346 IONIC SAMPLES: REVERSED-PHASE, ION-PAIR, AND ION-EXCHANGE CHROMATOGRAPHY 0 0246 246 Time (min) Time (min) 1 2 3 4 + 5 6 7 10% MeOH 1 2 3 4 5 7 6 6% ACN (a) (b) Figure 7.17 Solvent-type selectivity in the IPC separation of a catechol amine sample. Sample: 1, noradrenaline; 2, adrenaline; 3, octopamine; 4, 3,4-dihydroxyphenylalanine; 5, dopamine; 6, isoprenol; 7, tyrosine. Conditions: 150 × 4.6-mm C 18 column (5-μm particles); pH-2.5 phosphate buffer plus 2-mM octane sulfonate IPC reagent; 25 ◦ C;1mL/min.Adaptedfrom [50]. 7.4.2.3 Summary Developing an IPC separation can proceed as follows: 1. select initial conditions for RPC separation (Section 7.3.3.1) 2. vary %B as in RPC, in order to determine a value for an appropriate retention range (e.g., 1 ≤ k ≤ 10) 3. vary pH in order to tentatively identify various peaks in the chromatogram as acidic, basic, or neutral (unless peak identities are known by injecting standards) 4. at some stage of further RPC method development, consider the possible value of or need for IPC separation (Section 7.4) 5. if IPC separation is chosen, choose an IPC reagent and its initial concentra- tion as described in Section 7.4.2.1. A prior knowledge of the composition of the sample, or previous exper- iments where pH is varied (step 3) should indicate the choice of either a sulfonate IPC reagent (for increasing the retention of acidic solutes) or a quaternary ammonium salt (for basic or cationic solutes). Alternatively, for basic solutes, a chaotrope can be used as IPC reagent to increase their retention. 6. optimize relative retention (selectivity) Simultaneous changes in %B and temperature are expected to be highly effective. For further changes in selectivity, mobile-phase pH and the IPC reagent concentration can be varied (e.g., Figs. 7.13, 7.15). If an additional change in selectivity is needed (unlikely), vary other separation conditions listed at the beginning of Section 7.4.2.2. 7.4 ION-PAIR CHROMATOGRAPHY (IPC) 347 7. vary column conditions for further improvements in either resolution or run time (Section 2.5.3) 7.4.3 Special Problems The separation of ionic samples by IPC is subject to some of the same requirements as for RPC: • a need for the close control of mobile-phase pH in some cases (e.g., ±0.10 units or better) • a need for reproducible temperature control (more so than for RPC) In addition, there are certain problems in IPC that are either absent from RPC separation or differ in some respect for IPC: • greater complexity of operation and more challenging interpretation of results •artifactpeaks • slow column equilibration after changing the mobile phase • poor peak shape for poorly understood reasons The greater complexity of IPC compared to RPC has been noted. There are more variables to choose from in method development or to control during routine operation. While this greater complexity can be manageable, it is nevertheless a distraction that tends to make IPC less attractive. On the other hand, tailing peaks for protonated basic solutes are less likely in IPC (less ionic repulsion of adjacent molecules BH + in the stationary phase), and IPC is a more powerful means (when needed) for optimizing the relative retention of ionic samples. 7.4.3.1 Artifact Peaks Both positive and negative peaks are sometimes observed when the sample solvent (without sample) is injected in IPC (blank run). These artifact peaks can interfere with the development of an IPC method or its routine use. For this reason blank runs should be carried out both during method development and subsequent routine applications, in order to avoid any confusion due to artifact peaks. Problems with artifact peaks are usually the result of differences in composition of the mobile phase and sample solvent. Such problems can be magnified by the use of impure IPC reagents, buffers, or other mobile-phase additives. A good general rule in IPC is to match the compositions of the sample solvent and mobile phase as closely as possible (including, if necessary, the IPC reagent concentration). Smaller volume sample injections are also recommended (e.g., <25 μL if possible). If problems with artifact peaks persist, a different lot or source of the IPC reagent should be tried. For a general discussion of artifact peaks, see Section 17.4.5.2 and the discussion of [55, 56]. 7.4.3.2 Slow Column Equilibration When a new mobile phase is used, the column must be flushed with a sufficient volume to equilibrate the column (Section 2.7.1). In IPC, both the uptake and 348 IONIC SAMPLES: REVERSED-PHASE, ION-PAIR, AND ION-EXCHANGE CHROMATOGRAPHY release of the IPC reagent by the column can be slow under some circumstances, leading to incomplete equilibration of the column by the new mobile phase. For this reason it is essential to confirm that sample retention is reproducible after a change in the mobile phase, when either the old or new mobile phases contain an IPC reagent (several hours of flow of the new mobile phase may be required to confirm complete column equilibration; see the example below). Column equilibration can be especially slow when the IPC reagent is more hydrophobic (e.g., decane sulfonate vs. octane sulfonate), as well as when quaternary ammonium salts are used with type-A columns [47]. When an IPC reagent is to be replaced, it may be necessary to first remove the previous IPC reagent from the column with a special wash solvent (see below), followed by equilibration of the column with the new mobile phase. Anionic reagents (e.g., alkane sulfonates) can be removed with a wash solvent composed of 50–80% methanol-water. Quaternary ammonium salts and type-A columns require the use of 50% methanol-buffer (e.g., 100 mM potassium phosphate at pH 4–5; the added potassium phosphate serves to reduce the interaction of the quaternary ammonium group with ionized silanols in the stationary phase). In either case a minimum of 20 column volumes of wash solvent should be used before checking for retention reproducibility (that is, column equilibration) with the new mobile phase. The initial equilibration of the column with a mobile phase that contains an IPC reagent may prove to be unexpectedly slow. The IPC separation of Figure 7.10b was at first believed to equilibrate after washing the column with 20 to 30 column volumes of mobile phase [46], since the replicate injections indicated no significant change in retention times. When samples were subsequently run for an extended period, however, it was found that a very slow decrease in retention for basic compounds X–X 3 occurred over a period of 11 hours, suggesting a very slow approach to column equilibrium. To avoid the need for a 12-hour equilibration at the beginning of every new series of routine runs, it was necessary to store the column filled with mobile phase (containing the IPC reagent) upon completion of each series of runs. This expedient allowed much more rapid column equilibration during startup for assays by IPC, and this is a procedure that we recommend when a separation is to be repeated every day or two. Column lifetime may be reduced, however, when the column is stored in this way (Section 5.8). The slow equilibration of the column with more hydrophobic IPC reagents can create problems if gradient elution is used. Retention may be less reproducible, baselines can be erratic, and other separation problems may arise. For this reason gradient elution with an IPC reagent added to the mobile phase is usually not recommended, especially for more hydrophobic IPC reagents. An exception can be made for the weakly ion-pairing buffer trifluoroacetic acid (TFA), and for chaotropes such as ClO − 4 ,BF − 4 , and PF − 6 , since all of these are less susceptible to slow column equilibration. Passage through a column of 10 to 20 column volumes of the mobile phase is usually adequate for mobile phases that contain TFA or chaotropic reagents. For this and other reasons the use of the latter ion-pair reagents is finding increasing application. Finally, because of the slow equilibration of the IPC reagent with the column, it is possible that not all of the IPC reagent will be washed from the column, even with aggressive washing procedures. For this reason we recommend that columns that have been used with IPC not be used subsequently for RPC separations without 7.5 ION-EXCHANGE CHROMATOGRAPHY (IEC) 349 IPC reagents (TFA and chaotropes represent an exception to this warning). A trace of IPC reagent remaining on such a column could cause differences in selectivity that would not be reproduced upon replacement with a new column. Changing from one IPC reagent to another, however, should be less problematic. 7.4.3.3 Poor Peak Shape Peak tailing of bases usually does not arise in IPC because type-B columns are generally used, and the alkylsulfonate IPC reagent can further minimize the effect of column silanols (the IPC reagent competes with ionized silanols for interaction with protonated bases). Some studies have found peak fronting in IPC to be corrected by operating at a higher column temperature [57]. In one case, conversely, IPC provided better peak shape at a lower temperature [45]. The separations were in each case carried out with type-A columns; it is reasonable to expect that peak shape in IPC will be less problematic, as when type-B columns are used. If poor peak shape and/or low values of the column plate number N are observed in IPC, a change in temperature (either lower or higher) should be explored. 7.5 ION-EXCHANGE CHROMATOGRAPHY (IEC) Ion-exchange chromatography (IEC) is an important separation technique, but for the most part, today, with limited applications: • mixtures of inorganic ions (ion chromatography) • biomolecules, including amino acids, peptides, proteins, and especially oligonucleotides (Sections 13.4.2, 13.5.1) • carbohydrates (Section 13.6.3) • carboxylic acids • sample preparation (Chapter 16) • two-dimensional separation (Sections 9.3.10, 13.4.5) In addition inadvertent ion-exchange interactions can occur and contribute to the separations of ionic samples by RPC (Eq. 7.7, Section 5.4.1). When HPLC became available in the late 1960s, IEC was a strong candidate for the analysis of any mixture that contained organic acids or bases. Together with liquid–liquid partition and adsorption chromatography, IEC then accounted for most of the reported separations by HPLC. Since that time, however, RPC has taken over most of these applications for small-molecule samples (molecu- lar weights <1000 Da). The reasons for this decline in the use of IEC include (1) lower plate numbers N compared to RPC, (2) greater user-familiarity with RPC separation, and (3) the increased complexity of IEC separations (compared to RPC). Additionally some HPLC equipment is less well suited for typical IEC conditions (high concentrations of salt in the mobile phase, salts such as halides, which are corrosive to stainless steel, etc.). We will first summarize the applications of IEC noted above and then discuss the general principles of IEC separation. Ion chromatography has represented a major application of IEC since its intro- duction by Small in 1974 [58]. Prior to that time the analysis of mixtures of inorganic 350 IONIC SAMPLES: REVERSED-PHASE, ION-PAIR, AND ION-EXCHANGE CHROMATOGRAPHY 0 2 4 6 8 10 12 14 16 Time (min) 1 2 3 4 5 6 ? 7 8 9 10 11 12 ? Figure 7.18 Separation of carboxylic acids mixture by ion-exclusion chromatography. Sample: 1, oxalic; 2, maleic; 3,citric;4,tartaric;5, gluconic; 6, maliic; 7, succinic; 8,lactic; 9,glutaric;10, acetic; 11, levulinic; 12, propionic. Conditions: 300 × 7.8-mm cation-exchange column (9-μm particles); 0.006 N sulfuric acid-water mobile phase; 65 ◦ C; 0.8 mL/min. Chro- matogram redrawn from [57]. ions by other means was tedious and required specialized equipment of limited gen- eral applicability. The attempted use of HPLC for separating inorganic solutes was constrained mainly by the lack of a suitably sensitive detector. Ion chromatography overcame this difficulty by the use of ion suppression with conductivity detection. In this book we will not provide a further discussion of ion chromatography; the reader is instead referred to several books on the technique [59–61]. The separation of biomolecules by IEC predates the introduction of HPLC by about a decade. During the 1970s HPLC columns were introduced that permitted fast, high-resolution separations of amino acids, peptides, proteins, nucleotides, oligonucleotides, and nucleic acids by means of IEC. These important applications of IEC are discussed in Chapter 13. Separations of carbohydrates by HPLC are possible by means of either hydrophilic interaction chromatography (HILIC, Section 8.6) or by anion exchange chromatography (Section 7.5.7). As discussed below, the latter technique, with amperometric detection, is generally preferred, especially when a greater detection sensitivity is required. See also the analysis of carbohydrate fragments from the digestion of glycosylated proteins (Section 13.6.3). Carboxylic acids are often separated on IEC columns by ion-exclusion, using acidified water as mobile phase in order to suppress solute ionization. These separations do not involve ion exchange but instead are based on a partition process similar to that involved in RPC. An example of such a separation is provided in Figure 7.18. Relatively hydrophilic samples of this kind are retained weakly on most RPC columns, but more-polar IEC columns are able to provide stronger retention and acceptable k values. Ion-exclusion chromatography (by means of ion-exchange columns) continues to be popular for the assay of samples that contain carboxylic acids, as illustrated by several examples described in [62]. For a further discussion of how experimental conditions affect ion-exclusion separations of carboxylic acids, see [63]. Sample preparation (Section 16.6.5.1) remains a very important application of IEC, albeit as a low-efficiency (non-HPLC) supplement to analysis by RPC or other procedures. The effective use of IEC for sample preparation requires a basic understanding of how retention depends on separation conditions, as briefly reviewed in this section. Two-dimensional separation is mentioned in Section 2.7.3, and further dis- cussed in Sections 9.3.10 and 13.4.5. This technique is reserved for very complex 7.5 ION-EXCHANGE CHROMATOGRAPHY (IEC) 351 samples that contain too many components to be separated in a single HPLC run. The principle of operation is the use of a first HPLC separation to achieve partial separation of the sample, followed by injection of fractions into a second column for the further resolution of individual solutes. If IEC with an aqueous mobile phase is used for the first separation, the resulting aqueous fractions can be injected directly onto a second RPC column. 7.5.1 Basis of Retention IEC separations are carried out on columns with ionized or ionizable groups attached to the stationary-phase surface (Section 7.5.4). For example, cation-exchange columns for the IEC retention of protonated bases (BH + ) might contain sulfonate groups –SO − 3 of opposite charge. Similarly anion-exchange columns for the reten- tion of ionized acids (A − ) might be substituted with quaternary ammonium groups such as –N(CH 3 ) + 3 . Retention in IEC is governed by a competition between sam- ple ions and mobile-phase counter-ions for interaction with stationary-phase ionic groups of opposite charge. IEC retention can be illustrated by the cation-exchange retention of a protonated basic solute BH + with K + as the counter-ion: BH + + R − K + ⇔ K + + R − BH + (7.10) Here R − refers to an anionic group attached to the column packing (e.g., –SO − 3 ), which can bind either the sample ion BH + or a mobile-phase counter-ion K + by coulombic attraction. Equation (7.10) can be generalized for both acidic and basic sample ions that have an absolute charge |z|≡m (e.g., fully ionized oxalic acid as solute, − OOC–COO − ,withz =−2, m = 2). For a cationic solute X +m ,anda counter-ion Y + , retention is described by X +m + m(R − Y + ) ⇔ X +m R − m + mY + (7.11) Here the stationary-phase group R − refers to an anion-exchange group R − (e.g., –SO − 3 ). For an anionic solute X −m , Equation (7.11) becomes X −m + m(R + Y − ) ⇔ X −m R + m + mY − (7.12) Values of the retention factor k in IEC for a univalent counter-ion Y + or Y − in cation- or anion-exchange respectively can be derived from the equilibrium of either Equation (7.11) or (7.12): log k = a − m log C (7.13) where C is the molar concentration of the counter-ion Y + or Y − in the mobile phase, a is a constant (equal to log k for C = 1M), and m is the absolute value of the charge z on the solute molecule X; a and m are constants for a given sample compound, column, salt, buffer, mobile phase pH, and temperature. An illustration of Equation (7.13b) is shown in Figure 7.19 for the anion-exchange separation of four polyphosphates with z equal −3, −4, −6, and −8 (tri-, tetra-, hexa-, and octa-phosphates, respectively). Numerous examples of the validity of Equation (7.13b) for isocratic IEC have been reported (e.g., [64, 65]). 352 IONIC SAMPLES: REVERSED-PHASE, ION-PAIR, AND ION-EXCHANGE CHROMATOGRAPHY Na 3 P 3 0 9 (“tri”) Na 4 P 4 0 12 (“tetra”) Na 6 P 6 0 18 (“hexa”) Na 8 P 8 0 24 (“octa”) 2.0 1.5 1.0 0.5 log k −0.5 −0.4 3 lo g C k*= 20 10 5 Figure 7.19 Illustration of the dependence of log k on counter-ion concentration (log C)in isocratic IEC. Sample: four polyphosphates described in figure; conditions: 500 × 4.0-mm TSKgel SAX anion exchange column; aqueous KCl salt solutions (buffered at pH-10.2 with EDTA) as mobile phase; 30 ◦ C. Adapted from [64]. In Equations (7.10) to (7.13) we treat ion exchange as involving a stoichiometric process, where one molecule of retained solute displaces a certain number of counter ions that are tightly held by the individual charges on the stationary phase surface. While these relationships are adequately reliable in practice, they nevertheless represent a simplification of the actual ion-exchange process. For details concerning the fundamental nature and theory of ion-exchange retention, see [66]. 7.5.2 Role of the Counter-Ion Mobile phases for IEC usually consist of water, a buffer to control pH, and a salt (or counter-ion) to adjust sample retention (solvent-strength control). Because solute retention with IEC columns is usually the result of both IEC and RPC interactions with the column, the addition of methanol or acetonitrile to the mobile phase can further increase solvent strength. However, the primary control of retention is usually accomplished by changing the concentration of the counter-ion (C); an increase in C results in a decrease in retention for solutes that are ionized and retained (Eq. 7.13). For univalent solutes where m = 1, an increase in C leads to a proportional decrease in values of k. When the charge m on the solute is larger, a faster decrease in k results as C is increased. Thus, when two solutes have different values of m, a change in C will result in changes in relative retention (with possible peak reversals). This is illustrated in Figure 7.20 for the sample described in Figure 7.19. As the concentration of Cl − changes from 0.32 to 0.35 to 0.40 M, 7.5 ION-EXCHANGE CHROMATOGRAPHY (IEC) 353 02040 020 Time (min) Time (min) 0 Time (min) 0.32M KCl 0.35M 0.40M (a) (b) (c) tri tetra hexa octa 10 Figure 7.20 Examples of a change in counter-ion (KCl) concentration for the separations of Figure 7.19. Recreated separations for data of [64], assuming a 150 × 4.6-mm column (5-μm particles), 2.0 mL/min, and N = 1000. a decrease in the retention time results for each peak, but at the same time the ‘‘octa’’ peak (shaded) moves toward the front of the chromatogram—with a change in retention order (compare Figs. 7.20 and 7.19). Different mobile-phase counter-ions are retained more or less strongly by ion exchange, so that a change in the counter-ion can also be used to increase or decrease solvent strength and overall sample retention. Generally, counter-ions with a higher charge will be more effective at reducing sample retention. The relative ability of an ion to bind more strongly, suppress sample retention, and provide smaller values of k increases in the following order: (anion exchange) F − (larger values of k for solutes) < OH − < acetate − < Cl − < SCN − < Br − < NO − 3 < I − < oxalate −2 < SO −2 2 < citrate −3 (smaller values of k) (cation exchange) Li + (larger values of k for solutes) < H + < Na + < NH + 4 < K + < Rb + < Cs + < Ag + < Mg +2 < Zn 2+ < Co 2+ < Cu 2+ < Cd 2+ < Ni 2+ < Ca 2+ < Pb 2+ < Ba 2+ (smaller values of k) A change in counter-ion (e.g., from Cl − to NO − 3 ) can also affect values of a in Equation (7.13b), in turn leading to possible changes in selectivity. 354 IONIC SAMPLES: REVERSED-PHASE, ION-PAIR, AND ION-EXCHANGE CHROMATOGRAPHY 7.5.3 Mobile-Phase pH IEC is typically used for acidic or basic samples. As only the charged (ionized) molecule is retained by ion exchange, values of k for a monovalent solute (m = 1) will be proportional to the ionization of the solute; for example, as mobile-phase pH is decreased so that an acid goes from fully ionized to half ionized, the value of k will decrease by half. Similarly the ionization and retention of bases will be decreased as mobile-phase pH increases. This behavior is the opposite of retention changes with pH in RPC (Fig. 7.11a) but is the same as in IPC (Fig. 7.11b). 7.5.4 IEC Columns Based on the kind of ionic group R ± that forms part of the stationary phase, four general kinds of IEC columns are available: strong and weak anion exchangers (SAX, WAX), and strong and weak cation exchangers (SCX, WCX). Strong IEC columns contain groups R ± that are completely ionized over the usual pH range of interest (2 ≤ pH ≤ 13). For strong anion-exchange columns, the most commonly used group R + is –N(CH 3 ) + 3 ; for strong cation-exchange columns, the most common group R − is –SO − 3 . Weak IEC columns contain groups R ± with pK a values in an intermediate range (e.g., 4 ≤ pK a ≤ 10); consequently the ionization of these groups (and the ion- exchange capacity of the column) can change with mobile-phase pH (see Fig. 13.16). Because the mobile-phase pH for IEC is chosen for solute ionization, and this pH may be outside the 2 < pH < 8 region recommended for the operation of most silica-based columns, most IEC columns use a polymeric support, such as methacrylate or styrene-divinylbenzene polymers. Weak anion-exchange columns are commonly substituted with amine groups (e.g., –NH 2 ); these columns begin to lose their charge and ion-exchange capacity when the mobile-phase pH increases much above the pK a value of the amine group (5 ≤ pK a ≤ 10; Table 7.2). Weak anion-exchangers are therefore used primarily with acidic mobile phases (pH ≤ 6) that can significantly protonate the amine groups. Weak cation-exchangers are commonly substituted with carboxyl groups (–COOH), with pK a ≈ 5, so their ionization begins to decrease when the mobile-phase pH drops below 7. Weak cation-exchangers are used primarily with basic mobile phases (e.g., pH ≥ 8). Because the ionization and ion-exchange capacity of weak IEC columns can be reduced by the use of an appropriate mobile-phase pH, solutes that are strongly retained and might be difficult to remove from a strong IEC column can be eluted more easily from weak IEC columns by a change in mobile-phase pH. Column selectivity will also differ for weak versus strong ion exchangers, which can be another reason for their use with a particular sample. See the further discussion of Section 13.4.2.1. 7.5.5 Role of Other Conditions Other IEC conditions are varied primarily for a change in relative retention or selectivity: • salt or buffer type • addition of organic solvent to the mobile phase • temperature 7.5 ION-EXCHANGE CHROMATOGRAPHY (IEC) 355 For the IEC separation of small organic solutes, few generalizations have been offered for the effect of the above conditions on selectivity. However, it is known that changes in each condition can affect selectivity. 7.5.6 Method Development The usual goal of IEC separation is the retention and resolution of a mixture of either anions (ionized acids) or cations (protonated bases). When IEC is used for sample pretreatment, conditions usually are selected for the selective capture of acids or bases for further analysis by HPLC. If the goal is the high-performance separation and analysis of the sample by IEC, then either acids or bases can be resolved and analyzed—but not both simultaneously. If separation involves retention of acids, an anion-exchange column should be selected. For bases, a cation-exchange column will be used. Usually a strong ion-exchange column is preferred (at least initially). As in the development of all HPLC methods, experiments for the determination of optimum IEC conditions should start with a new (unused) column. The general approach used for RPC method development (Sections 6.4, 7.3.3) also can be followed for IEC separation. An aqueous mobile phase will be used initially, with addition of 1 to 5 mM of a suitable buffer plus a variable concentration of some salt (e.g., NaCl). The concentration of the salt (or counter-ion) is then varied by trial and error (or by gradient elution) in order to achieve a desirable retention range (e.g., 1 ≤ k ≤ 10). Changes in selectivity can then be investigated as discussed above. 7.5.7 Separations of Carbohydrates Carbohydrate mixtures can be separated by either hydrophilic interaction chro- matography (HILIC, Section 8.6) or by IEC. Carbohydrates have pK a values of about 12, which means that high-pH mobile phases can effect their ionization and allow their separation by anion-exchange chromatography (AEC). AEC separations of carbohydrates is now generally preferred because of the greater sensitivity of amperometric detection (detection limits<1 nanomole [67]), combined with the pos- sibility of influencing selectivity by small changes in mobile-phase pH. An example of such a separation is shown in Figure 7.21 for the separation of a sample that contains 11 different sugars. Other studies have demonstrated a significant role for temperature in affecting peak spacing and resolution for these separations [69]. See also Section 13.6. 7.5.8 Mixed-Mode Separations Mixed-mode columns can be thought of as hydrophobic ion-exchangers—in contrast to the more hydrophilic columns used for conventional IEC. As a result these columns exhibit both RPC and IEC behaviors. The original use of these columns was suggested by their different selectivity, compared to either RPC or IEC columns, and this feature continues as a reason for their use. An example is shown in Figure 7.22, where separations by RPC (Fig. 7.22a) and mixed-mode cation-exchange (Fig. 7.22b)are compared, for a nitrogen-mustard mixture (small, hydrophilic amines). The better retention (and resolution) of early-eluting peaks 1 to 6 in Figure 7.22b is obvious, . the analysis of any mixture that contained organic acids or bases. Together with liquid liquid partition and adsorption chromatography, IEC then accounted for most of the reported separations. concentration of Cl − changes from 0.32 to 0.35 to 0 .40 M, 7.5 ION-EXCHANGE CHROMATOGRAPHY (IEC) 353 02 040 020 Time (min) Time (min) 0 Time (min) 0.32M KCl 0.35M 0.40M (a) (b) (c) tri tetra hexa octa 10 Figure. ION-EXCHANGE CHROMATOGRAPHY (IEC) 351 samples that contain too many components to be separated in a single HPLC run. The principle of operation is the use of a first HPLC separation to achieve partial separation