Introduction to Modern Liquid Chromatography, Third Edition part 36 pptx

a Ionization of HA and B as a function of mobile-phase pH and effect on k; b sample separation as a function of mobile-phase pH; values of k0for HA and B are assumed equal, k±= 0, and t0

0 20

40

60

80

100

50% ionized

k

%

ioniized

pH-4

pH-5

pH-6

pH-7

pH-8

pH-9

pH-10

(a)

(b)

pK a HA

pK a B

B

B

B

B + HA HA

HA

HA

HA

HA

HA

B

B

B

t0

10

8

6 4

2 0 pH

Figure7.1 Hypothetical illustration of the RPC separation of an acidic compound HA

from a basic compound B as a function of pH (a) Ionization of HA and B as a function of mobile-phase pH and effect on k; (b) sample separation as a function of mobile-phase pH; values of k0for HA and B are assumed equal, k±= 0, and t0 = 1.0 min.

When pH= pKa(or [H+]= Ka), a compound is half ionized (F± = 0.5).

Figure 7.1b shows chromatograms for the separation of HA and B as a function

of mobile-phase pH As the pH of the mobile phase increases from 4 to 10, the acid HA (shaded peak) becomes more ionized and less retained, while the base B eventually becomes less ionized and more retained (for pH= 7, k = 0.1 for each

peak) It can be appreciated from this example that a change in mobile-phase pH can be a powerful means of controlling relative retention (selectivity) and separation for samples that contain acids and/or bases The relative retention of two acids (or bases) can vary with pH when their values of pKa, k0, and/or k±differ (usually the case) Some authors have claimed that the separation of partly ionized solutes (e.g., where significant amounts of both HA and A− are present at the same time) will

necessarily lead to poor peak shape, but there is no empirical or theoretical basis for this belief

A similar representation of RPC retention behavior as a function of pH is shown

in Figure 7.2, for the variation of retention time tRas a function of mobile-phase pH for a hypothetical, weakly basic solute with pKa = 5.0 (e.g., a substituted aniline or

pyridine) When pH is varied over a sufficiently wide range, solute retention exhibits

a characteristic S-shaped plot as shown; this retention plot mirrors the ionization of

the sample as in Figure 7.1a At the midpoint of this retention versus pH curve (solid

circle in Fig 7.2), the mobile-phase pH is equal to the pKa value of the solute The mobile-phase pH is often chosen in order to control selectivity and resolution When the mobile-phase pH ≈ pKa for a critical compound or compounds, a change in

pH will provide a maximum change in retention and separation Thus mobile-phase

pH should fall within region ‘‘II’’ of Figure 7.2 (pH= pKa± 1.0), if we want to

change selectivity and resolution by varying pH However, as discussed below, a mobile-phase pH that allows a greater control over selectivity (i.e., region II) can mean a less reproducible separation—one of many necessary compromises in HPLC method development

When an acid or base is half-ionized, a change in pH of 0.1 unit will result in a

change of k by about 10% For typical separation conditions, a 10% change in k for

a solute can result in a change in resolution of as much as±2.5Rsunits, meaning a

possible change in separation from baseline resolution (Rs> 1.5) to complete overlap (Rs= 0) Thus, if a solute is half-ionized, a change in mobile-phase pH by 0.1 unit can cause a complete loss of resolution This suggests that mobile-phase pH may need

to be controlled within about 0.02 units for such a separation, which could prove difficult for many laboratories (see Section 7.3.4.1) In order to avoid pH-related variations in retention, the mobile-phase pH can be selected to be different from the

pKavalues of all sample components, by at least±1.5 pH-units (regions I and III of Fig 7.2) As the majority of compounds have pKa values> 4, low-pH separations

(2≤ pH ≤ 3) are more likely to be less sensitive to small changes in pH—which

is one reason for beginning method development with a low-pH mobile phase (as recommended in this chapter) Separations at high pH (≥ 10) can also be used for this purpose, although special columns are required which are stable under these conditions (Sections 5.2.5, 5.3.2)

pKa± 1.0

pKa= 5

pH I

III

Figure7.2 RPC retention as a function of pH A basic solute with pKa = 5.0 is assumed.

A further illustration of the dependence of separation on pH is provided by

Figure 7.3, for several compounds of varying acidity or basicity Figure 7.3a maps

retention time versus mobile-phase pH for five solutes: compound 1 is salicylic acid (a relatively strong carboxylic acid), compound 2 is phenobarbital (a weak acid), compound 3 is phenacetin (a neutral compound in this pH range), compound 4 is nicotine (a weak base), and compound 5 is methylamphetamine (a strong base)

Figure 7.3b–e shows the corresponding chromatograms for the separation of this

sample as a function of mobile-phase pH Note, for example, the relative (and absolute) change in retention for strongly basic compound 5 (shaded peak); as pH increases, compound 5 becomes less ionized, and more retained

Several points are worth making about the example of Figure 7.3 First, for this mixture of acids, bases, and neutrals, a change in pH is a powerful means of varying relative retention and thereby optimizing resolution A maximum resolution

of Rs= 7.2 can be obtained for this sample at pH-8.3 (Fig 7.3f) in a time of

28 minutes Alternatively, baseline separation (Rs= 2.0) can be obtained in the shortest time (11 min) at pH-5 (Fig 7.3c) However, by reducing column length

from 300 to 50 mm for the separation at pH-8.3, and increasing flow rate from 2.0

to 5.0 mL/min, run time can be shortened to 2 minutes (Fig 7.3g), while maintaining

Rs≥ 2.0.

Second, this sample contains acids and bases with a wide range in pKa values (see following discussion) and therefore exhibits sizable changes in retention for small changes in pH throughout the range 3 < pH < 9 Consequently, either a

careful control of mobile pH will be required for the separation of this sample (Section 7.3.4.1) or conditions must be selected that provide excess resolution

(Rs 2) For example, the separation of Figure 7.3g with pH = 8.3 and Rs= 2.0 could be made more robust by using a 10-cm column (for Rs= 2.8 in a run time of

4 min), holding other conditions the same

Finally, the shape of a plot of retention versus pH for a peak allows a determination of its sample type (acid, base, or neutral), and a rough estimate of its

pKavalue Thus compounds in Figure 7.3a whose retention increases significantly as

30

20

10

0

pH

5 strong base (… )

4 weak base ( -)

3 neutral ( )

2 weak acid ( _ _ )

1 strong acid ( _ )

(a)

pH

Figure7.3 Effect of mobile-phase pH on RPC retention as a function of solute type Sample:

1, salicylic acid; 2, phenobartitone; 3, phenacetin; 4, nicotine; 5, methylamphetamine (shaded peak) Conditions for separations (a–f ): 300 × 4.0-mm C18column (10-μm particles); 40% methanol-phosphate buffer; ambient temperature; 2.0 mL/min Flow rate is 5.0 mL/min and

column length is 50-mm in (g) Adapted from [1], with chromatograms (b–g) recreated by

computer

pH increases are bases (4 and 5), those whose retention decreases with an increase

in pH are acids (1 and 2), and compounds that show little change in retention with

pH (3) are either neutral or are fully ionized over the pH range studied Compounds

2 and 4 are seen to have pKavalues of about 8 and 6.5, respectively While the pKa values of compounds 1 and 5 cannot be estimated accurately (a complete retention

vs pH curve is required), it is safe to say that pKa ≥ 9 for compound 5, and pKa≤ 3 for compound 1

The relationship between RPC retention and mobile-phase pH is more com-plicated for amphoteric compounds that contain both acidic and basic groups This

is illustrated in Figure 7.4 for the retention of two amino acids as a function of

pH A molecule of each compound contains both an acidic –COOH group and a basic –NH2 group As a result minimum retention is observed at intermediate pH values, because for 4< pH < 8 both the carboxyl and amine groups are ionized.

More precisely, the molecule is maximally ionized in this pH range, even though

the net charge is zero (different ionized groups within a molecule—even of different

sign—can each prefer the more polar mobile phase)

7.2.1 Choice of Buffers

Whenever acids or bases are separated, it is necessary to buffer the mobile phase in order to maintain a constant pH and reproducible retention during the separation The use of a pH meter to measure (and control) pH will be less precise when the mobile phase contains organic solvent because the electrode response tends to drift

pH-5

pH-7

pH-9

pH-8.3 (300-mm column)

pH-8.3 (50-mm column)

Time (min)

0

0

Time (min)

Time (min)

Time (min)

Time (min)

4

5

2

1 +

3

1

4

5

2 3

1

2

5

(b)

(c)

(d)

(e)

(f)

(g)

1

2

1

2

5

Time (min)

1

2

5

Figure7.3 (Continued)

for organic-water solutions Consequently we recommend that pH measurements

be carried out for the A-solvent (aqueous buffer) prior to the addition of organic to form the final mobile phase The pH of the final mobile phase (including organic

solvent) can then be equated to (or labeled as) that of the A-solvent, although the actual mobile-phase pH will be somewhat different (Section 7.2.3) This uncertainty

0 2 4 6 8 10 12 14

pH

25

20

15

10

5

k

70 60 50 40 30 20 10

RCHCOO− RCHCOO−

RCHCOOH

Figure7.4 Dependence of RPC retention on mobile-phase pH for amphoteric compounds Sample: phenylalanine (•), leucine (o) Conditions: polystyrene column, 40-mM phosphate buffer as mobile phase Adapted from [2]

concerning the final mobile-phase pH is unimportant for the routine application of RPC When the A-solvent is prepared in this way, different laboratories should be able to obtain the same final mobile-phase pH within±0.04 to 0.05 units [3] If a

closer control of mobile-phase pH is required, see Section 7.3.4.1 When we refer

to mobile-phase pH in this book, we will generally mean the pH of the A-solvent.

Directions for the preparation of buffer solutions of varied pH and buffer-type are given in Appendix II

In selecting a buffer for RPC separation, several buffer properties may prove relevant:

• pKaand buffer capacity

• solubility

• UV absorbance (when UV detection is used)

• volatility (when mass-spectrometric or evaporative light-scattering detection

is used)

• ion-pairing properties

• stability and compatibility with the equipment

The first four buffer properties are usually the most important

7.2.1.1 Buffer pKaand Capacity

‘‘Buffer capacity,’’ or the ability of the buffer to maintain a constant pH, depends on

• pKavalue of the buffer

• buffer concentration

• pH of the mobile phase

Just as for the ionization of a sample component in Figure 7.1a, the fractional

ionization of the buffer as a function of pH can be expressed by Equations (7.2) or (7.2a); that is, buffer and solute ionization are identical functions of mobile-phase

pH and pKa Maximum buffering occurs when the concentrations of the two forms

of the buffer (e.g., HA and A−) are equal; that is, when the buffer pKa equals the mobile-phase pH Buffer capacity decreases as values for the buffer pKa and mobile-phase pH become more different Consequently the first requirement of the buffer is that its pKavalue should be within±1.0 units of the selected mobile-phase

pH (this requirement can be relaxed to±1.5 unit for higher concentrations of the buffer) The buffer capacity of the mobile phase is proportional to buffer concentra-tion, which typically falls within a range of 5 to 25 mM To minimize the possibility

of inadequate buffering of the sample during RPC separation, it is generally desirable for the sample to be dissolved in the mobile phase (or buffered to the same pH as the

mobile phase); this practice becomes especially important for lower concentrations

of the mobile-phase buffer, and/or for larger volumes of injected sample

Table 7.1 provides a list of buffers that can be used in RPC, along with pertinent properties such as buffer pKaand the mobile-phase pH range in which the buffer is effective For separations with UV detection, and a mobile-phase pH≤ 8, popular buffers include phosphate, trifluoroacetate, acetate, and formate In addition ammonium bicarbonate can also be considered The pKa values of ammonia (9.2) and bicarbonate (10.3) overlap, hence somewhat extending the buffering range of ammonium bicarbonate (8.2 ≤ pH ≤ 11.3) This buffer is volatile and therefore compatible with LC-MS; however, when the buffer pH < 8.5, loss of CO2 (e.g., from excessive degassing) may lead to an unintended increase in pH Because of this instability it is recommended to prepare fresh ammonium bicarbonate buffers daily

The remaining discussion of this section (7.2.1.1) is more detailed The reader may wish to skip this digression, proceed to Section 7.2.1.2, and return to the present section as needed.

We can define the ‘‘effective buffer capacity’’ of the mobile phase to mean that

an increase in this quantity will result in fewer problems due to insufficient buffering The effective buffer capacity of the mobile phase increases for:

1 a smaller difference between values of the buffer pKa and the pH of the mobile phase (change either the buffer or pH)

2 a greater difference between the mobile-phase pH and the pKa of the solute (for large differences, the solute is either non-ionized or completely ionized; buffering is then much less important)

3 increased buffer concentration

4 smaller volumes of injected sample

5 a sample whose pH is adjusted to that of the mobile phase

An example of inadequate buffering is provided by the chromatograms of Figure 7.5 for the solute 3,5-dimethylaniline as a function of mobile-phase pH, using

a 25 mM potassium phosphate buffer Despite this sizable buffer concentration,

Table 7.1

Buffers for Use in HPLC Separation

(25 ◦ C) Buffer Range Cutoffb

Trifluoroacetic acid >2 1.5–2.5 210 nm (0.1 %) Ion-pairing, volatile

Bis-tris propane.HCle 6.8 75.5–8.0 215 nm (10 mM) Possibly unstablef

1-methylpiperidine.HCl 10.1 9.0– 11.5 215 (10 mM) Possibly unstablef

Triethylamine.HCl 11.0 9.5– 12.5 <200 (10 mM) Possibly unstablef

aBuffer composed of buffer acid plus ionized acid, such as for phosphoric acid, H 3 PO 4 and H 2 PO 4−.

bAqueous solutions; absorbance<0.5 AU at wavelengths above cutoff

cClaimed to attack stainless steel; we have experienced equipment problems associated with the long-term use of citrate buffer.

dUse of this buffer is impractical, because of loss of CO2from the reservoir.

e1,3-Bis[tris(hydroxymethyl)methylamino] propane.

fAmmonium carbonate is unstable at pH-7.0 (loss of CO 2 to atmosphere), but stable at pH-8.5 [11]; amine buffers tend to oxidize, with a large increase in UV absorbance.

gTris(hydroxymethyl)aminomethane.

a slight tailing of the peak is seen in Figure 7.5 for a mobile-phase pH= 3.0.

As the pH increases to 3.5 (Fig 7.5b) and 4.0 (Fig 7.5c), the peak progressively

broadens and becomes more distorted (peak fronting)— the result of decreased buffer capacity The pKa values of solute and buffer are 3.8 and 2.1, respectively,

so as pH increases above 3.0 in this example, the difference between the buffer pKa value and mobile-phase pH (factor 1 above) increases, while the difference between mobile-phase pH and the pKaof the solute decreases (factor 2 above) Together, this results in a decrease of the effective buffer capacity as the mobile-phase pH increases from 3 to 4—and a progressive deterioration of peak shape Poor peak shape in each of the examples of Figure 7.5 could be improved by any of the experimental factors 3 to 5 on p 312 (increased buffer concentration, etc.)

pH = 3.0 3.5 4.0

Figure7.5 Effect of insufficient buffer capacity on peak shape for 3,5-dimethylaniline

as solute Conditions: column, 250× 4.6-mm cyano column (5-μm particles); 25%

methanol-buffer, buffer is 50-mM potassium monophosphate; 35◦C; 1 mL/min Adapted from [4]

When we wish to reduce buffer concentration for any reason (limited buffer solubility, increased UV absorbance, etc.), we need to optimize other contributions to buffer capacity (factors 1, 2, 4, or 5 on p 312); for example, choose a mobile-phase

pH that provides either minimal or maximal ionization of the sample (factor 2) For further discussion of buffer capacity in RPC, see [5]

7.2.1.2 Other Buffer Properties

The remaining buffer properties listed at the end of Section 7.2.1 should also be considered when developing a RPC separation for an ionic sample

Buffer Solubility Organic buffers are usually adequately soluble in all

organic-water mobile phases (0–100% B), but many inorganic buffers have limited solubility

in mobile phases which are predominantly organic (high %B) Consequently there

is a danger that combining the A- and B-solvents may result in buffer precipitation, which could lead to blockage of the column or HPLC equipment If there is any doubt as to whether a mobile phase might precipitate, the complete solubility of the buffer in the final mobile phase should be confirmed first (over the intended

pH range), especially when the A- and B-solvents are mixed by the HPLC pumping system Thus varying proportions of the A- and B-solvents can be combined manually

in a container and observed over a period of 30 minutes or so If any cloudiness develops, or a precipitate is observed for a given mobile-phase composition (%B), mobile phases of that %B or higher should be excluded or the buffer concentration should be reduced (see the discussion of [5, 6] for further details) Buffer solubility

is of special concern for separations by gradient elution (Section 9.3.1)

Buffer solubility is affected by several separation conditions [6] The buffer counter-ion is one such factor; for acidic buffers such as phosphate, buffer solubility usually increases as

sodium salt (least soluble)< potassium salt < ammonium salt (most soluble)

Similarly buffer solubility varies with the organic solvent (B-solvent), provided that comparisons are made for the same %B and pH:

tetrahydrofuran (least soluble)< acetonitrile < methanol (most soluble)

Buffer solubility is also affected by the relative ionization of the buffer; as the charge

on the buffer ion increases (e.g., HPO4=vs H2PO4−), the buffer becomes less soluble

in high-%B mobile phases Thus the choice of buffer and other separation conditions permits a considerable control over buffer solubility Because most isocratic RPC separations of ionic samples are carried out with mobile phases of<60% B, however,

buffer solubility is usually not a problem To a lesser extent this is also true for gradient elution because, when buffer is added only to the A-solvent (the usual practice for inorganic buffers), buffer concentration in the mobile phase becomes inversely proportional to %B (also true for isocratic elution method development) Buffer solubility is usually only an issue for inorganic buffers, especially phosphate One study [6] has reported that potassium phosphate has an ambient-temperature solubility at pH-7 of 10 mM for either 85% MeOH-water or 75% ACN-water (with higher solubilities at lower %B, and vice versa for higher

%B) At pH-3, a solubility of 10 mM can be achieved with 85% MeOH or 85% ACN A phosphate-buffer concentration of 1 to 2 mM, combined with other favorable choices from the list ‘‘effective buffer capacity’’ of Section 7.2.1.1, should allow %B values as high as 90% for either methanol or acetonitrile as B-solvent Fortunately, %B-values this high are rarely required for ionized compounds, in which case buffer solubilty may no longer be an issue

Detector Requirements The absorbance of the buffer is proportional to buffer

concentration and adds to the absorbance of the water-organic mixture used for the mobile phase Table 7.1 provides a rough guide for assessing whether a given buffer will result in a significant increase in the UV absorbance of the mobile phase The influence of buffers on gradient baseline drift is illustrated in Figures 17.7 to 17.10 Additional information on buffer absorbance versus wavelength is provided

in Table I.2 of Appendix I

Mass spectrometric detection (LC-MS) requires a volatile buffer Common choices include trifluoroacetic acid (TFA), acetic acid, formic acid, and their ammo-nium salts For the separation of basic compounds at low pH, with volatile buffers such as formic or acetic acid, it is preferable to select a mobile phase with a higher ionic strength; that is, a higher buffer concentration and a pH where the buffer

is significantly ionized (i.e., choose a mobile-phase pH that is fairly close to the

pKa value of the buffer [7]) Otherwise, even small weights of injected sample can result in column overload and peak tailing because of the ionic repulsion of retained molecules of protonated bases BH+ (Section 15.3.2.1) For example, ammonium formate can be used as buffer at a pH of 3.5 to 4.0, whereas the use of formic

or acetic acid alone (at a lower pH) provides much less ionization of the buffer With reduced buffer ionization, column overload and peak tailing become more likely For additional information on the best choice of buffer for MS detection, see Section 4.14

Ion-Pairing by the Buffer So far we have assumed that the sole effect of the

buffer on sample retention and separation is to control mobile-phase pH and sample ionization Additionally an ionized buffer X can interact with an ionized solute