and chemical composition many biopolymers have an electrical charge and can therefore be fractionated by ion-exchange processes. This article briefly reviews the principles underlying biopolymer purification by ion exchange and addresses some of the process issues associated with their purification.
Ion-Exchange Chemistries Ion-exchange chromatography is routinely used for the separation of biopolymers at laboratory scale
through to process scale. Ion exchangers are available from a number of manufacturers each produced using their proprietary synthesis methods. For biopolymer separations there are typically four ionic functionalities available. In the case of anion exchange, there are so-called weak anion exchangers (WAX) often based on weak amines including aminoethyl, diethylaminoethyl, guanidoethyl, p-aminobenzyl, ECTEOLA, and polyethyleneimine, and strong anion exchangers often based on a quaternary amine. Examples of each functionality are represented in Figure 1. The choice of anion exchanger is process dependent and for high pH applications, i.e., pH48.5 the more ionized grade, e.g., N,N,N-trimethyl-2-hydroxypropyl amine, would be recommended. In the case of cation exchange there are so-called weak cation exchangers often based on a carboxylic acid and strong cation exchangers often based on a sulfonic acid. Examples of each functionality are represented in Figure 2. Choice of cation exchanger is also process dependent but for low pH applications, i.e., pHo4.5, the more ionized grades, e.g., sufoxyethyl or sulfopropyl, would be recommended.
ION EXCHANGE / Chelation Ion Chromatography 0.2 0.18 0.16 0.14 0.12 0.1 0.08 0.06 0.04 0.02 0
467
See also: Ion Exchange: Overview; Principles; Ion Chromatography Instrumentation; Chelation Ion Chromatography; Isolation of Biopolymers; Isotope Separation. Thiocyanate
Further Reading
0
1
2
3
4
5
6
7
8
9
10
Time (min) Figure 4 Analysis of urine for thiocyanate using ion interaction liquid chromatography. Lower trace: sample (heavy smoker) diluted 1:20. Upper trace (heavy smoker) diluted 1:20 and spiked with thiocyanate.
alkaline earth cations in saliva. The sample matrix is relatively simple compared to other biological fluids and can be analyzed directly or simply diluted prior to injection. IC methods have also been developed for the determination of sweat samples for ionic analytes. Methods looking at sulfate levels and also concentrations of sodium and potassium in sweat samples have been developed, with relative levels of the latter metal ions being useful indicators of several important diseases, one of which is cystic fibrosis.
Betti M (1997) Use of ion chromatography for the determination of fission products and actinides in nuclear applications. Journal of Chromatography A 789: 369–379. Buldini PL, Cavalli S, and Trifiro A (1997) State-ofthe-art ion chromatographic determination of inorganic ions in food. Journal of Chromatography A 789: 529–548. Haddad PR and Jackson PE (1990) Ion Chromatography. Principles and Applications. Amsterdam: Elsevier. Lopez-Ruiz B (2000) Advances in the determination of inorganic anions by ion chromatography. Journal of Chromatography A 881: 607–627. Singh RP, Smesko SA, and Abbas NM (1997) Ion chromatographic characterisation of toxic solutions: analysis and ion chemistry of biological liquids. Journal of Chromatography A 774: 21–35. Small H (1990) Ion Chromatography. New York: Plenum. Smith RE (1987) Ion Chromatography Applications. Boca Raton, FL: CRC Press. Vanatta LE (2001) Application of ion chromatography in the semiconductor industry. Trends in Analytical Chemistry 20: 336–345.
Chelation Ion Chromatography P Jones, University of Plymouth, Devon, UK P N Nesterenko, Lomonosov Moscow State University, Moscow, Russia & 2005, Elsevier Ltd. All Rights Reserved.
Introduction It is now over 60 years since the development of column ion-exchange chromatography using polymeric resins. Interestingly, little has changed since then in terms of the basic separation processes for inorganic anions and cations. The renaissance in the 1970s, where ion-exchange was the principal process in a group of techniques now known as ion chromatography (IC), was mainly associated with the improvement in efficiency of stationary phases and detection systems rather than new types of ionexchange groups or elution systems. In essence, the IC separation of metal ions principally involves the use of eluents containing complexing organic acids,
such as tartaric, citric, oxalic, combined with high efficiency stationary phases. The complexing strength of acid and concentration chosen depended on whether the ion-exchange substrate was cationic, anionic, or mixed. Although poly(styrene–divinylbenzene) (PS–DVB) based resins are still the most common substrate, silica-based materials are increasingly being used. There are a number of problems associated with present IC methods involving high efficiency ion-exchange separations of metal ions, the two principal ones being sensitivity to ionic strength and limited selectivity. The influence of ionic strength is particularly serious as a relatively large salt concentration in the sample can drastically affect the chromatography, in many cases making it impossible to resolve the analytes. Limited selectivity is also a restriction, as once the substrate is chosen for conventional ion-exchange separations there are only a small number of cases where changes in eluent composition can significantly alter separation order.
468 ION EXCHANGE / Chelation Ion Chromatography
There is an alternative to ion-exchange, known generally as chelation ion-exchange, giving greater control over selectivity and relatively unaffected by changes in ionic strength. Although related, chelation ion-exchange differs fundamentally from ionexchange in that it involves the formation of coordinate bonds between metal ions and a complexing ligand immobilized on the surface of the substrate. Thus, the sorption process in chelation ion-exchange depends mainly upon the values of the stability constants of the metal complexes formed on the stationary phase. Inevitably, because ionexchange and chelation ion-exchange arise from the same functional group, they can occur together. Nevertheless, conditions can be chosen where chelation ion-exchange is the dominant mechanism controlling separation, as explained in the next section. It can be seen that the greater versatility of chelation ion-exchange arises from the wide range of complexing ligands available, where selectivity can be altered to suit the trace metal composition of a particular interest and sample type. Chelation ionexchange is not a recently exploited technique, being used for nearly as long as ion-exchange itself. However, until recently, chelation ion-exchange columns have principally been used for matrix isolation and preconcentration of suites of metals rather than chromatographic separation. The isolation of trace metals from seawater using Chelex 100 (iminodiacetic acid functionalized PS-DVB) is a classic example. Later, chelating ion-exchangers such as 8-hydroxyquinolinol attached to silica (Table 1) have been extensively used in flow injection analysis for determination of metals. In the last 10 years or so, chelation ion-exchange columns have become available for use as precolumns in IC systems where matrix isolation and preconcentration occur online before the suite of metals is swept onto an ion-exchange column for separation (see later). This approach became known as chelation ion chromatography (CIC). Another use of chelation ion-exchange columns, the main focus of this article, is for the high-efficiency separation of metal ions on small particle size substrates. It is a relatively new approach and shows great potential for the IC determination of metals in a wide variety of complex samples, including those with very high ionic strengths. Although the term CIC can be used to describe both uses of chelation ion-exchange substrates, it should be noted that some workers have preferred to use the term high-performance chelation ion chromatography (HPCIC) to differentiate the high-efficiency separation technique from CIC methods where chelation ion-exchange is only
used for preconcentration or matrix isolation purposes. The principal purpose of this article is to describe the latest work involving high-efficiency chelation ion-exchange columns for determining trace metals in a wide variety of complex matrices. The basic principles relating to the sorption of metal ions will be described first, followed by illustrations of the different separation modes available, and, finally, key examples will be given of the wide range of samples analyzed by the technique.
Chelation Ion-Exchange Equilibria The efficient separation of metal cations can be achieved with three main methods: 1. using conventional cation-exchange based on electrostatic interactions; 2. by preconcentration of metal cations on a short column packed with chelating substrate with further separation by cation-exchange ion chromatography; 3. using the ability of chelating ion-exchangers to achieve analytical separations of metal ions with a single column. The last two methods can both be described as CIC as discussed earlier. However, it is only with approach (3), the principal concern of this article, is it important to fully understand the factors affecting efficiency and selectivity to obtain good chromatography. Therefore, the basic principles described below will focus on these aspects. Distribution Ratio
Pure sorption mechanisms are very rare in chromatography and chelation ion-exchange is no exception. As the chelating groups are usually charged, the chromatographic retention of alkaline-earth, transition, and heavy metal ions on such a column, in a noncomplexing eluent, occurs through the combination of electrostatic and coordination interactions between cations and chelating functional groups on the substrate. The distribution ratio DM of metal cation My þ between chelating ion-exchanger and the contacting mobile phase can be expressed by the following equation: yþ C I yþ DM ¼ ð½Myþ R þ ½MR Þ=½Maq
yþ
yþ
½1
where ½MR I and ½MR C are equilibrium concentrations of cation retained by the stationary phase due to electrostatic interactions (conventional ion-exchange) and chelation ion-exchange, respectively.
ION EXCHANGE / Chelation Ion Chromatography
469
Table 1 Selectivity of chelating ion-exchangers for HPCIC Functional groups
Selectivity
(A) Chemically bonded 8-Hydroxyquinolinol
O
Ni2 þ 4Co2 þ 4Zn2 þ 4Pb2 þ 4Cd2 þ 4Mn2 þ Yb3 þ 4Gd3 þ 4La3 þ
(CH2)3NHC(O)
Si
N
N
OH N
(a) Mg2 þ 4Ca2 þ 4Sr2 þ 4Ba2 þ (b) Fe3 þ 4Cu2 þ 4In3 þ 4UO22 þ 4Pb2 þ 4Ni2 þ 4Zn2 þ 4 Cd2 þ 4Co2 þ 4Be2 þ 4Fe2 þ 4Mn2 þ (c) Lu3 þ 4Yb3 þ 4Tm3 þ 4Er3 þ 4Ho3 þ 4Tb3 þ 4Eu3 þ 4 Gd3 þ 4Dy3 þ 4Sm3 þ 4Y3 þ 4Nd3 þ 4Pr3 þ 4Ce3 þ 4La3 þ
Iminodiacetic acid
CH2COOH O
Si
(CH2)3OCH2 CH(OH)CH2 N CH2 COOH
(a) Mg2 þ 4Ca2 þ 4Sr2 þ 4Ba2 þ
Aminophosphonic acid
(b) Mn2 þ 4Cd2 þ 4Pb2 þ 4Cu2 þ 4Zn2 þ 4Co2 þ 4Ni2 þ
O O
Si
(c) Lu3 þ 4La3 þ 4Be2 þ 4Al3 þ
(CH2)3NHCH2 −P−OH O− UO22 þ 4Cu2 þ 4Cd2 þ 4Co2 þ
Amidoxime
NOH O
Si
(CH2)3C NH2 Fe3 þ 4Cu2 þ 4Zn2 þ
p-Aminosulfosalycilic acid
OH O
Si
(CH2)3OCH2 CH(OH)CH2NH
COOH
K þ 4Rb þ 4Cs þ 4Na þ 4Li þ (as Cl salts)
Bis(benzo-15-crown-5)
Ba2 þ 4Sr2 þ 4Ca2 þ 4Mg2 þ (as Cl salts) I 4Br 4Cl (as K þ salts) O
Si
(CH2)2NH
(CH2)3N C
C
O
O
O
O O
O
O
O
O
O
O
O
Cu2 þ 4Co2 þ 4Zn2 þ 4Cd2 þ Fe2 þ 4Mg2 þ
Poly(amines)
O
Si
(CH2)3OCH2CH(OH)CH2N(CH2CH2NH)nH n = 0−4 Continued
470 ION EXCHANGE / Chelation Ion Chromatography Table 1 Continued Functional groups
Selectivity
Poly(butadienemaleic acid) PBDMA
Cu2 þ 4In3 þ 4UO22 þ 4Pb2 þ 4Ni2 þ 4Co2 þ 4Zn2 þ 4 Cd2 þ 4Mn2 þ 4Mg2 þ
COOH [
]
n
COOH Cu2 þ 4Pb2 þ 4Zn2 þ 4Ni2 þ 4Mn2 þ 4Cd2 þ 4Co2 þ 4 Mg2 þ 4Ca2 þ 4Ba2 þ
Aspartic acid
COOH O
(CH2)3OCH2CH(OH)CH2NHCH
Si
CH2CH2COOH (B) Precoated or impregnated substrates (a) Mg2 þ 4Ca2 þ 4Sr2 þ 4Ba2 þ
Xylenol Orange
(b) Cu2 þ 4Ni2 þ 4Zn2 þ 4Cd2 þ 4Mn2 þ CH2COOH
O
CH2NCH2COOH
CH3
CH3
C
OH
CH2NCH2COOH SO3H
CH2COOH
(a) In3 þ 4Ga3 þ 4Al3 þ
Aurin tricarboxylic acid
(b) Cu2 þ 4Pb2 þ 4Cd2 þ 4Ni2 þ 4Zn2 þ 4Co2 þ 4Mn2 þ O COOH
C
HOOC
COOH
HO
OH
Ca2 þ 4Mg2 þ 4Sr2 þ 4Ba2 þ
o-Cresolphthalein complexone O
CH2COOH CH2NCH2COOH
CH3
CH3
C
OH
CH2NCH2COOH COOH
CH2COOH
ION EXCHANGE / Chelation Ion Chromatography
471
Table 1 Continued Functional groups
Selectivity
Methyl thymol blue
Ca2 þ 4Mg2 þ 4Sr2 þ 4Ba2 þ CH2COOH
O
CH2NCH2COOH
(CH3)2CH
CH(CH3)2 CH3 C
OH
CH2NCH2COOH
CH3 SO3H
CH2COOH
Fe3 þ 4Al3 þ
Chrome Azurol S O COOH
CH3 Cl
SO3H
C HOOC Cl HO CH3
(C) Dynamically coated Methyl thymol blue (see structure in section 1b)
(a) Cu2 þ 4UO22 þ 4Mg2 þ 4Ca2 þ (b) Lu3 þ 4Tm3 þ 4Tb3 þ 4Sm3 þ 4Nd3 þ 4Ce3 þ 4La3 þ 4 Cu2 þ 4Pb2 þ 4Cd2 þ 4Zn2 þ 4Co2 þ 4Mn2 þ 4Mg2 þ (a) Cd2 þ 4Pb2 þ 4Zn2 þ 4Ni2 þ 4Cu2 þ 4Mn2 þ (b) Zr(IV)4Hf(IV)4U(VI)4Bi(III)4V(V)4Th(IV)4Fe(III)
Dipicolinic acid
N
HOOC
COOH (a) Cd2 þ 4Pb2 þ 4Cu2 þ 4Zn2 þ 4Ni2 þ 4Co2 þ 4Mn2 þ (b) UO22 þ 4Fe3 þ 4Lu3 þ 4La3 þ 4Al3 þ
4-Chlorodipicolinic acid
Cl
N
HOOC
COOH (a) Cu2 þ 4Ni2 þ 4Co2 þ 4Zn2 þ 4Cd2 þ 4Pb2 þ 4Mn2 þ (b) Lu3 þ 4La3 þ
Quinaldic acid
N
COOH
472 ION EXCHANGE / Chelation Ion Chromatography
½Myþ aq is the concentration of cation in the mobile phase. The retention factor k can be expressed as k ¼ ðt t0 Þ=t0 ¼ DM j
½2
where t is retention time, t0 the void time, and j is a characteristic constant for a given chromatographic column expressed as the ratio of the volumes of mobile Vaq and stationary VR phases: j ¼ Vaq =VR
½3
As suitable chromatographic separation can be achieved at values of ko10–15 and the formation of complexes at the surface with more than one ligand is for simplification assumed not to take place due to thermodynamic and steric restrictions; then, x Myþ ¼ MLðyxÞþ aq þ L
½4
ðyxÞþ C
Thus, ½MR
can be expressed as follows: ðyxÞþ C
n ¼ b1 ½Myþ aq ½L
½MR
½5
where [Ln ] is the concentration of functional groups and b1 is the stability constant of complex ML(y–x) þ formed at the surface in accordance with (4). Selectivity Ratio
The ion-exchange process of cation My þ and alkali metal cation B þ as competing cation at constant pH of the eluent on the chelating ion-exchanger can be expressed as follows: yþ yþ þ yBþ R þ Maq ¼ yBaq þ MR
½6
where the subscript ‘aq’ denotes the mobile phase and ‘R’ denotes the stationary phase. The following selectivity ratio is given by KBM ¼
þ y ½Myþ R ½Baq þ y ½Myþ aq ½BR
½7
Taking into account the two possible types of interactions in accordance with [1], the retention factor k of metal cation My þ can be expressed as k¼
KBM
! y ½Bþ n R y þ b1 ½L j ½Bþ aq
½8
The first member of the sum in brackets expresses the impact of conventional ion-exchange interactions on retention, and the second member expresses the impact of chelation on retention. Several conclusions can be formulated from eqn [8]. Assuming little change in b1 with high ionic strength (40.1), the retention of a metal ion due to
chelation should not depend upon the concentration þ ], but solely on the conof alkali metal cations [Baq centration of functional groups in the chelating ion-exchanger. In practice, it means electrostatic interactions need to be suppressed for chelation to be the dominant sorption mechanism. The retention of M due to conventional ion-exþ ]. change can be suppressed by increase of [Baq Thus, in the case of domination by chelation y þ y (b1 ½LcKBM ð½Bþ R =½Baq Þ at high concentration level þ of Baq in the eluent) the separation selectivity a is defined by ratio of corresponding stability constants of metal chelates formed at the surface: M1 a ¼ k2 =k1 ¼ bM2 1 =b1
½9
Strictly, it should be the ratio of conditional stability constants, but for a given ligand with no hydrolysis of the metal ions, the ratio is the same as the thermodynamic stability constants. Temperature Effects
As well as varying the ionic strength of the eluent, the separation selectivity KBM in CIC can be changed by variation of column temperature. Sorption heat relates the retention factor k with the column temperature by the van’t Hoff equation: ln k ¼ DH=RT þ DS=R þ ln j
½10
where DH and DS are sorption enthalpy and entropy, respectively, and j is the phase volume ratio. The temperature effects are exothermic (negative values of DH) in the case of conventional ion-exchange. However, the heat values do not exceed 8–13 kJ mol 1 and therefore have little influence on retention. In contrast, for stationary phases where metal ion complexation is present, the sorption process will normally exhibit much higher values of DH in both exothermic and endothermic conditions. Thus, temperature effects will be significant and some regulation of selectivity is possible.
Factors Affecting the Efficiency of Chelation Ion-Exchange It is very important to understand the factors controlling the kinetics of the sorption process as they will have a major bearing on separation efficiency. There are three factors affecting the kinetics of chelating ion-exchange: 1. transport of metal ion from the mobile phase to the surface of chelating ion-exchanger;
ION EXCHANGE / Chelation Ion Chromatography
2. diffusion of the ions inside of the stationary phase; and 3. kinetics of chelation of metal ion with functional groups. The first two factors are similar to those occurring in simple conventional ion-exchange so they will not be discussed further. The resulting kinetics of chelation depends upon the chemical nature of the functional groups and the structure of the chelating ion-exchanger, with the former being the most crucial for good separations. Chemical Nature of Functional Groups
Charge on the functional groups It was empirically established that better kinetics of CIC arise when negatively charged functional groups are present. Obviously, negatively charged groups at the surface, attracting oppositely charged cations from the mobile phase, will provide better transport of metal ions from the mobile phase than neutral or positively charged chelating surfaces. Dentation of the ligand It would be expected that the higher the denticity of the ligand, the slower the kinetics as more groups need to be dissociated and more coordinate bonds formed for each metal exchange reaction. In practice, polydentate ligands are usually necessary to achieve adequate retention and selectivity. Fortunately, for most ligands with a denticity not greater than 3, the slower kinetic effect in most cases appears to be relatively minor, as long as the eluent conditions are chosen to ensure small conditional constants. Magnitude of the conditional constants Of all the ligand properties affecting chelation ion-exchange kinetics, the value of the metal/ligand conditional constant has the largest influence. In simple terms, the magnitude of a stability or conditional constant is governed by the ratio of the velocity of the forward reaction (association) to the velocity of the back reaction (dissociation). Except for a small number of metal ion/ligating atom combinations (see later), the velocity of the forward reaction is very fast. Thus, the kinetics of chelation ion-exchange will be controlled by the value of the velocity of the back reaction (dissociation), which will increase as the conditional constant decreases. However, the retention factor k is directly proportional to the value of the conditional constant (eqn [8]). Therefore, to achieve optimum efficiency of separation, the conditional constants should be as low as possible, but not so low as to cause serious overlap of peaks close to
473
the solvent front. As most ligands arise from dissociation of an acid group, change in pH is the simplest way of changing the conditional constant and hence k. It should be noted that this property is very important for the chromatographic separation of metal cations, but not so critical when used for preconcentration or matrix isolation of groups of metal ions. Type of ligating atom Nitrogen and oxygen are the most common ligating atoms in the chelating groups used in CIC. Sulfur-containing ones have been investigated, but are inherently unstable due to air oxidation. Nitrogen-ligating atoms can give slower kinetics than oxygen atoms with certain common metals, nickel being most notable. Even N, O, O chelaters like iminodiacetic acid (IDA) give broad nickel peaks unless the conditional constant is very low and the nickel is close to the solvent front. Structural Properties of the Chelating Exchanger
Conformational mobility of the bonded ligand Chelating ion-exchangers having functional groups chemically attached as a monolayer to the surface through flexible long linkers will provide better selectivity and separation efficiency as they are more easily accessible to metal ions. On the other hand, chelating substrates formed by immobilizing ligands in micropores will be more sterically hidden, subsequently decreasing the kinetics of metal ion association. The distribution density of functional groups at the surface A very high concentration (capacity) of functional groups can lead to nonuniform complexation at the surface of the chelating ion-exchanger. Perhaps a more important consideration is that close proximity of ligands allows the theoretical possibility of formation of ML2 and even ML3 complexes with much less favorable kinetics of dissociation, causing poor (broad and tailing) peak shapes in CIC. It should be noted that for some chelating substrates such as silica bonded IDA, due to thermodynamic considerations, ML2 and ML3 complexes are not formed at the typical low pHs used for separations. It is interesting to note that, depending on the distribution and density of the functional groups, weak cation-exchangers with carboxylic and phosphonic acid functional groups could serve as chelating ionexchangers as two or more groups in close proximity may be able to form chelate-related structures with metal ions, though this may have a bearing on peak broadening as discussed above.
474 ION EXCHANGE / Chelation Ion Chromatography
Pore size The structure of the substrate can also affect the stoichiometry of complexes at the surface. In narrow pores the coordination of two attached ligands is much more probable.
Main Types of Chelation Ion-Exchangers Rigidity and mechanical stability of the substrate under conditions of high-performance liquid chromatography are important and microspherical particles of macroporous highly crosslinked PS-DVB or poly(metacrylate) and silica are well suited for CIC. There are three main methods of producing a chelating ion-exchange surface. Chemically Bonded
This is the most common approach to producing a chelating surface as can be seen from the selection shown in Table 1A. Although they are all capable of producing separations, only a small number appear to have sufficiently good kinetics for chromatographic separations, the most popular being IDA and aminophosphonic acid. Impregnation
Relatively large chelating dyestuff molecules can form an essentially permanent coating by becoming ‘trapped’ in the pores of the substrate. PS-DVB resins are the most successful for this mode as p–p interactions between the aromatic rings of the dye and the resin help to hold the chelating molecule in place. Table 1B shows some representative examples of those used for chromatographic separations. Those containing the IDA group appear to give the best efficiency, though not as good as the chemically bonded type, presumably due to the chelating groups being more sterically hindered in the pore structure. Dynamic Coating
Dynamic coating or modification involves creating a chelating surface by having a ligand continuously present in the eluent. Conditions are chosen so that no permanent impregnation occurs, but rather a dynamic equilibrium is established between a sorbed layer of ligand on the surface of the substrate and the concentration of the ligand in the mobile phase. Due to the preferred adsorption of the ligand, its concentration at the surface is much higher than in the eluent, thus producing a chelation ion-exchange mechanism. Altering such parameters as the concentration of ligand in the eluent, mobile phase pH, and the nature of the stationary phase allows the dynamic
equilibrium to change, thus affecting metal selectivity. This is a relatively new approach and some representative examples are given in Table 1C. Although large molecules such as chelating dyestuffs have been successfully used in the dynamic mode for chromatographic separations, most work has concentrated on small molecules with aromatic groups such as dipicolinic acid.
Practical Aspects of Chelation Ion-Chromatography Precolumn Systems with Common Chromatographic Equipment
The original and most widespread application of chelating ion-exchangers is in the selective adsorption of one or more metal ions of interest from diluted solutions followed by sensitive determination. The short columns or cartridges are not designed for the efficient chromatographic separation of metal ions, but rather for collection on the column before being swept off as a concentrated ‘plug’. By this means matrix isolation and/or preconcentration can be carried out before further analysis online or offline. For CIC, one approach, as explained previously, is to use a chelating precolumn as part of an online IC system and can be considered as one constituent of the overall separation/determination process. Conventional ion-exchange columns, connected in series with the chelating column, are used for the analytical separation of metal ions before postcolumn reaction detection. The most common chelating columns are based on IDA or 8-hydroxyquinoline, bonded to controlled pore glass, silica, or PS-DVB resins. Some are available commercially, though many ‘home made’ columns are described in the literature. These systems have been used successfully to determine trace metals in a number of complex samples, though of necessity the set-up can be rather complex. One commercially available system, for example, involves three columns (the first one being the chelating ion-exchange column) several pumps, eluents, and switching valves. High-Performance Chelation Ion Chromatography
A relatively recent approach in CIC involves more efficient, small particle size substrates as the chelating surface. The use of these high-performance stationary phases means that reasonably fast analytical separations of groups of metal ions can be obtained similar to conventional high-performance ion-exchange. Matrix isolation is not normally required as the chelating column is relatively insensitive to concentrated salt solutions. Preconcentration, if necessary, can also
ION EXCHANGE / Chelation Ion Chromatography
be carried out using a step pH gradient. Thus, matrix isolation, preconcentration, and analytical separation can be achieved using a single high-efficiency chelating column, removing the need for a complex multicolumn system as described in the previous section. The sorption process controlling separation can be almost pure chelation or a combination of chelation and simple ion-exchange as discussed in detail in the section on chelation ion-exchange equilibria. A straightforward way of controlling the relative contribution of chelation and simple ion-exchange is
475
through the ionic strength of the eluent. Increasing the concentration of an alkali metal salt such as potassium nitrate in the eluent will suppress simple ionexchange interactions. Concentrations as high as one molar have been used to achieve almost complete suppression of ion-exchange for many metal ions. The chromatograms in Figures 1–6 were selected to show the kind of analytical separations and selectivities available for groups of metal ions for the three types of chelating surface: bonded,
La
658 nm 0.002 AU
0.10 Mn Y Nd Pr
Absorbance
0.08 Cd
0.06
Ce
Zn
Sm Eu Gd Tb
Co
Dy Ho Er
0.04
Tm
Yb
Lu
0.02 0
0 0
4
2
(A) 0.35
6 8 Time (min)
10
12
Mn
Absorbance
0.25 Co
0.20 0.15 Zn
0.10
40
50
60
70
Time (min)
:——————————————————————
0.00 0
2
4
6
8 10 12 14 Time (min)
(B)
16
Co Mn
1050 Absorbance (mAU)
30
Pb
0.05
650 Cd
250
Zn
Pb
−150 0 (C)
20
Figure 2 Isocratic separation of standard mixture of 14 lanthanides and yttrium on a 250 mm 4 mm column, packed with 5 mm silica IDA. Eluent: 1.6 10 2 mol l 1 HNO3 with 0.5 mol l 1 KNO3; flow rate 1.0 ml min 1; column temperature 651C, sample volume 20 ml, sample concentration of each metal was 4 ppm in 0.2% HNO3. Detection, Arsenazo III postcolumn reaction at 658 nm. (Reprinted with permission from Nesterenko PN and Jones P (1998) Isocratic separation of lanthanides and yttrium by high performance chelation ion chromatography. Journal of Chromatography A 804: 223–231; & Elsevier.)
Cu
0.30
10
2
4 6 Time (min)
8
10
18 20
Figure 1 Separations on silica IDA columns with different elution protocols. (A) Isocratic separation of four metal ions on a 100 mm 4 mm column packed with 5 mm IDA silica. Eluent, 10 mmol l 1 nitric acid. Detection, PAR postcolumn reaction at 510 nm. (Unpublished work, Nesterenko PN and Jones P.) (B) Isocratic separation of five metal ions on a 250 mm 4 mm column packed with 5 mm IDA silica. Eluent, 0.5 mol l 1 KCl, 20 mmol l 1 picolinic acid, and 12.5 mmol l 1 nitric acid. Detection, PAR postcolumn reaction at 510 nm. (Unpublished work, Nesterenko PN and Jones P.) (C) Step gradient separation of Mn(II), Cd(II), Co(II), Zn(II), and Pb(II). Eluent conditions: 0.1 mol l 1 NaCl (pH 2.6) switched to 0.1 mol l 1 NaCl (pH 1.6) at time ¼ 3 min prior to standard injection. Column, 250 mm 4 mm, packed with 8 mm silica IDA. Detection, PAR postcolumn reaction at 495 nm. (Reprinted with permission from Bashir W and Paull B (2002) Ionic strength, pH and temperature effects upon selectivity for transition and heavy metal ions when using chelation ion chromatography with an iminodiacetic acid bonded silica get column and simple eluents. Journal of Chromatography 942: 73–82; & Elsevier.)
476 ION EXCHANGE / Chelation Ion Chromatography 0.05
Absorbance
0.04 Ba
0.03
Mn
Zn
Pb Cd
Mn Cu
0.02 0.01
0.04
−0.01
0
2
4
6
8
10
12
14
16
18
20
Time (min) Figure 3 The separation of Ba(II) 1.5 ppm, Ni(II) 1 ppm, Co(II) 2 ppm, Zn(II) 1 ppm, Pb(II) 5 ppm, Cd(II) 2.5 ppm, Mn(II) 1.5 ppm, and Cu(II) 5 ppm on the aminophosphonic acid functionalized silica column, 250 mm 4.6 mm. Eluent 1 mol l 1 KNO3, 5 mmol l 1 HNO3. Detection, PAR/ZnEDTA postcolumn reaction at 495 nm. (Reprinted with permission from Nesterenko PN, Shaw MJ, Hill S, and Jones P (1999) Aminophosphonatefunctionalised silica: A versatile chromatographic stationary phase for high performance chelation ion chromatography. Microchemical Journal 62: 58–69; & Elsevier.)
Cu Zn Cd
Co 0.02
0
0.28
0
Zn(II)
0.18 Pb(II)
0.13
Cu(II)
Mn(II) Cd(II)
0.08
0.03
0
2
4
6 Time (min)
5
10 15 Retention time (min)
20
Figure 5 The separation of Mn(II) 0.5 mg l 1, Co(II) 0.5 mg l 1, Ni(II) 0.5 mg l 1, Zn(II) 2 mg l 1, Cu(II) 1 mg l 1, Pb(II) 10 mg l 1, and Cd(II) 20 mg l 1 on a 300 4.6 mm PRP-1 7 mm PS-DVB dynamically coated column. Eluent: 1 mol l 1 potassium nitrate, 0.25 mmol l 1 chlorodipicolinic acid, and 6.25 mmol l 1 nitric acid (pH 2.2). Detection: PAR at 520 nm. (Reprinted with permission from Shaw MJ, Jones P, and Nesterenko PN (2002) Dynamic chelation ion chromatography of transition and heavy metals using a mobile phase containing 4-chlorodipicolinic acid. Journal of Chromatography A 953: 141–150; & Elsevier.)
0.23
−0.02
Ni
Absorbance
0.00
Absorbance (AU)
Pb
0.06
Ni Co
8
10
12
Figure 4 Gradient elution of a 0.5 mg l 1 Mn(II), 20 mg l 1 Cd(II), 10 mg l 1 Zn(II), 10 mg l 1 Pb(II), and 1 mg l 1 Cu(II) mixture on the 100 mm 4.6 mm Xylenol Orange impregnated Hamilton resin column. Injection volume used was 100 ml with detection at 520 nm with PAR postcolumn reaction. (Reprinted with permission from the Ph.D. dissertation of James Cowan (2002) The development and study of chelating substrates for the separation of metal ions in complex matrices. University of Plymouth, Figure 2.9, p. 84.)
impregnated, and dynamically loaded. For many chelating substrates the selectivity factors for metal ions in simple eluents can vary over a large range. This means that to separate four or more metal ions isocratically in a reasonable time can be a problem. Figure 1 illustrates this situation quite clearly for silica IDA columns, where a simple dilute nitric acid eluent can separate four relatively weakly retained metal ions, but the more strongly held metal ions such as copper or lead require complexing eluents and/or gradients to elute them more quickly. Although the application of gradients is more complex, gradients have the advantage of sharpening later eluting peaks and allowing on-column preconcentration if required. Two exceptions to this general situation have been found so far. The first involves aminophosphonate bonded silica (Figure 3) where eight common metal ions can be separated isocratically in
ION EXCHANGE / Chelation Ion Chromatography 0.006
477
Th(IV) Sr
Ca+Mg
Absorbance
0.005 0.004 U(VI)
Bi(III)
0.003
Zr(IV) Hf(IV) Absorbance
0.002 Fe(III)
0.001 0 0
5
10 Time (min)
15
20
Figure 6 Chromatogram showing the separation of six acid hydrolyzing metal ions on a dynamically coated column (100 4.6 mm ID), packed with 5 mm PLRPS polystyrene divinylbenzene resin. Eluent, 1 mol l 1 potassium nitrate and 0.1 mmol l 1 dipicolinic acid in 0.5 mol l 1 nitric acid. Detection, Arsenazo III postcolumn reaction monitored at 654 nm. Sample injection, 100 ml of a mixture of 20 ppm Fe(III), 0.5 ppm Th(IV), 20 ppm Bi(III), 0.5 ppm U(VI), 25 ppm Hf(IV), and 5 ppm Zr(IV). (The ion chromatographic separation of high valence metal cations using a neutral polystyrene resin dynamically modified with dipicolinic acid. Cowan J, Shaw MJ, Achterberg EP, Nesterenko PN, and Jones P (2000) The analyst communication. Analyst 125: 2157–2159; reproduced by permission of The Royal Society of Chemistry.)
Cu(II)
Zn(II) Cd(II) Co(II)
Ni(II)
Absorbance, 490 nm
0.1 AU
15 ppb Co(II) 60 ppb Ni(II) 15 ppb Cd(II)
20 ppb Co(II) 40 ppb Ni(II) 10 ppb Cd(II)
Sample
Ba 60
0 Time (min)
Figure 8 Chromatogram showing the separation of barium and strontium from magnesium and calcium in an oil-well brine (sample 1, diluted 1:1 v/v with double distilled water) using a Methylthymol Blue impregnated column (pH 9.2). (Determination of alkaline earth metals in offshore oil-well brines using high performance chelation ion chromatography. Paull B, Foulkes M, and Jones P (1994) Analytical Proceedings 31: 209–211; reproduced by permission of The Royal Society of Chemistry.)
16 min using dilute nitric acid eluent, though iron(III) is strongly retained under these conditions. The second exception concerns the separation of the lanthanides and yttrium on IDA bonded silica (Figure 2). The separation of such a large number of metals isocratically is possible because of the relatively small differences in stability constants and hence k values between the individual lanthanides. In contrast, this very difficult lanthanide separation is impossible to achieve isocratically by conventional ion-exchange in a realistic time frame and all published methods use gradients with complexing eluents. Detection in Chelation Ion Chromatography
Blank run 0
20
40
60
Time (min) Figure 7 Determination of trace metals in seawater showing two standard additions. Column, 250 4.0 mm, 6.5 mm silica IDA. Step gradient: 0–10 min, 0.5 mmol l 1 nitric acid–0.5 mol l 1 KCl; 10–30 min, 80 mmol l 1 tartaric acid; 30–50 min, 10 mmol l 1 picolinic acid. Flow rate, 0.8 ml min 1. Detection, 490 nm, postcolumn reaction with PAR/NH3/HNO3 reagent. Sample, 6 ml Carnon estuary water. (From Nesterenko PN and Jones P, unpublished data.)
The main mode of detection of eluted metal species is the same as that used in conventional high-performance ion-exchange chromatography, so the reader is referred to the Further Reading section for more detailed information. Essentially, postcolumn reactions involving colorimetric reagents are employed to achieve the highest sensitivity detection. 4-(2pyridylazo)resorcinol (PAR) is the most commonly used colorimetric reagent, reacting sensitively with a wide range of metals. However, it should be pointed
Table 2 Practical applications of HPCIC for the determination of trace metals Functional groups
Sorbent (particle size, pore Column (mm) diameter, surface area, and capacity)
(A) Chemically bonded ligands Iminodiacetic acid 8 mm silica, 13 nm, 350 m2 g 1, 320 mmol g 1
Aminomethylphosphonic acid 2-Pyridinecarboxyaldehyde phenylhydrazone Amidoxime
10 mm polymer, 100 nm, 20 mmol ml 1
75 7.5
5 mm silica, 13 nm, 350 m2 g 1, 100 mmol g 1 5 mm silica, 12 nm, 400 m2 g 1, 18 mmol g 1 5 mm silica, 13 nm, 350 m2 g 1, 130 mmol g 1
50 4.6
(B) Precoated or impregnated substrates Xylenol Orange 8 mm PS-DVB, 10 nm, 414 m2 g 1, 31 mmol g 1
Methyl thymol Blue
250 4.0
8.8 mm PS-DVB, 12 nm, 470 m2 g 1, 48 mmol g 1 5 mm PS-DVB, 10 nm
250 4.6
Mobile phase
Metal ions separated (in order of retention)
Photometric detection
Application
Gradient elution 0.035 mol l 1 KCl– 0.065 mol l 1 KNO3, pH 2.5 1 mol l 1 KNO3, pH 4.9
Mg, Ca, Mn, Cd, Co, Zn, Ni, Pb, Cu Mg, Ca, Mn, Cd, Co, Zn, Pb, Ni, Cu Mg, Ba, Sr, Ca, Be
PAR, 550 nm PAR, 550 nm
0.4 mol l 1 KNO3, pH 2.5
Mg, Ba, Sr, Ca, Be
CAS, 590 nm
1 mmol l 1 dipicolinic acid, pH 3.0 0.2 mol l 1 KCl, 50 mmol l 1 phosphate buffer, 0.1 mmol l 1 CPC, pH 5.3 1 mol l 1 KNO3, 0.5 mol l 1 HNO3, 0.08 mol l 1 ascorbic acid Oxalate buffer, pH 4.5
Cu, Zn, Pb, Co, Cd
Direct UV, 290 nm Direct with buffering, 575 nm CAS, 560 nm
Mg, Ca, Mn, Cd, Co, Zn, Ni, Cu in seawater Mn, Cd, Co, Zn in freshwater Mg, Ca in NaCl and KCl brine solutions; eyewash saline solution Be in tap water, waste water, seawater Zn, Pb in waste waters of galvanic bath Mg, Ca in coastal seawater
Mg, Sr, Ca
Ni, Zn, Cu, Cd, Mn, Al, Be, La, Lu
o-CPC, 572 nm
Mn, Fe, Cd, Zn, Co, Pb, Cu
PAR, 550 nm
Be in stream sediment
Mn, Fe, Zn, Cu in tomato leaves and vitamin tablets Transition metals in seawater
50 3
5 mmol l 1 oxalic acid, pH 2.2
Cd, Pb, Co, Zn, Ni, Cu
PAR, 540 nm
100 4.6
1 mol l 1 KNO3, pH 7.7
Ba, Sr, Mg, Ca
PAR-ZnEDTA, 490 nm
Alkaline-earth metals in KCl or NaCl brines
1 mol l 1 KNO3, pH gradient
Ba, Sr, Mg, Ca, Mn, Cd, Zn, Ni, Cu, Fe
PAR-ZnEDTA, 490 nm
1 mol l 1 KNO3, 0.05 mol l 1 lactic acid, pH 8.5
Ba, Sr, Mg, Ca
PAR-ZnEDTA, 490 nm
Transition metals in coastal or estuarine seawater, inorganic chemicals Ba, Sr in mineral water
1 mol l 1 KNO3, pH 7.9
Ba, Sr, Mg, Ca
0.5 mol l 1 KNO3, pH 1.2
Ca, Mg, U, Cu
PAR-ZnEDTA, 490 nm Arsenazo III, 600 nm
100 4.6
150 4.1
Ba, Sr, Mg, Ca in oil-well brines U in saline lake water
100 4.6
1 mol l 1 KNO3, 0.05 mol l 1 lactic acid, pH 9.8
Ba, Sr, Mg, Ca
PAR-ZnEDTA, 490 nm
Sr in milk powder
100 4.6
1 mol l 1 KNO3, pH step gradient from 4.0 to 1.1
Al, Fe
PCV, 580 nm
Al in seawater
Aurin tricarboxylic acid (ATA)
8.8 mm PS-DVB, 12 nm, 470 m2 g 1, 48 mmol g 1 8.8 mm PS-DVB, 12 nm, 470 m2 g 1, 35 mmol g 1 7 mm PS-DVB, 10 nm, 150 mmol g 1
250 4.6
1 mol l 1 KNO3, 50 mmol l 1 acetic acid, pH step gradient from 4.5 to 1.0
Mn, Co, Ni, Cd, Pb, Cu, Ga, In
PAR, 520 nm
Pb, Cd, Cu in highly mineralized water
(C) Dynamically coated Dipicolinic acid
5 mm PS-DVB
100 4.6
Fe, Th, V, Bi, U, Hf, Zr
7 mm PS-DVB, 10 nm
300 4.6
Arsenazo III, 654 nm or PCV, 585 nm PAR, 520 nm
U, Bi, Zr in sediment. U in mineral water, seawater
4-Chlorodipicolinic acid
Pb, Cd, Cu in rice flour
o-Cresolphtalein complexone (CPC)
5 mm porous graphitic carbon, 25 nm, 120 m2 g 1 5 mm PS-DVB, 10 nm
100 4.6
1 mol l 1 KNO3, 0.5 mol l 1 HNO3, 0.1 mmol l 1 dipicolinic acid 1 mol l 1 KNO3, 0.25 mmol l 1 chlorodipicolinic acid, pH 1.5 45–58% MeOH, 0.4 mmol l 1 CPC, pH 10.0–10.5
Mg, Ca
Direct detection, 600 nm
Mg, Ca in seawater, saline lake water
Ba, Sr, Ca, Mg
CPC, 575 nm
Sr in Antarctic saline lake water
Methyl thymol Blue (MTB)
5 mm PS-DVB, 10 nm
150 4.1
0.5 mol l 1 KNO3, 0.2 mmol l 1 CPC, 20 mmol l 1 borate, pH 9.5 0.5 mol l 1 KNO3, 0.2 mmol l 1 MTB, pH 1.2
Mg, Mn, Zn, Cd, Pb
Zn in industrial gypsum
N-n-dodecyliminodiacetic acid
5 mm ODS-silica, 14 nm, 300 m2 g 1
150 4.0
75 mmol l 1 tartrate buffer, pH 5.5
Ca, Sr, Mg, Ba
Buffering at pH 5.9 with NH4OAc, 600 nm CPC, 575 nm or HQS-MgEDTA, fluorimetric 405/525 nm
o-Cresolphtalein complexone Chrome Azurol S
150 4.1
Mn, Co, Ni, Zn, Cu, Pb, Cd, Al, La, Lu, Fe, U
Mg, Ca in coastal seawater
Postcolumn reagents: PAR – 4-(2-pyridylazo)resorcinol; o-CPC – o-cresolphthalein complexone; CAS – chrome azurol S; PCV – pyrocatechol violet; HQS – 8-hydroxyquinoline-5-sulfonic acid.
480 ION EXCHANGE / Chelation Ion Chromatography 0.018
Mn
Cu 0.016 0.004
0.014
Cd
Absorbance
Absorbance
0.012 0.01 0.008
Zn Pb 0.006
Cu
0.004
Ni
0.002
Pb Cd
0 0
2
4
6
8
10
12
Retention time (min)
5
10
15
20 min
Figure 9 Chromatogram showing the preconcentration and separation of trace metals in 1 mol l 1 Na2SO4 sample spiked with 10 mg l 1 Mn2 þ , 25 mg l 1 Zn2 þ , and 0.1 mg l 1 Cd2 þ , Pb2 þ , Ni2 þ , and Cu2 þ . Sample volume was 10 ml adjusted to pH 6. Column, 100 mm 4.6 mm, packed with Xylenol Orange impregnated 8 mm PLRPS resin. (Reprinted with permission from Challenger OJ, Hill SJ, and Jones P (1993) Separation and determination of trace metals in concentrated salt solutions using chelation ion chromatography. Journal of Chromatography 639: 197–205; & Elsevier.)
out that lower limits of detection can be achieved using CIC compared to conventional IC separations. This is because many IC methods use strong complexing acids at relatively high concentrations in the eluents, which compete with colorimetric reagents such as PAR, reducing optimum sensitivity. In contrast, most CIC methods use either noncomplexing eluents or eluents containing relatively weak complexing acids. ——————————————————————9 Figure 11 Isocratic separation of Bi(III), U(VI), and Zr(IV) in GBW07311 sediment sample at pH 0 on the 15 cm PLRP-S column dynamically modified with 0.1 mmol l 1 dipicolinic acid. Injection volume used was 500 ml with detection at 654 nm with Arsenazo III postcolumn reaction. (Reproduced with permission from the Ph.D. dissertation of James Cowan (2002) The development and study of chelating substrates for the separation of metal ions in complex sample matrices. University of Plymouth, Figure 5.15, p. 218.)
0.001 9
0.001 4 Absorbance (AU)
0
Figure 10 The separation of Cd(II), Pb(II), and Cu(II) from matrix interferences in the certified rice flour GBW08502 on the 100 4.6 mm PRP-1 7 mm PS-DVB column. Eluent: 1 mol l 1 potassium nitrate, 30 mmol l 1 nitric acid, and 0.25 mmol l 1 chlorodipicolinic acid. Detection: PAR at 520 nm. (Reprinted with permission from Shaw MJ, Jones P, and Nesterenko PN (2002) Dynamic chelation ion chromatography of transition and heavy metals using a mobile phase containing 4-chlorodipicolinic acid. Journal of Chromatography A 953: 141–150; & Elsevier.)
0.000 9 U(VI)
Zr(IV)
0.000 4 Bi(III)
−0.000 1 0
5
10
15
Time (min)
20
25
ION EXCHANGE / Isolation of Biopolymers 481
Applications In this section, examples of applications involving high-efficiency CIC columns have been selected to demonstrate the wide range of complex samples analyzed using this technique (Table 2). The table is divided into three sections in terms of how the chelating substrate is formed, namely, bonded, impregnated, and dynamically coated. The insensitivity to ionic strength and special selectivity of these columns can be especially seen in the ability to analyze highly saline samples and digested sediment and mineral samples containing relatively enormous amounts of matrix metals. Figures 7–11 have been selected to illustrate some of the actual chromatographic separations obtained under these conditions. See also: Ion Exchange: Overview; Principles; Ion Chromatography Instrumentation; Ion Chromatography Applications; Isotope Separation. Liquid Chromatography: Ion Pair.
Further Reading Fritz JS and Gjerde DT (2000) Ion Chromatography, 3rd edn. Heidelberg and New York: Huethig.
Haddad PR and Jackson PE (1990) Ion Chromatography, Principles and Applications. Amsterdam: Elsevier. Jones P and Nesterenko PN (1997) High performance chelation ion chromatography: a new dimension in the separation and determination of trace metals. Journal of Chromatography 789: 413–435. Paull B and Haddad PR (1999) Chelation ion chromatography of trace metal ions using metallochromic ligands. Trends in Analytical Chemistry 18: 107–114. Paull B and Jones P (1996) A comparative study of the metal selective properties of chelating dye impregnated resins for the IC separation of trace metals. Chromatographia 42: 528–538. Sarzanini C (1999) Liquid chromatography: a tool for the analysis of metal species. Journal of Chromatography A 850: 213–228. Sarzanini C and Mentasi E (1997) Determination and speciation of metals by liquid chromatography. Journal of Chromatography A 789: 301–321. Timerbaev AR and Bonn GK (1997) Complexation ion chromatography – an overview of developments and trends in trace metal analysis. Journal of Chromatography 640: 195–206. Weiss J (1995) Ion Chromatography. Weinheim: VCH.
Isolation of Biopolymers P R Levison, Pall Europe Ltd., Portsmouth, UK & 2005, Elsevier Ltd. All Rights Reserved.
Introduction Biopolymers, the so-called ‘building blocks of nature’, are found in all living matter be it of animal, microbial, or vegetable origin. Biopolymers include proteins (polymers of amino acids), genetic material (polymers of nucleic acids), glycoforms (carbohydrates and glycosylated molecules), metabolites, and other structural molecules. By their very sequence and chemical composition many biopolymers have an electrical charge and can therefore be fractionated by ion-exchange processes. This article briefly reviews the principles underlying biopolymer purification by ion exchange and addresses some of the process issues associated with their purification.
Ion-Exchange Chemistries Ion-exchange chromatography is routinely used for the separation of biopolymers at laboratory scale
through to process scale. Ion exchangers are available from a number of manufacturers each produced using their proprietary synthesis methods. For biopolymer separations there are typically four ionic functionalities available. In the case of anion exchange, there are so-called weak anion exchangers (WAX) often based on weak amines including aminoethyl, diethylaminoethyl, guanidoethyl, p-aminobenzyl, ECTEOLA, and polyethyleneimine, and strong anion exchangers often based on a quaternary amine. Examples of each functionality are represented in Figure 1. The choice of anion exchanger is process dependent and for high pH applications, i.e., pH48.5 the more ionized grade, e.g., N,N,N-trimethyl-2-hydroxypropyl amine, would be recommended. In the case of cation exchange there are so-called weak cation exchangers often based on a carboxylic acid and strong cation exchangers often based on a sulfonic acid. Examples of each functionality are represented in Figure 2. Choice of cation exchanger is also process dependent but for low pH applications, i.e., pHo4.5, the more ionized grades, e.g., sufoxyethyl or sulfopropyl, would be recommended.