Introduction to Modern Liquid Chromatography, Third Edition part 39 pptx

7.4.1.2 Ion-Pair Reagent: Concentration and Type It is possible to continuously vary the nature of retention, from RPC retention as in Figure 7.11a to IPC retention as in Figure 7.11b, b

sorbed TBA+ For examples of these generalizations, see the discussion of Figure 7.12

in the following section

7.4.1.2 Ion-Pair Reagent: Concentration and Type

It is possible to continuously vary the nature of retention, from RPC retention as

in Figure 7.11a to IPC retention as in Figure 7.11b, by varying the concentration

of the IPC reagent in the stationary phase The concentration of the reagent in the stationary phase can be varied by changing its concentration in the mobile phase We will use the example of Figure 7.12 to illustrate the effect of IPC reagent concentration on solute retention In this example a tetrabutylammonium phosphate IPC reagent (TBA+) is assumed initially, with a C8 column Consider first the equilibrium uptake of TBA+≡ R+ by the stationary phase (solid curve for TBA+

at the bottom of Fig 7.12a) The concentration of R+in the stationary phase [R+]s

is plotted against its concentration in the mobile phase [R+]m, showing a continued increase in stationary phase concentration as [R+]m increases, until the stationary phase becomes saturated with R+(with no further change in concentration of R+in the stationary phase for further increase in [R+]m) The form of this plot is typical

of the uptake of sample or other molecules by a RPC column, when the sample concentration in the mobile phase is increased (so-called Langmuir adsorption; Section 15.3.1.1 and Eq 15.1)

Two different plots of [R+]sversus [R+]m are shown in Figure 7.12a: a solid

curve for TBA+, and a dashed curve for tetraethylammonium (TEA+) Because TBA+ is the more hydrophobic of the two IPC reagents, it is retained by the stationary phase more strongly and saturates the column at a lower concentration

of R+ in the mobile phase The extent of ion-pairing will depend on the fractional saturation of the stationary phase, and this is seen to depend on (1) the IPC reagent concentration in the mobile phase and (2) the hydrophobicity or retention of the IPC reagent IPC reagent hydrophobicity and retention will increase for an increase in the carbon number of the reagent (number of CH3- plus CH2- groups in the reagent molecule), making TBA+ (with 16 carbons) more hydrophobic and more retained than TEA+(with 8 carbons) We also see in Figure 7.12a (dotted lines) that a larger concentration y of a less hydrophobic reagent (TEA+) can result in the same uptake [R+]s of reagent by the column as a lower concentration x of a more hydrophobic

IPC reagent (TBA+), consequently resulting in similar ion pairing and retention of the solute A−(e.g., see later Fig 7.15 and the accompanying discussion)

Consider next the change in sample retention k as the IPC reagent concentration increases (Fig 7.12b) Assume a fully ionized acidic solute RCOO−, whose RPC retention is essentially zero for some value of %B When an IPC reagent is added to

the mobile phase, k will increase initially as a result of the interaction of RCOO−

with R+ in the stationary phase Once the stationary phase is saturated with R+,

no further increase in retention of RCOO− can occur; however, a further increase

in the mobile-phase concentration of R+ is accompanied by an increase in the concentration of its counter-ion X− (e.g., H2PO4−) An increase in the H2PO4− concentration [H2PO4−]mthen competes with RCOO− for ion-exchange retention

(Eq 7.9a), leading to a gradual decrease in k as shown in Figure 7.12b Thus

preferred concentrations of the IPC reagent in the mobile phase should not exceed a value that begins to saturate the column—so that solute retention then declines

(b) Retention of ionized acid RCOO− vs concentration of R+

in mobile phase [R+]

m

mobile phase

stationary phase

(a) Uptake of IPC reagent R+ by the stationary phase

+ +

TBA + H 2 PO4+

TBA + H 2 PO4− TBA + H 2 PO4−

TEA + TBA +

H2PO4+

C8

C8 C8 C8 C8

C8 C8 C8 C8 C8 C8 C8

C8 C8 C8 TBA + RCOO−

RCOO−

k

[R +] m

[R +] m

[R +] s

IPC retention

competition with H2PO4−

Figure7.12 Representation of the uptake of IPC reagent by the column, and its effect on solute retention

7.4.1.3 Simultaneous Changes in pH and Ion Pairing

When mobile-phase pH and the concentration of the IPC reagent are varied simulta-neously, a remarkable control is possible over retention range and relative retention for ionic samples (as anticipated by Fig 7.10) This can be visualized for an

indi-vidual solute from the plots of k against either pH or IPC reagent concentration

in Figures 7.11b and 7.12b Now consider that similar plots will result for other solutes, but with the possibility of different dependencies of k on pH and IPC

reagent concentration An example of separation as a function of change in both

0 2

Time (min)

Time (min)

Time (min)

B

HA1 +

HA2 N

B

N

HA2

HA2

HA1

HA1

N

B

(a)

pH-2.5

RPC

(b)

pH-5.0

RPC

(c)

pH-7.5

RPC

Time (min)

HA2

HA1 N

B

pH-5.0 IPC (R −)

(d)

Figure7.13 Example of the separation of an ionic sample where both mobile-phase pH and IPC reagent concentration are varied Sample: B, pseudoephedrine; N, glycerol guaicolate (a neutral compound, shaded); HA1, sodium benzoate; HA2, methylparaben (a phenol) Conditions: 150× 4.6-mm C8column (5-μm particles); 30% methanol-citrate buffer with 130-mM hexane sulfonate for the IPC mobile phase; 50◦C; 3.0 mL/min Adapted from [51]

mobile-phase pH and IPC reagent concentration is illustrated in Figure 7.13 The sample consists of a neutral compound (N; shaded peak), a weakly basic compound (B), an acidic compound (HA1) and a weakly acidic compound (HA2) Three

separa-tions were carried out (Fig 7.13a–c), with pH varying but without any IPC reagent

in the mobile phase The dotted lines track changes in relative retention for each

peak A fourth separation (Fig 7.13d) uses a mobile phase with intermediate pH

plus added hexane sulfonate (R−) as the IPC reagent

Consider first the retention of the neutral compound N (shaded peak) As pH is

varied in the separations of Figure 7.13a–c, there is little change in its retention—as

expected When the IPC reagent (R−) is added in Figure 7.13d, the retention of

N is reduced as a result of partial blockage of the stationary-phase surface by sorbed

R− Next consider the retention of weakly basic compound B Its retention increases for pH> 5, and the addition of IPC reagent in Figure 7.13d (pH = 5.0), increases

retention even more (the arrows in Fig 7.13d indicate changes in retention vs the separation of Fig 7.13b [same pH]) The various changes in the retention of B are

the result of a decreasing ionization of this basic solute (BH+ → B) as pH increases, while addition of the IPC reagent R− confers a negative charge on the column that attracts the positively charged BH+ Finally, acidic compounds HA1and HA2show decreased retention with increase in pH, and their retention is further decreased by

the addition of the IPC reagent R− (because of ionic repulsion between A− and

R−in the stationary phase, plus partial coverage of the stationary phase by sorbed molecules of R−)

Finally, we can see that as a result of change in both mobile-phase pH and IPC reagent concentration, major changes in relative retention result for each of the conditions of Figure 7.13:

Low pH, no ion pairing (a):

B< N < AH1= AH2

Intermediate pH, no ion pairing (b):

B< HA1< N < HA2

High pH, no ion pairing (c):

HA1< N < B < HA2

Intermediate pH, ion pairing, d:

HA1< N < HA2< B

The best separation for this sample is seen in Figure 7.13d, using an IPC

reagent at pH-5.0

To summarize, the concentration and type of the IPC reagent can be varied for systematic changes in ion pairing, with predictable effects on retention range and relative retention When an anionic IPC reagent (e.g., an alkylsulfonate) is added to the mobile phase, the retention of ionized basic compounds will be increased, and the retention of neutral and (especially) acidic compounds will be decreased When

a cationic IPC reagent (e.g., a tetraalkylammonium salt) is used, the retention of ionized acidic compounds will be increased, while that of neutral and (especially) basic solutes will be decreased These changes in retention will be greater for larger concentrations of the IPC reagent in the mobile phase Changes in mobile-phase pH that increase the ionization of a compound will increase the effect of the IPC reagent

on separation

7.4.2 Method Development

IPC method development is similar to that for the RPC separation of ionic samples

(Section 7.3.3) The same seven method-development steps listed in Figure 6.21a

for neutral samples still apply, with only step 3 (‘‘choosing separation condition’’) differing for IPC separation As in the case of RPC method development, the choice

of separation conditions for IPC includes the following steps:

1 choose starting conditions

2 select %B for 1≤ k ≤ 10 (with IPC reagent in the mobile phase)

3 adjust conditions for improved selectivity and resolution

4 vary column conditions for best compromise between resolution and run

time

In most cases, an RPC separation (without IPC reagent present) will have

been attempted initially, including a study of changes in mobile-phase pH (our

recommendation) Consequently it is likely that different peaks in the chromatogram

can be assigned as neutrals, acids, or bases (as in the example of Fig 7.3) This approach also explores the possibility of a non-IPC, RPC separation—with a simpler mobile phase and one less likely to have IPC-related problems (Section 7.4.3) Even when IPC separation is anticipated at the beginning of method development, initial experiments should proceed in similar fashion as described in Section 7.3.3 (development of an RPC method for ionic samples)—without addition of the IPC reagent to the mobile phase The latter experiments will define the approximate

%B required for an acceptable retention range (e.g., 1< k < 10) or at least a %B

value for an average value of k that falls within this range As in the case of

RPC method development for ionic samples, only steps 1 and 3 above differ for IPC

7.4.2.1 Choice of Initial Conditions (Step 1)

The requirement of both a buffer and an IPC reagent in the mobile phase may favor the use of methanol as B-solvent, because of the greater solubility of these additives

in methanol (TFA and HFBA, however, have adequate solubility in acetonitrile but are weaker IPC reagents) If acetonitrile is used in preliminary RPC experiments (our recommendation), and if solubility problems are subsequently encountered with this solvent, methanol can be substituted (guided by the solvent strength nomograph

of Fig 6.11) In most cases an alkylsulfonate will be chosen as IPC reagent for samples that contain basic compounds, while a tetraalkylammonium salt will be used for acidic samples When both acids and bases are present in the sample,

either type of IPC reagent may prove useful, but it is not recommended to add both

reagents to the mobile phase The reagents tend to ion-pair with each other, with cancellation of their net effect on separation; the use of two IPC reagents would also complicate method development An alkylsulfonate is preferred when it is necessary

to selectively increase the retention of basic solutes, while a tetraalkylammonium salt can increase the retention of acidic solutes

For mixtures of acids and bases, a low-pH mobile phase plus an alkylsulfonate IPC reagent is a good starting point because the pH suppresses ionization of acids and the IPC reagent retains the bases The final choice of one or the other of these reagents can be determined from information acquired during preliminary RPC experiments; specifically, retention as a function of mobile-phase pH Both sulfonates and quaternary ammonium salts can be used with UV detection at a wavelength of 210 nm or higher

The discussion of Section 7.4.1.2 and Figure 7.12a suggest that similar

separations can be obtained with different concentrations of two different alkyl-sulfonates (or quaternary ammonium salts), for example, a lower concentration

of a C8-sulfonate, or a higher concentration of a C6-sulfonate This is generally correct [47, 48], but the practical question is then: Which IPC reagent and which concentration should be used for an initial IPC experiment? One study [49] has provided an approximate answer to this question, as summarized in Figure 7.14

10

1

100 80

60 40

20 0

% MeOH

100 80

60 40

20 0

% MeOH

C4

(C1)4 (C2)4

(C3)4 (C

4 )4

(C5)4

8

C10

C12

100

10

(a) Recommended concentrations of different alkyl sulfonates

(b) Recommended concentrations of different tetraalkylammonium salts

[R −]

m

[R +]

m

Figure7.14 Recommended IPC reagents and concentrations as a function of mobile-phase

%B: (a) sulfonate IPC reagent; (b) quaternary ammonium IPC reagent Adapted from [49].

Conditions corresponding to shaded regions are not recommended

The recommended starting concentrations of different sulfonates (Fig 7.14a) or quaternary ammonium salts (Fig 7.14b) are plotted against %-MeOH in the mobile

phase For example, given a mobile phase of 40%B, and the planned addition

of an alkylsulfonate as IPC reagent, Figure 7.14a suggests that either 75 mM of

octanesulfonate (C8) or 15 mM of decanesulfonate (C10) would be a suitable starting

concentration Alternatively, for the use of a tetraalkylammonium salt (Fig 7.14b)

with a mobile phase of 40% B, either 70 mM of tetrabutylammonium (C4) or

20 mM of tetrapentylammonium (C5) is recommended It is desirable to select an initial concentration of the IPC reagent between 5 and 100 mM, as indicated by the

unshaded regions of Figure 7.14a, b.

The (initial) concentrations recommended in Figure 7.14 will provide about

1/3 surface coverage by the reagent Varying the concentration up or down from this initial value then allows a significant change in reagent uptake by the column, with predictable changes in relative retention (Section 7.4.1.2) If acetonitrile is used

as B-solvent for IPC separation, Figure 7.14 can still be used for estimates of IPC reagent type and concentration, but the equivalent value of %B for MeOH should

be (very approximately) doubled Thus, if the mobile phase consists of 20% ACN,

a value of 2× 20 ≈ 40% MeOH should be used in Figure 7.14 [50] for the purpose

of selecting an IPC reagent and its initial concentration That is, acetonitrile is a stronger solvent than methanol, so a lower value of %-ACN is equivalent to a higher value of %-MeOH

An example that illustrates some of the principles above is provided by Figure 7.15, where the separation of a mixture of water-soluble vitamins is exam-ined as a function of IPC reagent concentration and type As the sample includes compounds with both acidic (anionic) and basic (cationic) character, either a sulfonate or quaternary ammonium salt could be used for ion pairing If a sul-fonate is selected, Figure 7.14 suggests for this mobile phase (15% methanol-buffer [pH-3.2]) the use of hexane, heptane, or octane sulfonate as IPC reagent Sepa-rations of the sample with varying concentSepa-rations of hexane sulfonate are shown

in Figure 7.15a–c Peaks 1 to 3 exhibit little change in retention with changing

reagent concentration and can be regarded as effectively neutral (neither anionic

or cationic) Peaks 4 and 6 show an increase in retention as the reagent concen-tration increases, so these peaks must be cationic (protonated bases or quaternary

Time (min)

Time (min)

Time (min)

Time (min)

4

4

1

2

4

4 +

4 +

(a)

(b)

(c)

(d)

(e)

(f )

Figure7.15 IPC separation of a sample of water-soluble vitamins as a function of IPC reagent

concentration and type Sample: 1, ascorbic acid; 2, niacin; 3, niacinamide; 4, pyridoxine;

5, folic acid; 6, thiamine; 7, riboflavin Conditions; 83 × 4.6-mm C8column (3-μm parti-cles); 15% methanol- buffer (pH-3.2); 35◦C; 2.0 mL/min Chromatograms recreated from data in [48]

ammonium compounds) Similarly peaks 5 and 7 exhibit decreased retention as the reagent concentration increases and are therefore anionic (ionized acids) From

these initial experiments (Fig 7.15a–c), it appears that a hexane sulfonate

concen-tration of 6 to 7 mM would provide maximum resolution of peaks 4 to 7 (placing peak 5 midway between peaks 4 and 6, but without moving peak 7 too close to peak 6)

Figure 7.15d –f shows corresponding separations with heptane sulfonate as

IPC reagent; in each case, the reagent concentration is reduced fourfold compared

to separations with hexane sulfonate as IPC reagent The resulting separations for

Figure 7.15a and d,b and e, or c and f , are each quite similar (but not identical) That is, essentially the same separation can be achieved for this sample with a lower

concentration of a more hydrophobic IPC reagent, but the selectivity may not be

exactly the same The arbitrary substitution of one IPC reagent for another in a

previously developed method is therefore not recommended.

Inorganic reagents (or ‘‘chaotropes’’) such as ClO4−, BF4−, and PF6− have also been used in IPC [52, 53], in place of the usual alkane sufonates Because of the lesser retention of inorganic reagents, it is likely that the retention mechanism is based on Equation (7.9)— ion-pairing in the mobile phase—rather than Equation (7.9a)—sorption of the IPC reagent Chaotropes are advantageous in being better suited for gradient elution (less baseline noise and drift) and are more soluble in mobile phases with larger values of %B The relative ion-pairing strength of various anions (including both buffers and IPC reagents) increases in the following order:

H2PO4− < HCOO−< CH3SO3−< Cl−< NO3−

CF3COO−< BF −

4 < ClO4−< PF −

6

Only the last four anions are useful for IPC Because inorganic IPC reagents (chaotropes) are less strongly retained by the stationary phase, this can mean faster equilibration of the column when changing the mobile phase (Section 7.4.3.2) The effect of chaotropes in altering the retention of protonated bases appears much more pronounced for acetonitrile as B-solvent, compared to methanol or tetrahydrofuran [54]

7.4.2.2 Control of Selectivity (Step 3)

The separation conditions available for the control of selectivity in IPC include:

• pH

• IPC reagent type (sulfonate, quaternary ammonium salt, chaotrope)

• IPC reagent concentration

• solvent strength (%B)

• solvent type (ACN, MeOH, etc.)

• temperature

• column type

• buffer type and concentration

Despite the large number of variables that can affect selectivity in IPC, usually only

a few of these conditions need to be investigated during method development Fur-thermore the effects on retention of several of the conditions above are interrelated Thus a change in mobile-phase pH will, in some cases, give similar results as a change in IPC reagent concentration; for example, an increase in pH or an increase

in the concentration of an alkylsulfonate IPC reagent will in each case result in an increase in the retention of basic solutes (and a decrease in retention for acids) Also

we have seen that the primary effect of a change in %B or temperature may be the result of associated changes in the ‘‘effective’’ pH of the mobile phase—hence providing similar changes in relative retention as for a change in mobile-phase pH Other examples of this kind are noted below

Mobile Phase pH and IPC Reagent Type or Concentration The combined effects

of these conditions on IPC separation were discussed in detail above (Section 7.4.1), and are best investigated first during IPC method development It is more convenient

to vary the concentration of the IPC reagent (as in Fig 7.15a–c), than to change the IPC reagent (as in Fig 7.15f vs a).

Solvent Strength When %B is varied for the RPC separation of ionic samples

(Section 7.3.2.2; Fig 7.7), changes in both absolute and relative retention can be expected In some cases these change in retention can be related to corresponding changes in the ‘‘apparent’’ pH of the mobile phase (or values of pKafor the solute; Section 7.2.3) Corresponding changes in relative retention with %B should also occur for IPC separation, but with an added feature Thus, if %B is increased, the uptake of the IPC reagent by the column will decrease, just as for the case of sample molecules Consequently a change in %B should lead to predictable changes

in relative retention for peaks that are strongly affected by ion pairing An example

is presented in Figure 7.16 for the same sample of Figure 7.10 An initial separation

with 40% MeOH and octanesulfonate as IPC reagent is shown in Figure 7.16a, with

the four protonated bases (X, X1–X3) distinguished as shaded peaks (remaining peaks correspond to either neutral or acidic solutes) When the mobile phase is

changed to 45% MeOH in Figure 7.16b, and 50% MeOH in Figure 7.16c, the

retention of all peaks decreases (solvent strength effect), but the four bases become

even less retained relative to the remaining neutral and acidic peak (they move

toward the front of the chromatogram) This behavior is predictable, as the increase

in %MeOH will result in a decrease in the retention of the IPC reagent (R−) by the stationary phase The reduced concentration of R− in the stationary phase means a reduction in ion-pairing for the cationic species X, X1–X3, and therefore

their reduced retention—apart from the general decrease in retention for all peaks

when %MeOH is increased The variation of %MeOH in this example shows the exceptional power of a change in %B in IPC to affect band spacing and resolution

(note the optimized separation for 45% MeOH in Fig 7.16b).

Solvent Type A change in solvent type usually leads to changes in IPC

selectivity, for either neutral samples (Section 6.3.2; Fig 6.9) or (especially) ionic samples (Section 7.3.2.3) Because of the added effect of the B-solvent on the uptake of IPC reagent by the column, a change of solvent type in IPC can result

in even larger changes in selectivity than in RPC Figure 7.17 provides a striking

B

X + MP

PP

Time (min)

Time (min)

Time (min)

HB

B

X

X1

X1

X1

X2

X2

X2

X3

X3

X3

PP MP

(a)

(b)

(c)

40% MeOH

45% MeOH

50% MeOH

HB

B MP X

PP

Figure7.16 Solvent-strength selectivity in IPC separation Sample and conditions as in Figure

7.10b, except for indicated % methanol Peaks for protonated bases X, X1–X3 shaded.

Adapted from [45]

example of solvent-type selectivity in IPC, for a change of B-solvent from MeOH in

Figure 7.17a to ACN in Figure 7.17b, with use of the solvent nomograph of Figure

6.11 A combined variation of mobile-phase pH, IPC reagent concentration, and solvent type should prove especially effective for the separation of challenging ionic samples [48, 50]

Temperature A change in temperature for IPC should also have a pronounced

effect on relative retention Temperature will alter the amount of IPC reagent held by

the column For this reason temperature control during IPC separation is especially

important.

Column Type and Buffer We have seen that column type can have a major

effect on selectivity in RPC separations of ionic samples, and it seems likely that this will also be true for IPC separation However, the partial coverage of the stationary phase surface by IPC reagent may tend to mask the contribution of the column per se to sample retention In view of the many other ways in which selectivity can

be controlled in IPC, the use of column type for this purpose should not be a first choice, nor is it likely to be especially promising Likewise the effect of buffer type and concentration on IPC separation can be significant, but larger, more predictable changes in selectivity can be obtained by varying pH, %B, temperature, and/or the type and concentration of the IPC reagent