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Chapter 8 Miscellaneous Separation Methods
223
Chapter 8 Miscellaneous Separation Methods 8.1
INTRODUCTION
In addition to the mainstream separation methods discussed thus far, we will also consider a number of alternative approaches which can be used for the separation of inorganic ions and carboxylic acids. In the strict sense, none of these methods can be defined accurately as IC, yet the fact that they can be employed for the same solutes as those normally encountered in IC suggests that a brief discussion is merited. The purpose of this discussion will be to indicate the operating principles of each approach and to provide some representative chromatograms in order to facilitate comparison with the ion-exchange, ion-interaction and ion-exclusion methods discussed in Chapters 2-7. Fig. 8.1 provides an overview of the separation methods which will be considered.
Reversed-
- phase
HPLC
Miscellaneous separation methods
-
Chelating stationary phases
Coordination compounds
i -r
Organometallics Carboxylic acids (Ion-suppression) Chemically bound ligands
L Crown ether stationary phases Anions
chromatography
Cations
Fig. 8.2 Overview of miscellaneous separation methods.
Chapter 8
224
8.2
REVERSED-PHASE LIQUID CHROMATOGRAPHY
Ionic or partially ionized solutes are generally retained only weakly on conventional c18 HPLC stationary phases. We have already seen in Chapter 6 that retention of these solutes can be increased through the use of an ion-interaction reagent added to the eluent. In this Section, we now turn to the use of non-polar stationary phases for the separation of coordination compounds, organometallics and carboxylic acids.
Coordination compounds
8.2.1
A convenient and frequently used method for the determination of metal ions is to first complex the metal ions with a suitable ligand, and to then separate the resultant coordination compounds by conventional reversed-phase or normal-phase HPLC. The complexes formed are often uncharged and this permits separation to be achieved on Cis or silica stationary phases. Several comprehensive reviews on HPLC of coordination compounds are available 11-61, from which it can be seen that there are a number of desirable properties of both the ligand and the chelate. These include [3-51:
(i) (ii)
(iii) (iv)
(v)
The ligand should form neutral complexes with a large number of metals, using relatively simple preparation methods. The complexes formed should be coordination saturated, since this gives the greatest probability of separation of complexes formed from different metals. Moreover, the ligand should not be too large, so that specific properties of the central metal atom are retained in the coordination complex. The donor atoms in the ligand should have low total electronegativity to minimize adsorption effects on silica-based reversed-phases. Preferred donor atoms are N-. 0- and S-. Separation selectivity increases when ligand substituents do not have large induction or steric effects, and also when electronegative atoms exist in close proximity to the chelate ring. The complexes should have high stability, good detectability and high solubility in non-polar organic solvents.
Many ligands have found application in HPLC separations of metal chelates. These ligands include dithiocarbamates (71, 8-hydroxyquinoline [8, 91, P-diketones [lo]. 4-(2pyridylaz0)-naphthol [ 11],4-(2-pyridylazo)-resinol [ 121, dialkyldithiophosphates[ 13, 141, xanthates [ 151, 2.3-diaminonaphthalene [16], pyrazolones [17] and hydrazones [18. 191. No single ligand is suitable for all metal ions and typically only a few metals are determined in a single chromatographic separation. In most cases, water-insoluble chelates are formed and these must be extracted into a suitable organic solvent, prior to the chromatographic separation step. This sometimes involves extraction with solvents which cannot be injected directly into a reversed-phase HPLC system, so that evaporation and redissolution become necessary. Alternatively, complexes can be formed in-situ by injecting metal ions into a mobile phase which contains the ligand and
Miscellaneous Separation Methods
225
an appropriate buffer [20] or through the use of solid-phase reaction on a suitable precolumn 1141. The stability of the metal complexes is also of great importance because these complexes are generally injected at very low concentrations and are therefore prone to dissociation as they traverse the chromatographic system. This is particularly true of complexes which may undergo ligand-exchange reactions at the surfaces of metallic chromatographic components, such as the injector, interconnecting tubing and the inlet and outlet frits in the column. Kinetic stability is of more importance than thermodynamic stability, since kinetically inert complexes are more likely to pass intact through the chromatographic system. The large volume of literature on HPLC of metal chelates precludes a comprehensive discussion of this topic. Table 8.1 provides a selected listing of some applications of HPLC of metal chelates. To illustrate the utility of this technique, we will focus attention only on the use of dithiocarbamate ligands, since these reflect most of the trends which exist for other ligands. Dithiocarbamate complexes Alkyldithiocarbamate ligands form complexes with a very wide range of metal ions and therefore offer the potential for separation of a larger number of metal ions than any other ligand. Diethyldithiocarbamate (DEDTC) complexes have been studied extensively and Fig. 8.2 shows a typical separation of metal-DEDTC complexes achieved on a reversed-phase column with a ternary eluent comprising water, methanol and acetonitrile [7]. Two important characteristics are evident from this chromatogram. First, there is a peak due to the ligand oxidation product, bis(diethy1thio-carbamy1)disulfide (usually referred to as disulfiram), which results either from excess ligand in the extracting solution, or from ligand produced by dissociation of labile complexes. Second, the peak shape for Pb(I1) is very poor, due to the low kinetic stability of this complex and the resultant likelihood of dissociation or ligand-exchange reactions. This behaviour occurs with other unstable complexes, such as those of Cd(II), Fe(II1) and Zn(II), and is especially evident when columns with stainless-steel frits are used. The porous nature and high surface areas of these frits provide ideal sites for ligand-exchange reactions to occur between the injected metal complexes and metal ions produced from the oxidation of stainless steel components, especially nickel. These reactions can be minimized either by using columns without porous metallic frits, such as radial compression columns [7], by deactivating the frits with an organosilane [25],or by addition of EDTA to the mobile phase 171. Formation of the chelate It should be stressed that it is the metal chelates which are separated in the above example, rather than the metal ions themselves. Therefore, these chelates must be formed before the sample reaches the analytical column. Chelate formation is usually achieved by buffering the sample and then extracting with a solution of the ligand in a suitable organic solvent. This process can be automated using the apparatus shown in Fig. 8.3, wherein the sample is added to a solution stream of ligand (diethyldithiocarbamate) in acetonitrile [26]. The mixture is then passed, in turn, to a heated
TABLE 8.1 TYPICAL DETERMINATIONS OF METAL CHELATES BY REVERSED-PHASEHPLC Solute(s)
Liganda
Stationary phase
Al(III), In(1LI) As(III), Sb(III), Bi(II1) Cd(II), PWII), Ni(II), Co(III), Hg(I1). Cr(III), Se(IV), Cu(II), Te(W Cd(II), Co(II), Pb(II), Ni(II), Wrr)
PMBP DEDTP DEDTC BHEDC BHEDC Oxine HFAA Oxine DEDTC
sew) Ti(IV), FeOII), U(VI), V(V)
DAN DAPMP, DAPMT
Detection modec
Detection limitd
Ref
Shim-Pack CLC-ODs ACN-MeOH Hypersil ODS 10 mM DEDTP in ACN Waters C18 Rad-Pak 40:35:25 MeOH-ACNwater
S (290 nm) S (280 nm) S (254 nm)
21-121 ng 2 ng 0.5
17 14 7
O.lmM HEDC in 40:a MeOH-water with 0.1 m~ Zn2+ SUPelCO cl8 25 mM TEA acetate Cg Reversed-phase 1 mM oxine in borate buffer (pH 9) 100% cHZC12 Silica c18 reversed-phase 10 mM oxine, ACN an^ acetate buffer (pH 6) 0.05% DEDTC in waterHypersil ODs MeOH-cHC13 60:40MeOH-water pBondapak C18 Polymer Labs PLRP-S 10%-40% ACN-water gradients
S (300 nm)
7-53 ppb
21
S (255 nm) S (254 nm)
5 PPb
22 23
DCP S (400 nm)
n.s. 100 PI--
24 8
S (350 nm)
0.5 ppm
20
S (254 nm), Fluor S (340 nm)
10 ppb n.s.
16
pBondapak cl8
Mobile phaseb
n.s.
19
PMBP = l-phenyl-3-methyl-4-benzoyl-5-pyrazolone, DEDTP = diethyldithiophosphate, DEDTC = diethyldithiocarbamate, BHEDC = bis(2hydroxyethy1)dithiocarbamate. Oxine = 8-hydroxyquinoline, HFAA = hexafluoroacetylacetone, DAN = 2,3 diaminonaphthalene, DAPMP = 2,6diacetylpyridine bis (N-methylenepyridiniohydrazone),DAPMT = 2,kliacetylpyridine bis(N-methylene-N,N,N,-trimethylammoniohydnuone). MeOH = methanol, ACN = acetonitrile, TEA = tetraethylarmnonium. S = spectrophotometry, Fluor = fluorhetry, DCP = direct current plasma atomic emission spectrometry. n.s = not stated. a
5
00
Miscellaneous Separation Methods
227
I
0.005 Ahsorhancc
Fig. 8.2 Separation of a mixture of diethyldithiocarbamate complexes by reversed-phase HPLC. The mobile phase comprised 40:35:25 methanol-acetonitrile-waterand a Waters c18 Rad-Pak was used as the column. The flow-rate was 2.0 rnl/rnin and the detection wavelength was 254 nm. Peak identities: A-disulfiram, B-Cd(II), C-Pb(II), D-Ni(II), E-Co(III), F-Cr(III), G-Se(IV), HCu(II), I-Hg(II), J-Te(1V). Reprinted from [7] with permission.
reaction coil, a bubble capture device and to the sampling loop of an auto-injector. Excess ligand in the solution is removed by an anion-exchange guard column placed before the c18 analytical column. An alternative method for the formation of the dithiocarbamate complexes is to inject the metal ions into an eluent which contains the ligand. On-column complex formation provides a potentially quick and easy method for multi-element identification and determination. When DEDTC is added to the mobile phase, Cd(II), Pb(II), Co(II), Hg(I1) and Cu(I1) can be separated [20, 271. The chief problem encountered with oncolumn complexation is the high background detector signal produced by the presence of the ligand in the mobile phase. This requires that a selective wavelength be used in the case of spectrophotometric detection, or alternatively, amperometric detection must be used [28]. A further problem is the poor solubility of many dithiocarbamate complexes in typical mobile phases for reversed-phase HPLC, but this may be overcome either by the addition of a small amount of chloroform to the mobile phase [20], or through the use of a ligand which forms water-soluble complexes [21]. An example of the latter approach is the use of bis(2-hydroxyethy1)dithiocarbamate (BHEDTC), in which the
228
Ckpter 8
prewure
DEDTC in acetonitrile Sample
Fig. 8.3 Schematic diagram of apparatus for automated pre-column formation of diethyldithiocarbamate (DEDTC)complexes, prior to separation by reversed-phase HPLC. Reprinted from [26] with permission.
hydroxy groups on the ligand cause metal complexes to be water-soluble at low concentrations [22]. Fig. 8.4 compares chromatograms obtained using pre-column (Fig. 8 4 a ) ) and on-column (Fig. 8 4 b ) ) complex formation with BHEDTC. 8.2.2 Organometallic compounds
One of the factors which limits the applicability of HPLC analysis of metal chelates is the necessity to form the chelate itself. This limitation does not exist for many of the organometallic species which are amenable to chromatographic analysis, since these species often occur in a wide range of samples. The more important organometallic species which can be analyzed by HPLC include alkyllead, alkylmercury, alkylarsenic and alkyltin compounds. Of these, the organoarsenic species are the most widely studied. Monomethylarsonate, dimethylarsinate and phenylarsonate (as well as the inorganic ions arsenate and arsenite) are formed by the action of many common yeasts, fungi and bacteria on arsenic present in soils. The high toxicities of these compounds necessitate their accurate determination, especially in water samples. Separation can be accomplished by reved-phase chromatography [29], as well as by anion-exchange [30, 311 or ion-interaction 132, 331 methods. Organomercury compounds, such as methylmercury and ethylmercury, have also been separated by reversed-phase HPLC [34]. In most of the above examples, atomic spectroscopic detection methods have been employed Table 8.2 lists some further applications of the determination of organometallic species by reversed-phase HPLC, and Fig 8.5 shows chromatograms obtained for organomercury and organotin compounds.
% c;.
TABLE 8.2 TYPICALDETERMINATIONS OF ORGANOMETMC SPECIES BY REVERSED-PHASE HPLC Solute(s)
Sample
Stationary phase
Eluent
Detection modes
Detection limit
Alkyl Hg compounds Alkyl Pb compounds Ethyl Sn compounds Fe, Mo carbonyl complexes Methylmucury, ethylmacury Methyl Sn compounds Organo As compounds Organo Hg compounds
n.s. PeIml
ICP ICP
Reactionmixtures
HyperSd c18 H m f i c18 Spherisorb S5W ODs ZarbaXCg
ICP
n.s. 35 llppb 35 50-100pg 36 1 PPb 35
Tuna
Waters pic0 Tag
1:2 EtOH-0.05 M NaBr 75% EtOH-water 7030 acetone-pentane 7030 EtOH-water or J3OH-water gradients 6omManrmoniumacetate, 0.005% 2-mer~aptOethan01 6040 acctone-pentane 100% MeOH 4050 MeOH-water + 0.06 M ~ O A + C 0.01% 2-mexaptOethanol 90:lO MeOH-water 7525 MeOH-water
ICP-MS
1 PPb
34
Hydride AAS GFAAS
2-2opg 5 ng 2 PPb
37 38 39
Tetraphenyl Pb Transition metal cluster complexes
n.s. n.s.
480pg n.s.
40 41
Water
n.s. n.s. Fish
FtI
DP Amp ZeemanAAS
spec
I B = inductively coupled plasma atomic emission spe!cmehy, Hydxide AAS = hydxide generation atomic absorption spectrometry, GFAAS = graphite furnace atomic absorption specaometry,RI = refractive index, MS = mass spectrometry,DP Amp = differential pulse. ampemmefry, Spec = specfrophotomefry. n.s. = not stated. a
Ref
is
E
8e
I
230
Chapter8 CO2'
0
L
8
l i m e (minl (a)
12
-
0
5 10 lime (minl (bl
15
Fig. 8.4 Comparison of chromatograms obtained using (a) pre-column and (b) on-column formation of bis(2-hydroxyethy1)dithiocarbamate (BHEDTC) complexes. (a) The pre-formed BHEDTC complexes were injected onto a Supelcosil c18 column using an eluent comprising 4060 methanol-water containing 25 mM triethylammonium acetate. Detection was by spectrophotometry at 255 nm. Reprinted from [22]with permission. (b) Metal ions were injected onto a Waters WBondapak cl8 column using an eluent comprising 4050 methanol-water containing 0.1 mM BHEDTC and 0.1 mM a*+. Detection was by spectrophotometry at 300 nm. Reprinted from [21] with permission.
8.2.3
Carboxylic acids (Ion-suppression)
A further strategy which can be employed in the determination of ionizable solutes by reversed-phase HPLC is to suppress the ionization of these solutes by adding a buffer of appropriate pH to the eluent. Retention of the solutes on non-polar stationary phases is therefore increased and separation can then be accomplished. Acidic buffers are used for the separation of weak acids, whilst alkaline buffers are used for the separation of weak bases. This method, often described as "ion-suppression",is generally considered to be applicable only to those weak acids and bases for which the ionization can be suppressed using buffers having pH values in the range 3 - 8 [42]. The reason for this is that C1g stationary phases are unstable outside this pH range. Although this is undoubtedly a major limitation, many weak acids may separated on C18 columns, as demonstrated by Skelly [43]. Restrictions in eluent pH do not apply to the use of non-
23 1
Miscellaneous Separation Methods
MeSn( EtSnCI: Me2SnCI; Me3SnCI MebSr
#Hg+
I I
1
1
1
1
.L 1
1
1
1
0 2 4 6 8 10 12 14 16 Time (min) (a)
1 1 1 T
0 1 2 3 Time (min)
(bl
-
0 1 2 3 1 5 Time (min) (C 1
Fig. 8.5 Separation of (a) organomercury, (b) methyltin and (c) ethyltin compounds by reversedphase HPLC. (a) A Spherisorb ODS column was used with an eluent comprising 40% methanolwater, 0.06 M NH4OAc (pH 5.5) and 0.01% 2-mercaptoethanol. Detection was by differential pulse amperometry. MeHg+ = methylmercury, EtHg+ = ethylmercury, @Hg+= phenylmercury. Reprinted from [39] with permission. (b) A Spherisorb ODs column was used with 60:40 acetonepentane as the eluent and hydride generation atomic absorption spectrometricdetection. Reprinted from [37] with permission. (c) Conditions as for (b) except that a 70:30 acetone-pentane eluent was used. Reprinted from [37] with permission.
polar polymeric stationary phases, so these materials can therefore be employed for the separation of a wider range of solutes using the ion-suppression technique than is possible with c18 stationary phases. The utility of ion-suppression on polymeric stationary phases can be appreciated by considering the separation of the homologous series of aliphatic carboxylic acids. Neither ion-exchange nor ion-exclusion chromatography yields a complete separation of these species. However, ion-suppression coupled with gradient elution and suppressed conductivity detection enables the separation of butyric through to stearic acid, as illustrated in Fig. 8.6. The gradient used involved an increase in the percentage of organic modifier in the eluent and a decrease in eluent pH. Carboxylic acids more hydrophilic than butyric acid were eluted as a single peak at the column void volume.
232
Chupter8
ityric
Lauric
Capric
I I
0
I
5
I
10
I
15
I
I
20 25 lime (min)
I
30
I
35
1
LO
I
45
Fig. 8.6 Gradient elution ion-suppression chromatogram of carboxylic acids, obtained on a polymeric reversed-phasecolumn. A Dionex MPIC-NS1 column was used with a gradient of 100% eluent A ( t 4 ) to 100% eluent B (t=20 min), with maintenance of eluent B after this time. Eluent A is 24% acetonitrile and 6%methanol in 0.03 mM HC1. Eluent B is 60% acetonitrile and 24% methanol in 0.05 mM HCl. Detection was by suppressed conductivity. The baseline conductance for a blank w e n t has been subtracted in the chromatogram shown. Reprinted from I441 with permission.
8.3
CHELATING STATIONARY PHASES
8.3.1 Chemically-bound ligands Metal ions may be separated on a stationary phase in which a suitable ligand is immobilized onto the stationary phase. Numerous chelating stationary phases have been synthesized using stryene-divinylbenzenepolymers or silica as the support material. In each case, the ligand is chemically bound to the support using an appropriate reaction, such as silylation reactions with silica. Some examples of the ligands which can be bound in this way include iminodiacetate (Chelex 100, Dow Chemical Company), propylene-diaminetetraacetate 1451. P-diketones (e.g. trifluoroacetoacetate [461), 8hydroxyquinoline [47], isothiuronium (481, hydroxamic acids [49], dithiocarbamates (501, phenylhydrazones [51] and dithizone [52]. Solute retention can be manipulated by varying the eluent pH or through the addition of a competing ligand to the eluent. The key factor in the success of the above materials as chromatographic stationary phases is the rate at which the metal-ligand complex is formed and dissociated. Slow rates will lead to poor peak shape in the chromatogram. An evaluation of the literature suggests that most ligands give unacceptably slow rates of reaction, so that chromatograms are typically characterized by very broad peaks. A recent study has compared 9 different chelating stationary phases and has shown that some useful separations can be achieved on dithizone silica gels, as illustrated in Fig. 8.7. It is interesting to note that the same metal ions shown in Fig. 8.7 can be well separated using either ion-exchange or ion-interaction chromatography (e.g. see Figs. 4.17 and 6.3).
Miscellaneous Separation Methotlr
233
Pb2
1 1 1 1 1
0
10 20 Time (mid
Separation of metal ions on a stationary phase formed by binding dithizone functionalities to silica gel. The eluent was 15 mM tartrate at pH 4.0. Detection was by specaophotometry after post-column reaction with 4-(2-pyridylazo)-resinol. Reprinted from [52] with permission.
Fig. 8.7
8.3.2 Crown ether stationary phases
Considerable effort has been expended over recent years on the development of stationary phases in which a crown ether is chemically bound to a suitable support, such as silica or an appropriate polymer. Crown ethers (or cyclic polyethers) are cyclic compounds which possess an inner cavity, generally consisting of oxygen atoms linked by ethylene bridges. Fig. 8.8 shows two examples of these compounds. Crown ethers are non-systematically named according to the total number of atoms in the ring, the number of oxygen atoms and any substituents on the ring. Thus the fiist crown ether in Fig. 8.8 is called 18-crown-6 (total of 18 atoms in the ring, with 6 oxygen atoms), whilst the second is called benzo-18-crown-6 (to indicate the benzene ring substituent). Cryptands are related compounds having two interconnected rings which produce a three dimensional cavity. The final compound in Fig. 8.8 is one such cryptand.
Synthesis of stationary phases Crown ether stationary phases may be synthesized in three ways. The simplest approach is to impregnate a silica particle with a solution of a suitable crown ether in formic acid, followed by cross-linking with formaldehyde [53], and is illustrated schematically for dibenzo-18-crown-6 in Fig. 8.9(a). The resultant material is
234
Chapter 8
18-crown-6
Benzo- 18-crow n-6
O
d
Cryptand-n-decyl-2.2.2 Fig. 8.8 Structures of some cyclic polyethers
Fig. 8.9 Structures of some crown ether stationary phases. (a) The crown ether is coated onto silica and then polymerized. (b) Typical bonding arrangement of a crown ether onto silica. (c) Typical bonding arrangement of a crown ether onto a resin. Reprinted from [53, 581 with permission.
Miscellaneouy Separation Methods
235
stable mechanically and is resistant to hydrolysis. Alternatively, the crown ether can be chemically bound to silica by silyl ether linkages using conventional silylation reactions. An example of the resultant stationary phase structure is shown in Fig. 8.9(b) for benzo18-crown-6. Finally, similar bonding reactions can be performed using resins as the support and a typical stationary phase structure is depicted in Fig. 8.9(c), again using benzo- 18-crown-6 as the ligand. Chromatographic properties The chromatographic utility of crown ether stationary phases rests in their ability to complex cations of a specified size. As the size of the cavity in the crown ether is altered, so too does the selectivity of the stationary phase. For example, Li+, Na+ and K+ are bound preferentially to 12-crown-4, 15-crown-5 and 18-crown-6 stationary phases, respectively, by virtue of the increasing cavity size. However, it is not essential for the solute cation to reside within the cavity since layered structures in which the solute is located between two cavities can also be formed [54]. The bound metal ion imparts a positive charge to the crown ether and this is balanced by a suitable counter anion. The binding of metal ions on crown ether stationary phases is .dependent on the following factors: (i) The size of the cation. (ii) The nature of the associated anion. (iii) The organic modifier content of the eluent. The influence of cation size has been discussed above. The most common elution trend for alkali metal ions on benzo-18-crown-6 stationary phases (which show preference for K+) is for K+ to be eluted last, with the remainder being eluted in order of size. That is, retention usually follows the sequence:
The nature of the associated anion can sometimes exert an influence on retention which dominates all other effects. "Soft" (polarizable) anions such I- and SCN- are more strongly associated with the bound cation than are "hard" anions, such as S04*-and C1-. It has been demonstrated that, when a mixture of anions and cations is injected, the most strongly retained species will be the preferred cation associated with the softest anion [55]. For example, injection of a mixture of Li+, K+,C1-, Br- onto a dibenzo-18-crown6 stationary phase gives four peaks corresponding to LiCI, KCI, LiBr and KBr [56]. The first eluted peak (LiCI) contains the least preferred cation (Li+) and the hardest anion (Cl-), whilst the last eluted peak (KBr) contains the preferred cation (K+) and the softest anion (Br-). Water alone can be used as an eluent in this form of chromatography, but the separations which are obtained are often relatively poor. The reason for this is that water complexes the solute cations and competes effectively with the crown ether. Addition of an organic modifier, such as methanol, to the eluent lowers its polarity and
236
Chapter8
5Br
KBI
Liar
KBr
1
0
I
5
1
I
1
10 15 20 Time (min) (a 1
I
25
-
0 10 20 30 LO Time (min) (b)
Fig. 8.10 Separation of (a) cations and (b) anions on crown ether stationary phases. (a) A paly(benzo-l5-crown-5)-modifiedsilica was used as stationary phase with water as eluent. Detection was by conductivity. Reprinted from [64]with permission. (b) A benzo-18-crown-6 modified silica stationary phase was used with water as eluent. Conductivity detection was used. Reprinted from [65] with permission.
decreases complexation with the solutes. This, in turn, results in increased solute retention and therefore improved separation. However, detection is more simple with a water eluent than with aqueous-organic mixtures, so the former eluent is preferable.
Applications Crown ether stationary phases can be used in two ways. First, a series of alkali metal salts with a common anion can be separated. These salts will be eluted with the cations following the sequence determined by the preference of the particular crown ether used 153, 57-62]. An example of such a separation on silica coated with poly(benzo-t5-crown-5) is shown in Fig. 8.lqa). We note that the cations must be present as their Br- salts for the separation to be reproducible. An alternative application is the separation of anions, using the cation bound to the crown ether as a site for selective retention of anions. The salts will be eluted in the sequence of reducing hardness of the anions [53,56, 57, 59, 60,62, 631. A typical separation by this method is given in Fig. g.lO(b). We again note that the separation is dependent on each anion being present as the K+ salt. If other cations are present, the elution order could vary.
MiscellaneousSeparation Methods
237
The separations shown in Fig. 8.10 are of limited practical applicability because of the need to specify a particular counter-anion or counter-cation for the solute ions. It has been suggested that a sample could be converted to the correct form by passage through a suitable ion-exchange column prior to analysis [54], but no results using this approach have been reported. A more attractive alternative is to use a sufficiently high concentration of the desired counter-cation or anion in the eluent so that the sample counter-ions do not influence solute retention. This approach has been reported for anion determinations using a stationary phase comprising the macrocycle cryptand ndecyl 2.2.2, which has the structure shown in Fig. 8.8, coated onto reversed-phase supports with alkali metal hydroxides as eluents [66, 671. The cryptand is hydrophobically bound to the support in the same manner as used in "permanentcoating" ion-interaction chromatography (see Section 6.3.2). Excellent chromatographic efficiencies were obtained and solute retention was not dependent on the cations present in the sample.
Gradient elution on macrocyclic stationary phases The above-mentioned studies with the cryptand n-decyl 2.2.2 have opened a new possibility for gradient elution of anions. The cation bound to the macrocycle acts as an anion-exchange site for solute anions. The number of bound cations, and hence the anion-exchange capacity of the column, is dependent on the identity of the alkali metal hydroxide used as eluent. The highest anion-exchange capacity is produced with KOH eluents (because of the preferential binding of K+ to the macrocycle), whereas the smallest capacity results when LiOH is used as the eluent. Thus, a gradient in which the eluent is changed from, for example, 30 mM NaOH to 30 mM LiOH will therefore result in a progressive decrease in column ion-exchange capacity. The change in baseline conductivity in such a system will be minimal, especially if a suppressor is employed. Fig. 8.1 1 shows a chromatogram obtained using this method, which has been called "gradient capacity IC" [67]. This method has considerable promise, especially in view of the ease with which the stationary phase can be prepared. 8.4 8.4.1
MICELLE EXCLUSION CHROMATOGRAPHY
Introduction
Many surfactant molecules form micelles in concentrated solution. Micellar chromatography [68, 691 utilizes micellar eluents as a means of improvement of the chromatographic selectivity. We have seen earlier (Section 6.3.2, Fig. 6.4) that micellar eluents can be employed,in ion-interaction chromatography of inorganic anions. The chromatograms obtained show good chromatographic efficiency, but the elution order of solutes (and hence the selectivity of the method) is the same as that for conventional ionexchange chromatography. This can be explained by the fact that in this particular case, the surfactant was strongly adsorbed onto the reversed-phase column which was used, so that ion-exchange was the dominant retention mechanism. On this basis, use of a different type of stationary phase, such as a size-exclusion material, could lead to changes in chromatographic selectivity. Okada [70,711 has investigated this approach
238
Chapter 8
F-
p-
Acct, SC N-
I r
0
I
10
1
20 Time (min)
30 1
LO I
Fig. 8.11 Gradient elution on a macrocyclic stationary phase using variation of the ion-exchange
capacity of the column. A Dionex MPIC-NS1 column was used after conditioning with cryptand-ndecyl-2.2.2. The eluent was a linear gradient (over 20 min) from 30 mM NaOH to 30 mM LiOH. Detection was by suppressed conductivity. Chromatogram courtesy of John D. Lamb. for the separation of inorganic anions and cations. The eluent consists of an aqueousorganic mixture containing a micellar solution of a surfactant of appropriate charge. The micelles in the eluent are excluded from part of the stationary phase, whilst the solute ions partition between the micelle and the bulk solvent in the eluent, and also between the stationary phase and the eluent, This method has been entitled "micelle exclusion chromatography" on the basis of this separation mechanism. 8.4.2
Micelle exclusion chromatography of anions
When anionic solutes are to be separated, the surfactant in the eluent should be positively charged. Hexadecyltrimethylammonium chloride (HTAC) and dodecyltrimethylammonium chloride (DTAC) are therefore suitable surfactants [71]. Use of a size-exclusion stationary phase means that the aggregated surfactant forming the micelle
Miscellaneous Separation Methodr
239 Mnll
L I I'
0
30
Time (rnin) (01
I
LO
r
0
I
10
I
20 lime (rnin)
I
30
1
LO
(bl
Fig. 8.12 Determination of (a) inorganic anions and (b) inorganic cations by micelle exclusion chromatography. (a) An Asahipak GC910H (poly(viny1 alcohol)) size-exclusion column was used with 0.01 M hexadecyltrimethylammonium chloride as eluent. Detection was by spectrophotometry at 210 nm. Reprinted from [70]with permission. (b) The same column as for (a) was used, with a 10 min gradient from 100%eluent A (commencing at t = 10 min) to 100%eluent B. Eluent A was 25 mM sodium dodecyl sulfate (SDS) and 80 m M a-hydroxyisobutyric acid at pH 4.0. Eluent B was 25 mM SDS and 60 mM tartaric acid at pH 4.0.Detection was by spectrophotometry after post-column reaction with 4-(2-pyridylazo)-resorcinol.Reprinted from [71] with permission.
can penetrate only partially into the pores of the stationary phase, whereas the monomeric surfactant can penetrate fully by virtue of its smaller size. The small solute anions can also penetrate into the pores and are therefore retained in this region of the stationary phase by ion-interaction chromatography. Solute anions will also partition between the bulk eluent and the surfactant micelles, and between the bulk eluent and the liquid inside the pores of the column. Retention is therefore based on three distinct processes. Fig. 8.1 2(a) shows a chromatogram obtained using HTAC as the surfactant. The elution order in Fig. 8.12(a) is different from that exhibited by conventional ionexchange or ion-interaction systems, particularly for I- and Br-. The retention order and
240
Chaprer 8
retention times observed in this system are dependent on a number of parameters, including: (i) (ii) (iii) (iv) (v)
The concentration of surfactant The nature of the surfactant used The nature of the surfactant counter-anion The Concentration of added salt The concentration of organic solvent in the eluent
All of these parameters also affect retention in ion-interaction chromatography, which supports the hypothesis that this process plays a significant role in the mechanism of micelle exclusion chromatography. However, the observed chromatographic selectivity shows that there are additional conmbutions to solute retention which are attributable to the partitioning processes described above.
8.4.3 Micelle exclusion chromatography of cations Micelle exclusion chromatography of cations requires an anionic surfactant, such as sodium dodecylsulfate (SDS),and a size-exclusion stationary phase [71]. Again, the surfactant micelles are partially excluded from the pores of the stationary phase, whereas monomeric surfactant and solute cations can penetrate fully. Retention occurs by the same mechanisms outlined above for anions and the retention times are dependent on the same parameters as listed earlier. As with ion-exchange and ion-interaction methods, further control over solute retention can be accomplished through the addition of a complexing agent, such as citrate, to the eluent. Moreover, gradient elution can be achieved by changing the nature of the complexing agent whilst keeping other eluent parameters constant. Fig. 8.12(b) shows a chromatogram obtained using this approach.
8.5
REFERENCES
1 2
Nickless G., J . Chromurogr.. 313 (1985) 129. Krull I.S., in Bemhard M.,BMkman F.E. and S d e r P.J. (Eds.), The fmponance of Chemical "Speciation" in Environmenral Processes, Springer-VerlagBerlin, Heidelberg,
3 4 5 6
Timerbaev A.R., Petrukhin O.M.and Zolotov Yu.A., Fres. 2. Anal. Chem., 327 (1987) 87. Steinbrech B., J. Liq. Chromurogr., 10 (1987) 1. OLaughlin J.W.. J . Liq. Chromurogr.,7 (1984) 127. Machnald J.C., in MacDonald J.C. (Ed.) Inorganic Chromatographic Analysis, WileyInterscience, 1985, p. 285. Hutchins S.R., Haddad P.R. and Dilli S., J. Chromufogr.,252 (1982) 185. Mooney J.P., Meaney M., Smyth M.R., Leonard R.G. and Wallace G.G., Analyst
1986, p. 579.
7 8 9 10 11
12
(London), 112 (1987) 1555.
Heisz O., GfT Fuchz. Lob., 29 (1985) 113. Gierira R.C. and Can P.W., J. Chromurogr.Sci., 20 (1982) 461. Schwedt G. and Rudde R.,Chromographia, 15 (1982) 527. Roston D.A., Anal. Chem., 56 (1984) 241.
Miscellaneous Separation Methodr
13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50
51 52 53
241
Cardwell T.J. and Caridi D.. J. Chromatogr., 193 (1980) 53. Irth H., Brouwer E.. De Jong G.J., Brinkman U.A.T. and Frei R.W., J. Chromatogr.,439 (1988) 63. Eggers H. and Russel H.A.. Chromatographia, 17 (1983) 486. Wheeler G.L. and Lott P.F., Microchem. J., 19 (1974) 390. Tong A., Akarna Y. and Tanaka S., J. Chromatogr.478 (1989) 408. Casoli A., Mangia A. and Predieri G., Anal. Chem., 57 (1985) 561.. Main M.V. and Fritz J.S., Anal. Chem., 61 (1989) 1272. Smith R.M., Butt A.M. and Thakur A.. Analyst (London), 110 (1985) 35. Ge H. and Wallace G.G.. Anal. Chem., 60 (1988) 830. King J.N. and Fritz J.S.. Anal. Chem., 59 (1987) 703. Hoffman B.W. and Schwedt G., J. HRC & CC, 5 (1982) 439. Mazzo D.J..Elliott W.G., Uden P.C. and Barnes R.M., Appl. Spectrosc., 38 (1984) 585. Shih Y-T. and Carr P.W., Talanta, 28 (1981) 41 1. Bond A.M., Garrard W.N.C.. Heritage I.G., Majewski T.P.. Wallace G.G., McBumey M.J.P., Crosher E.T. and McLachlan L.S., Anal. Chem., 60 (1988) 1357. Smith R.M. and Yankey L.E., Analyst (London), 107 (1982) 744. Bond A.M., Knight R.W., Reust J.B.. Tucker D.J. and Wallace G.G., Anal. Chim Acta, 182 (1986) 47. Irgolic K.J., Stockton R.A.. Chakraborti D. and Beyer W., Spectrochim Acta, 38B (1983) 437. Fish R.H., Brinkman F.E. and Jewett K.L., Environ. Sci. Technol., 16 (1982) 174. Haswell S.J.. ONiell P. and Bancroft K.C.C.. Talanta, 32 (1985) 69. Lawrence K.L., Rice G.W. and Fassel V.A., Anal. Chem.. 56 (1984) 289. LaFreniere K.E., FasseI V.A., and Eckels D.E., Anal. Chem., 59 (1987) 879. Bushee D.S.. Analyst (London), 113 (1988) 1167. Cast C.H., Kraak J.C., Poppe H. and Maessen F.J.M.. J. Chromatogr.. 185 (1979) 549. Burns D.T., Glockling F. and Harriott M., J. Chromatogr.. 200 (1980) 305. Burns D.T.. Clockling F. and Haniott M..Analyst (London), 106 (1981) 921. Brinkman, F.E., Blair W.R., Jewett K.L. and Iverson W.P.. J. Chromatogr. Sci., 15 (1977) 493. MacCrehan W.A. and Durst R.A., Anal. Chem.. 50 (1978) 2108. Vickrey T.M.. Howell H.E. and Paradise M.T., Anal. Chem., 51 (1979) 1880. Enos C.T., Geoffroy G.L. and Risby T.H., J. Chromatogr. Sci.. 15 (1977) 83. Bidlingmeyer B.A.. J. Chromatogr. Sci., 18 (1980) 525. Skelly N.E., J. Chromatogr.,250 (1982) 134. Slingsby R.W., J. Chromatogr.. 371 (1986) 373. Moyers E.M. and Fritz J.S., AnaL Chem., 49 (1977) 418. DenBleyker K.T., Arbogast J.K. and Sweet T.R., Chromatographia,8 (1983) 449. Jezorek J.R and Freiser J.R., Anal. Chem., 5 1 (1979) 366. Koster G. and Schmuckler G., Anal. Chim.Acta, 38 (1967) 179. Phillips R.J. and Fritz J.S.. Anal. Chim Acta. 121 (1980) 225. Raja R.. Am. Lab., 14 (1982) 35. Simonzadeh N. and Schilt A.A., Talunta, 35 (1988) 187. Faltynski K.H. and Jezorek J.R.. Chromatographia,22 (1986) 5. Blasius E. and Janzen K.P., Israel J. Chem. 26 (1985) 25.
242 54
55
56 57 58 59 60 61 62 63
64 65 66 67 68 69 70 71
Chapter 8 Dasgupta P.K., in Tarter J.G. (Ed.), Ion Chromurogruphy, Marcel Dekker, Inc.. New York, NY, 1987, p. 191. Blasius E., Janzen K-P., Klein W., Nguyen V.B., Nguyen-Tien T., Pfeiffer R., Scholten G.,Simon H., Stockemer H. and Touissant A.. J . Chromutogr.. 201 (1980) 147. Igawa M., Saito K., Tsukamoto J. and Tanaka M., A d . Chem., 53 (1981) 1942. Lauth M. and Gramain P.,J. Liq. Chromutogr., 8 (1985) 2403. Iwachido T., Naito H., Samukawa F., Ishimaru K. and Toei K., Bull. Chem. SOC.Jap., 59 (1986) 1475. Igawa M., Ito I. and Tanaka M., Bumeki Kuguku, 29 (1980) 580. Nakajima M., Kimura K. and Shono T., Bull. Chem. SOC.Jup., 56 (1983) 3052. Nakajima M., Kimura K., Hayata E. and Shono T., J. Liq. Chromatogr., 7 (1984) 21 15. Blasius E., Janzen K-P.. Simon H. and Zender J., Fres. 2.A d . Chem., 320 (1985) 435. Igawa M., Saito K., Tanaka M. and Yamabe T., Bumeki Kuguku, 32 (1983) E137. Nakajima M., Kimura K. and Shono T., Anal. Chem., 55 (1983) 463. Lauth M. and Gramain P.,J. Chromutogr., 395 (1987) 153. Lamb J.D. and Drake P.A., J. Chromatogr.,482 (1989) 367. Lamb J.D., Drake P.A. and Wooley K.E., 2nd Int. lon Chromutogr. Forum, Boston, September (1989). Armstrong D.W. and Nome F., Anal. Chem., 53 (1981) 1662. Armstrong D.W. and Fendler J.H., Biochim. Biophys. Acta, 478 (1977) 74. Okada T., Anal. Chem., 60 (1988) 1511. Okada T., Anal. Chem., 60 (1988) 21 16.
Chapter 8 Miscellaneous Separation Methods 8.1
INTRODUCTION
In addition to the mainstream separation methods discussed thus far, we will also consider a number of alternative approaches which can be used for the separation of inorganic ions and carboxylic acids. In the strict sense, none of these methods can be defined accurately as IC, yet the fact that they can be employed for the same solutes as those normally encountered in IC suggests that a brief discussion is merited. The purpose of this discussion will be to indicate the operating principles of each approach and to provide some representative chromatograms in order to facilitate comparison with the ion-exchange, ion-interaction and ion-exclusion methods discussed in Chapters 2-7. Fig. 8.1 provides an overview of the separation methods which will be considered.
Reversed-
- phase
HPLC
Miscellaneous separation methods
-
Chelating stationary phases
Coordination compounds
i -r
Organometallics Carboxylic acids (Ion-suppression) Chemically bound ligands
L Crown ether stationary phases Anions
chromatography
Cations
Fig. 8.2 Overview of miscellaneous separation methods.
Chapter 8
224
8.2
REVERSED-PHASE LIQUID CHROMATOGRAPHY
Ionic or partially ionized solutes are generally retained only weakly on conventional c18 HPLC stationary phases. We have already seen in Chapter 6 that retention of these solutes can be increased through the use of an ion-interaction reagent added to the eluent. In this Section, we now turn to the use of non-polar stationary phases for the separation of coordination compounds, organometallics and carboxylic acids.
Coordination compounds
8.2.1
A convenient and frequently used method for the determination of metal ions is to first complex the metal ions with a suitable ligand, and to then separate the resultant coordination compounds by conventional reversed-phase or normal-phase HPLC. The complexes formed are often uncharged and this permits separation to be achieved on Cis or silica stationary phases. Several comprehensive reviews on HPLC of coordination compounds are available 11-61, from which it can be seen that there are a number of desirable properties of both the ligand and the chelate. These include [3-51:
(i) (ii)
(iii) (iv)
(v)
The ligand should form neutral complexes with a large number of metals, using relatively simple preparation methods. The complexes formed should be coordination saturated, since this gives the greatest probability of separation of complexes formed from different metals. Moreover, the ligand should not be too large, so that specific properties of the central metal atom are retained in the coordination complex. The donor atoms in the ligand should have low total electronegativity to minimize adsorption effects on silica-based reversed-phases. Preferred donor atoms are N-. 0- and S-. Separation selectivity increases when ligand substituents do not have large induction or steric effects, and also when electronegative atoms exist in close proximity to the chelate ring. The complexes should have high stability, good detectability and high solubility in non-polar organic solvents.
Many ligands have found application in HPLC separations of metal chelates. These ligands include dithiocarbamates (71, 8-hydroxyquinoline [8, 91, P-diketones [lo]. 4-(2pyridylaz0)-naphthol [ 11],4-(2-pyridylazo)-resinol [ 121, dialkyldithiophosphates[ 13, 141, xanthates [ 151, 2.3-diaminonaphthalene [16], pyrazolones [17] and hydrazones [18. 191. No single ligand is suitable for all metal ions and typically only a few metals are determined in a single chromatographic separation. In most cases, water-insoluble chelates are formed and these must be extracted into a suitable organic solvent, prior to the chromatographic separation step. This sometimes involves extraction with solvents which cannot be injected directly into a reversed-phase HPLC system, so that evaporation and redissolution become necessary. Alternatively, complexes can be formed in-situ by injecting metal ions into a mobile phase which contains the ligand and
Miscellaneous Separation Methods
225
an appropriate buffer [20] or through the use of solid-phase reaction on a suitable precolumn 1141. The stability of the metal complexes is also of great importance because these complexes are generally injected at very low concentrations and are therefore prone to dissociation as they traverse the chromatographic system. This is particularly true of complexes which may undergo ligand-exchange reactions at the surfaces of metallic chromatographic components, such as the injector, interconnecting tubing and the inlet and outlet frits in the column. Kinetic stability is of more importance than thermodynamic stability, since kinetically inert complexes are more likely to pass intact through the chromatographic system. The large volume of literature on HPLC of metal chelates precludes a comprehensive discussion of this topic. Table 8.1 provides a selected listing of some applications of HPLC of metal chelates. To illustrate the utility of this technique, we will focus attention only on the use of dithiocarbamate ligands, since these reflect most of the trends which exist for other ligands. Dithiocarbamate complexes Alkyldithiocarbamate ligands form complexes with a very wide range of metal ions and therefore offer the potential for separation of a larger number of metal ions than any other ligand. Diethyldithiocarbamate (DEDTC) complexes have been studied extensively and Fig. 8.2 shows a typical separation of metal-DEDTC complexes achieved on a reversed-phase column with a ternary eluent comprising water, methanol and acetonitrile [7]. Two important characteristics are evident from this chromatogram. First, there is a peak due to the ligand oxidation product, bis(diethy1thio-carbamy1)disulfide (usually referred to as disulfiram), which results either from excess ligand in the extracting solution, or from ligand produced by dissociation of labile complexes. Second, the peak shape for Pb(I1) is very poor, due to the low kinetic stability of this complex and the resultant likelihood of dissociation or ligand-exchange reactions. This behaviour occurs with other unstable complexes, such as those of Cd(II), Fe(II1) and Zn(II), and is especially evident when columns with stainless-steel frits are used. The porous nature and high surface areas of these frits provide ideal sites for ligand-exchange reactions to occur between the injected metal complexes and metal ions produced from the oxidation of stainless steel components, especially nickel. These reactions can be minimized either by using columns without porous metallic frits, such as radial compression columns [7], by deactivating the frits with an organosilane [25],or by addition of EDTA to the mobile phase 171. Formation of the chelate It should be stressed that it is the metal chelates which are separated in the above example, rather than the metal ions themselves. Therefore, these chelates must be formed before the sample reaches the analytical column. Chelate formation is usually achieved by buffering the sample and then extracting with a solution of the ligand in a suitable organic solvent. This process can be automated using the apparatus shown in Fig. 8.3, wherein the sample is added to a solution stream of ligand (diethyldithiocarbamate) in acetonitrile [26]. The mixture is then passed, in turn, to a heated
TABLE 8.1 TYPICAL DETERMINATIONS OF METAL CHELATES BY REVERSED-PHASEHPLC Solute(s)
Liganda
Stationary phase
Al(III), In(1LI) As(III), Sb(III), Bi(II1) Cd(II), PWII), Ni(II), Co(III), Hg(I1). Cr(III), Se(IV), Cu(II), Te(W Cd(II), Co(II), Pb(II), Ni(II), Wrr)
PMBP DEDTP DEDTC BHEDC BHEDC Oxine HFAA Oxine DEDTC
sew) Ti(IV), FeOII), U(VI), V(V)
DAN DAPMP, DAPMT
Detection modec
Detection limitd
Ref
Shim-Pack CLC-ODs ACN-MeOH Hypersil ODS 10 mM DEDTP in ACN Waters C18 Rad-Pak 40:35:25 MeOH-ACNwater
S (290 nm) S (280 nm) S (254 nm)
21-121 ng 2 ng 0.5
17 14 7
O.lmM HEDC in 40:a MeOH-water with 0.1 m~ Zn2+ SUPelCO cl8 25 mM TEA acetate Cg Reversed-phase 1 mM oxine in borate buffer (pH 9) 100% cHZC12 Silica c18 reversed-phase 10 mM oxine, ACN an^ acetate buffer (pH 6) 0.05% DEDTC in waterHypersil ODs MeOH-cHC13 60:40MeOH-water pBondapak C18 Polymer Labs PLRP-S 10%-40% ACN-water gradients
S (300 nm)
7-53 ppb
21
S (255 nm) S (254 nm)
5 PPb
22 23
DCP S (400 nm)
n.s. 100 PI--
24 8
S (350 nm)
0.5 ppm
20
S (254 nm), Fluor S (340 nm)
10 ppb n.s.
16
pBondapak cl8
Mobile phaseb
n.s.
19
PMBP = l-phenyl-3-methyl-4-benzoyl-5-pyrazolone, DEDTP = diethyldithiophosphate, DEDTC = diethyldithiocarbamate, BHEDC = bis(2hydroxyethy1)dithiocarbamate. Oxine = 8-hydroxyquinoline, HFAA = hexafluoroacetylacetone, DAN = 2,3 diaminonaphthalene, DAPMP = 2,6diacetylpyridine bis (N-methylenepyridiniohydrazone),DAPMT = 2,kliacetylpyridine bis(N-methylene-N,N,N,-trimethylammoniohydnuone). MeOH = methanol, ACN = acetonitrile, TEA = tetraethylarmnonium. S = spectrophotometry, Fluor = fluorhetry, DCP = direct current plasma atomic emission spectrometry. n.s = not stated. a
5
00
Miscellaneous Separation Methods
227
I
0.005 Ahsorhancc
Fig. 8.2 Separation of a mixture of diethyldithiocarbamate complexes by reversed-phase HPLC. The mobile phase comprised 40:35:25 methanol-acetonitrile-waterand a Waters c18 Rad-Pak was used as the column. The flow-rate was 2.0 rnl/rnin and the detection wavelength was 254 nm. Peak identities: A-disulfiram, B-Cd(II), C-Pb(II), D-Ni(II), E-Co(III), F-Cr(III), G-Se(IV), HCu(II), I-Hg(II), J-Te(1V). Reprinted from [7] with permission.
reaction coil, a bubble capture device and to the sampling loop of an auto-injector. Excess ligand in the solution is removed by an anion-exchange guard column placed before the c18 analytical column. An alternative method for the formation of the dithiocarbamate complexes is to inject the metal ions into an eluent which contains the ligand. On-column complex formation provides a potentially quick and easy method for multi-element identification and determination. When DEDTC is added to the mobile phase, Cd(II), Pb(II), Co(II), Hg(I1) and Cu(I1) can be separated [20, 271. The chief problem encountered with oncolumn complexation is the high background detector signal produced by the presence of the ligand in the mobile phase. This requires that a selective wavelength be used in the case of spectrophotometric detection, or alternatively, amperometric detection must be used [28]. A further problem is the poor solubility of many dithiocarbamate complexes in typical mobile phases for reversed-phase HPLC, but this may be overcome either by the addition of a small amount of chloroform to the mobile phase [20], or through the use of a ligand which forms water-soluble complexes [21]. An example of the latter approach is the use of bis(2-hydroxyethy1)dithiocarbamate (BHEDTC), in which the
228
Ckpter 8
prewure
DEDTC in acetonitrile Sample
Fig. 8.3 Schematic diagram of apparatus for automated pre-column formation of diethyldithiocarbamate (DEDTC)complexes, prior to separation by reversed-phase HPLC. Reprinted from [26] with permission.
hydroxy groups on the ligand cause metal complexes to be water-soluble at low concentrations [22]. Fig. 8.4 compares chromatograms obtained using pre-column (Fig. 8 4 a ) ) and on-column (Fig. 8 4 b ) ) complex formation with BHEDTC. 8.2.2 Organometallic compounds
One of the factors which limits the applicability of HPLC analysis of metal chelates is the necessity to form the chelate itself. This limitation does not exist for many of the organometallic species which are amenable to chromatographic analysis, since these species often occur in a wide range of samples. The more important organometallic species which can be analyzed by HPLC include alkyllead, alkylmercury, alkylarsenic and alkyltin compounds. Of these, the organoarsenic species are the most widely studied. Monomethylarsonate, dimethylarsinate and phenylarsonate (as well as the inorganic ions arsenate and arsenite) are formed by the action of many common yeasts, fungi and bacteria on arsenic present in soils. The high toxicities of these compounds necessitate their accurate determination, especially in water samples. Separation can be accomplished by reved-phase chromatography [29], as well as by anion-exchange [30, 311 or ion-interaction 132, 331 methods. Organomercury compounds, such as methylmercury and ethylmercury, have also been separated by reversed-phase HPLC [34]. In most of the above examples, atomic spectroscopic detection methods have been employed Table 8.2 lists some further applications of the determination of organometallic species by reversed-phase HPLC, and Fig 8.5 shows chromatograms obtained for organomercury and organotin compounds.
% c;.
TABLE 8.2 TYPICALDETERMINATIONS OF ORGANOMETMC SPECIES BY REVERSED-PHASE HPLC Solute(s)
Sample
Stationary phase
Eluent
Detection modes
Detection limit
Alkyl Hg compounds Alkyl Pb compounds Ethyl Sn compounds Fe, Mo carbonyl complexes Methylmucury, ethylmacury Methyl Sn compounds Organo As compounds Organo Hg compounds
n.s. PeIml
ICP ICP
Reactionmixtures
HyperSd c18 H m f i c18 Spherisorb S5W ODs ZarbaXCg
ICP
n.s. 35 llppb 35 50-100pg 36 1 PPb 35
Tuna
Waters pic0 Tag
1:2 EtOH-0.05 M NaBr 75% EtOH-water 7030 acetone-pentane 7030 EtOH-water or J3OH-water gradients 6omManrmoniumacetate, 0.005% 2-mer~aptOethan01 6040 acctone-pentane 100% MeOH 4050 MeOH-water + 0.06 M ~ O A + C 0.01% 2-mexaptOethanol 90:lO MeOH-water 7525 MeOH-water
ICP-MS
1 PPb
34
Hydride AAS GFAAS
2-2opg 5 ng 2 PPb
37 38 39
Tetraphenyl Pb Transition metal cluster complexes
n.s. n.s.
480pg n.s.
40 41
Water
n.s. n.s. Fish
FtI
DP Amp ZeemanAAS
spec
I B = inductively coupled plasma atomic emission spe!cmehy, Hydxide AAS = hydxide generation atomic absorption spectrometry, GFAAS = graphite furnace atomic absorption specaometry,RI = refractive index, MS = mass spectrometry,DP Amp = differential pulse. ampemmefry, Spec = specfrophotomefry. n.s. = not stated. a
Ref
is
E
8e
I
230
Chapter8 CO2'
0
L
8
l i m e (minl (a)
12
-
0
5 10 lime (minl (bl
15
Fig. 8.4 Comparison of chromatograms obtained using (a) pre-column and (b) on-column formation of bis(2-hydroxyethy1)dithiocarbamate (BHEDTC) complexes. (a) The pre-formed BHEDTC complexes were injected onto a Supelcosil c18 column using an eluent comprising 4060 methanol-water containing 25 mM triethylammonium acetate. Detection was by spectrophotometry at 255 nm. Reprinted from [22]with permission. (b) Metal ions were injected onto a Waters WBondapak cl8 column using an eluent comprising 4050 methanol-water containing 0.1 mM BHEDTC and 0.1 mM a*+. Detection was by spectrophotometry at 300 nm. Reprinted from [21] with permission.
8.2.3
Carboxylic acids (Ion-suppression)
A further strategy which can be employed in the determination of ionizable solutes by reversed-phase HPLC is to suppress the ionization of these solutes by adding a buffer of appropriate pH to the eluent. Retention of the solutes on non-polar stationary phases is therefore increased and separation can then be accomplished. Acidic buffers are used for the separation of weak acids, whilst alkaline buffers are used for the separation of weak bases. This method, often described as "ion-suppression",is generally considered to be applicable only to those weak acids and bases for which the ionization can be suppressed using buffers having pH values in the range 3 - 8 [42]. The reason for this is that C1g stationary phases are unstable outside this pH range. Although this is undoubtedly a major limitation, many weak acids may separated on C18 columns, as demonstrated by Skelly [43]. Restrictions in eluent pH do not apply to the use of non-
23 1
Miscellaneous Separation Methods
MeSn( EtSnCI: Me2SnCI; Me3SnCI MebSr
#Hg+
I I
1
1
1
1
.L 1
1
1
1
0 2 4 6 8 10 12 14 16 Time (min) (a)
1 1 1 T
0 1 2 3 Time (min)
(bl
-
0 1 2 3 1 5 Time (min) (C 1
Fig. 8.5 Separation of (a) organomercury, (b) methyltin and (c) ethyltin compounds by reversedphase HPLC. (a) A Spherisorb ODS column was used with an eluent comprising 40% methanolwater, 0.06 M NH4OAc (pH 5.5) and 0.01% 2-mercaptoethanol. Detection was by differential pulse amperometry. MeHg+ = methylmercury, EtHg+ = ethylmercury, @Hg+= phenylmercury. Reprinted from [39] with permission. (b) A Spherisorb ODs column was used with 60:40 acetonepentane as the eluent and hydride generation atomic absorption spectrometricdetection. Reprinted from [37] with permission. (c) Conditions as for (b) except that a 70:30 acetone-pentane eluent was used. Reprinted from [37] with permission.
polar polymeric stationary phases, so these materials can therefore be employed for the separation of a wider range of solutes using the ion-suppression technique than is possible with c18 stationary phases. The utility of ion-suppression on polymeric stationary phases can be appreciated by considering the separation of the homologous series of aliphatic carboxylic acids. Neither ion-exchange nor ion-exclusion chromatography yields a complete separation of these species. However, ion-suppression coupled with gradient elution and suppressed conductivity detection enables the separation of butyric through to stearic acid, as illustrated in Fig. 8.6. The gradient used involved an increase in the percentage of organic modifier in the eluent and a decrease in eluent pH. Carboxylic acids more hydrophilic than butyric acid were eluted as a single peak at the column void volume.
232
Chupter8
ityric
Lauric
Capric
I I
0
I
5
I
10
I
15
I
I
20 25 lime (min)
I
30
I
35
1
LO
I
45
Fig. 8.6 Gradient elution ion-suppression chromatogram of carboxylic acids, obtained on a polymeric reversed-phasecolumn. A Dionex MPIC-NS1 column was used with a gradient of 100% eluent A ( t 4 ) to 100% eluent B (t=20 min), with maintenance of eluent B after this time. Eluent A is 24% acetonitrile and 6%methanol in 0.03 mM HC1. Eluent B is 60% acetonitrile and 24% methanol in 0.05 mM HCl. Detection was by suppressed conductivity. The baseline conductance for a blank w e n t has been subtracted in the chromatogram shown. Reprinted from I441 with permission.
8.3
CHELATING STATIONARY PHASES
8.3.1 Chemically-bound ligands Metal ions may be separated on a stationary phase in which a suitable ligand is immobilized onto the stationary phase. Numerous chelating stationary phases have been synthesized using stryene-divinylbenzenepolymers or silica as the support material. In each case, the ligand is chemically bound to the support using an appropriate reaction, such as silylation reactions with silica. Some examples of the ligands which can be bound in this way include iminodiacetate (Chelex 100, Dow Chemical Company), propylene-diaminetetraacetate 1451. P-diketones (e.g. trifluoroacetoacetate [461), 8hydroxyquinoline [47], isothiuronium (481, hydroxamic acids [49], dithiocarbamates (501, phenylhydrazones [51] and dithizone [52]. Solute retention can be manipulated by varying the eluent pH or through the addition of a competing ligand to the eluent. The key factor in the success of the above materials as chromatographic stationary phases is the rate at which the metal-ligand complex is formed and dissociated. Slow rates will lead to poor peak shape in the chromatogram. An evaluation of the literature suggests that most ligands give unacceptably slow rates of reaction, so that chromatograms are typically characterized by very broad peaks. A recent study has compared 9 different chelating stationary phases and has shown that some useful separations can be achieved on dithizone silica gels, as illustrated in Fig. 8.7. It is interesting to note that the same metal ions shown in Fig. 8.7 can be well separated using either ion-exchange or ion-interaction chromatography (e.g. see Figs. 4.17 and 6.3).
Miscellaneous Separation Methotlr
233
Pb2
1 1 1 1 1
0
10 20 Time (mid
Separation of metal ions on a stationary phase formed by binding dithizone functionalities to silica gel. The eluent was 15 mM tartrate at pH 4.0. Detection was by specaophotometry after post-column reaction with 4-(2-pyridylazo)-resinol. Reprinted from [52] with permission.
Fig. 8.7
8.3.2 Crown ether stationary phases
Considerable effort has been expended over recent years on the development of stationary phases in which a crown ether is chemically bound to a suitable support, such as silica or an appropriate polymer. Crown ethers (or cyclic polyethers) are cyclic compounds which possess an inner cavity, generally consisting of oxygen atoms linked by ethylene bridges. Fig. 8.8 shows two examples of these compounds. Crown ethers are non-systematically named according to the total number of atoms in the ring, the number of oxygen atoms and any substituents on the ring. Thus the fiist crown ether in Fig. 8.8 is called 18-crown-6 (total of 18 atoms in the ring, with 6 oxygen atoms), whilst the second is called benzo-18-crown-6 (to indicate the benzene ring substituent). Cryptands are related compounds having two interconnected rings which produce a three dimensional cavity. The final compound in Fig. 8.8 is one such cryptand.
Synthesis of stationary phases Crown ether stationary phases may be synthesized in three ways. The simplest approach is to impregnate a silica particle with a solution of a suitable crown ether in formic acid, followed by cross-linking with formaldehyde [53], and is illustrated schematically for dibenzo-18-crown-6 in Fig. 8.9(a). The resultant material is
234
Chapter 8
18-crown-6
Benzo- 18-crow n-6
O
d
Cryptand-n-decyl-2.2.2 Fig. 8.8 Structures of some cyclic polyethers
Fig. 8.9 Structures of some crown ether stationary phases. (a) The crown ether is coated onto silica and then polymerized. (b) Typical bonding arrangement of a crown ether onto silica. (c) Typical bonding arrangement of a crown ether onto a resin. Reprinted from [53, 581 with permission.
Miscellaneouy Separation Methods
235
stable mechanically and is resistant to hydrolysis. Alternatively, the crown ether can be chemically bound to silica by silyl ether linkages using conventional silylation reactions. An example of the resultant stationary phase structure is shown in Fig. 8.9(b) for benzo18-crown-6. Finally, similar bonding reactions can be performed using resins as the support and a typical stationary phase structure is depicted in Fig. 8.9(c), again using benzo- 18-crown-6 as the ligand. Chromatographic properties The chromatographic utility of crown ether stationary phases rests in their ability to complex cations of a specified size. As the size of the cavity in the crown ether is altered, so too does the selectivity of the stationary phase. For example, Li+, Na+ and K+ are bound preferentially to 12-crown-4, 15-crown-5 and 18-crown-6 stationary phases, respectively, by virtue of the increasing cavity size. However, it is not essential for the solute cation to reside within the cavity since layered structures in which the solute is located between two cavities can also be formed [54]. The bound metal ion imparts a positive charge to the crown ether and this is balanced by a suitable counter anion. The binding of metal ions on crown ether stationary phases is .dependent on the following factors: (i) The size of the cation. (ii) The nature of the associated anion. (iii) The organic modifier content of the eluent. The influence of cation size has been discussed above. The most common elution trend for alkali metal ions on benzo-18-crown-6 stationary phases (which show preference for K+) is for K+ to be eluted last, with the remainder being eluted in order of size. That is, retention usually follows the sequence:
The nature of the associated anion can sometimes exert an influence on retention which dominates all other effects. "Soft" (polarizable) anions such I- and SCN- are more strongly associated with the bound cation than are "hard" anions, such as S04*-and C1-. It has been demonstrated that, when a mixture of anions and cations is injected, the most strongly retained species will be the preferred cation associated with the softest anion [55]. For example, injection of a mixture of Li+, K+,C1-, Br- onto a dibenzo-18-crown6 stationary phase gives four peaks corresponding to LiCI, KCI, LiBr and KBr [56]. The first eluted peak (LiCI) contains the least preferred cation (Li+) and the hardest anion (Cl-), whilst the last eluted peak (KBr) contains the preferred cation (K+) and the softest anion (Br-). Water alone can be used as an eluent in this form of chromatography, but the separations which are obtained are often relatively poor. The reason for this is that water complexes the solute cations and competes effectively with the crown ether. Addition of an organic modifier, such as methanol, to the eluent lowers its polarity and
236
Chapter8
5Br
KBI
Liar
KBr
1
0
I
5
1
I
1
10 15 20 Time (min) (a 1
I
25
-
0 10 20 30 LO Time (min) (b)
Fig. 8.10 Separation of (a) cations and (b) anions on crown ether stationary phases. (a) A paly(benzo-l5-crown-5)-modifiedsilica was used as stationary phase with water as eluent. Detection was by conductivity. Reprinted from [64]with permission. (b) A benzo-18-crown-6 modified silica stationary phase was used with water as eluent. Conductivity detection was used. Reprinted from [65] with permission.
decreases complexation with the solutes. This, in turn, results in increased solute retention and therefore improved separation. However, detection is more simple with a water eluent than with aqueous-organic mixtures, so the former eluent is preferable.
Applications Crown ether stationary phases can be used in two ways. First, a series of alkali metal salts with a common anion can be separated. These salts will be eluted with the cations following the sequence determined by the preference of the particular crown ether used 153, 57-62]. An example of such a separation on silica coated with poly(benzo-t5-crown-5) is shown in Fig. 8.lqa). We note that the cations must be present as their Br- salts for the separation to be reproducible. An alternative application is the separation of anions, using the cation bound to the crown ether as a site for selective retention of anions. The salts will be eluted in the sequence of reducing hardness of the anions [53,56, 57, 59, 60,62, 631. A typical separation by this method is given in Fig. g.lO(b). We again note that the separation is dependent on each anion being present as the K+ salt. If other cations are present, the elution order could vary.
MiscellaneousSeparation Methods
237
The separations shown in Fig. 8.10 are of limited practical applicability because of the need to specify a particular counter-anion or counter-cation for the solute ions. It has been suggested that a sample could be converted to the correct form by passage through a suitable ion-exchange column prior to analysis [54], but no results using this approach have been reported. A more attractive alternative is to use a sufficiently high concentration of the desired counter-cation or anion in the eluent so that the sample counter-ions do not influence solute retention. This approach has been reported for anion determinations using a stationary phase comprising the macrocycle cryptand ndecyl 2.2.2, which has the structure shown in Fig. 8.8, coated onto reversed-phase supports with alkali metal hydroxides as eluents [66, 671. The cryptand is hydrophobically bound to the support in the same manner as used in "permanentcoating" ion-interaction chromatography (see Section 6.3.2). Excellent chromatographic efficiencies were obtained and solute retention was not dependent on the cations present in the sample.
Gradient elution on macrocyclic stationary phases The above-mentioned studies with the cryptand n-decyl 2.2.2 have opened a new possibility for gradient elution of anions. The cation bound to the macrocycle acts as an anion-exchange site for solute anions. The number of bound cations, and hence the anion-exchange capacity of the column, is dependent on the identity of the alkali metal hydroxide used as eluent. The highest anion-exchange capacity is produced with KOH eluents (because of the preferential binding of K+ to the macrocycle), whereas the smallest capacity results when LiOH is used as the eluent. Thus, a gradient in which the eluent is changed from, for example, 30 mM NaOH to 30 mM LiOH will therefore result in a progressive decrease in column ion-exchange capacity. The change in baseline conductivity in such a system will be minimal, especially if a suppressor is employed. Fig. 8.1 1 shows a chromatogram obtained using this method, which has been called "gradient capacity IC" [67]. This method has considerable promise, especially in view of the ease with which the stationary phase can be prepared. 8.4 8.4.1
MICELLE EXCLUSION CHROMATOGRAPHY
Introduction
Many surfactant molecules form micelles in concentrated solution. Micellar chromatography [68, 691 utilizes micellar eluents as a means of improvement of the chromatographic selectivity. We have seen earlier (Section 6.3.2, Fig. 6.4) that micellar eluents can be employed,in ion-interaction chromatography of inorganic anions. The chromatograms obtained show good chromatographic efficiency, but the elution order of solutes (and hence the selectivity of the method) is the same as that for conventional ionexchange chromatography. This can be explained by the fact that in this particular case, the surfactant was strongly adsorbed onto the reversed-phase column which was used, so that ion-exchange was the dominant retention mechanism. On this basis, use of a different type of stationary phase, such as a size-exclusion material, could lead to changes in chromatographic selectivity. Okada [70,711 has investigated this approach
238
Chapter 8
F-
p-
Acct, SC N-
I r
0
I
10
1
20 Time (min)
30 1
LO I
Fig. 8.11 Gradient elution on a macrocyclic stationary phase using variation of the ion-exchange
capacity of the column. A Dionex MPIC-NS1 column was used after conditioning with cryptand-ndecyl-2.2.2. The eluent was a linear gradient (over 20 min) from 30 mM NaOH to 30 mM LiOH. Detection was by suppressed conductivity. Chromatogram courtesy of John D. Lamb. for the separation of inorganic anions and cations. The eluent consists of an aqueousorganic mixture containing a micellar solution of a surfactant of appropriate charge. The micelles in the eluent are excluded from part of the stationary phase, whilst the solute ions partition between the micelle and the bulk solvent in the eluent, and also between the stationary phase and the eluent, This method has been entitled "micelle exclusion chromatography" on the basis of this separation mechanism. 8.4.2
Micelle exclusion chromatography of anions
When anionic solutes are to be separated, the surfactant in the eluent should be positively charged. Hexadecyltrimethylammonium chloride (HTAC) and dodecyltrimethylammonium chloride (DTAC) are therefore suitable surfactants [71]. Use of a size-exclusion stationary phase means that the aggregated surfactant forming the micelle
Miscellaneous Separation Methodr
239 Mnll
L I I'
0
30
Time (rnin) (01
I
LO
r
0
I
10
I
20 lime (rnin)
I
30
1
LO
(bl
Fig. 8.12 Determination of (a) inorganic anions and (b) inorganic cations by micelle exclusion chromatography. (a) An Asahipak GC910H (poly(viny1 alcohol)) size-exclusion column was used with 0.01 M hexadecyltrimethylammonium chloride as eluent. Detection was by spectrophotometry at 210 nm. Reprinted from [70]with permission. (b) The same column as for (a) was used, with a 10 min gradient from 100%eluent A (commencing at t = 10 min) to 100%eluent B. Eluent A was 25 mM sodium dodecyl sulfate (SDS) and 80 m M a-hydroxyisobutyric acid at pH 4.0. Eluent B was 25 mM SDS and 60 mM tartaric acid at pH 4.0.Detection was by spectrophotometry after post-column reaction with 4-(2-pyridylazo)-resorcinol.Reprinted from [71] with permission.
can penetrate only partially into the pores of the stationary phase, whereas the monomeric surfactant can penetrate fully by virtue of its smaller size. The small solute anions can also penetrate into the pores and are therefore retained in this region of the stationary phase by ion-interaction chromatography. Solute anions will also partition between the bulk eluent and the surfactant micelles, and between the bulk eluent and the liquid inside the pores of the column. Retention is therefore based on three distinct processes. Fig. 8.1 2(a) shows a chromatogram obtained using HTAC as the surfactant. The elution order in Fig. 8.12(a) is different from that exhibited by conventional ionexchange or ion-interaction systems, particularly for I- and Br-. The retention order and
240
Chaprer 8
retention times observed in this system are dependent on a number of parameters, including: (i) (ii) (iii) (iv) (v)
The concentration of surfactant The nature of the surfactant used The nature of the surfactant counter-anion The Concentration of added salt The concentration of organic solvent in the eluent
All of these parameters also affect retention in ion-interaction chromatography, which supports the hypothesis that this process plays a significant role in the mechanism of micelle exclusion chromatography. However, the observed chromatographic selectivity shows that there are additional conmbutions to solute retention which are attributable to the partitioning processes described above.
8.4.3 Micelle exclusion chromatography of cations Micelle exclusion chromatography of cations requires an anionic surfactant, such as sodium dodecylsulfate (SDS),and a size-exclusion stationary phase [71]. Again, the surfactant micelles are partially excluded from the pores of the stationary phase, whereas monomeric surfactant and solute cations can penetrate fully. Retention occurs by the same mechanisms outlined above for anions and the retention times are dependent on the same parameters as listed earlier. As with ion-exchange and ion-interaction methods, further control over solute retention can be accomplished through the addition of a complexing agent, such as citrate, to the eluent. Moreover, gradient elution can be achieved by changing the nature of the complexing agent whilst keeping other eluent parameters constant. Fig. 8.12(b) shows a chromatogram obtained using this approach.
8.5
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