Introduction to Modern Liquid Chromatography, Third Edition part 28 docx

In the remainder of Section 5.4, we will first discuss the basis of column selectivity, which can be attributed to different interactions between solute molecules and the column.. 5.4.1 B

C ≡N

CH3

(a) Alkylsilica columns

X

Phenyl columns Cyano column “Other”columns

Embedded-polar-group R-NH-(C=O)-O- Carbamate

R-NH-(C=O)-NH- Urea R-(C=O)-NH- Amide Fluoro columns

Perfluorophenyl (PFP)

-CF2CF2CF3 Fluoroalkyl

F

Figure5.19 RPC columns classified according to the ligand (figures omit the connecting silane group [–Si(CH3)2–])

simplified cartoons of Figure 5.19 (the—Si[CH3] group is omitted in Fig 5.19a–d).

The ligand of a RPC column is often an alkyl group, for example, C3, C8, C18

(Fig 5.19a) Alternatively, the ligand may consist of phenylpropyl or phenylhexyl, called phenyl columns (Fig 5.19b) If the ligand is –C3–C≡N (Fig 5.19c), we have a

cyano column The alkyl group may also be substituted by other functional groups

X (Fig 5.19d), and this gives rise to the additional column types listed at the bottom

of Figure 5.19 So-called embedded-polar-group (EPG) phases have been growing

in popularity, because of their compatibility with low %B mobile phases, their reduced silanol interactions, and unique selectivity (Section 5.4.1); peak shape for basic solutes is usually quite good with these columns The ligands in these phases contain amide, carbamate, urea (all of which are strong hydrogen-bond bases), or other polar functional groups embedded within the ligand structure Some EPG

packings tend to be less stable than comparable alkyl or aryl columns The nature

of the ligand mainly determines column selectivity, which is the subject of following Section 5.4

Column selectivity can be important for different reasons During method devel-opment a change of column may be necessary to improve selectivity and increase resolution (Sections 2.5.2, 5.4.3) For the latter application we must be able to

iden-tify a second column with quite different selectivity When a routine RPC procedure

is used at different times and places, a replacement column from the same source may not be immediately available locally, or too costly, or impractical for other reasons (Sections 5.4.2, 6.3.6.1) In this case we must identify a column of equivalent (or

at least similar) selectivity For either situation, we require a quantitative procedure that allows us to compare column selectivity Column selectivity is also related to certain problems that can arise during either method development or the routine use

of an RPC procedure: peak tailing, the deterioration of a column during use, and

‘‘de-wetting’’ of the column when used with mobile phases that are predominantly aqueous (Section 5.3.2.3) Finally, knowledge concerning column selectivity helps

us understand sample retention as a function of the column and solute molecular structure, in turn preparing us to better deal with various separation challenges

In the remainder of Section 5.4, we will first discuss the basis of column

selectivity, which can be attributed to different interactions between solute molecules and the column This will lead to quantitative values of those properties of a

column that determine its selectivity Finally, we will discuss the use of these

column-selectivity properties for both method development and the routine use of

an RPC procedure

5.4.1 Basis of RPC Column Selectivity

As discussed in Section 2.3.2.1, solute retention is determined by various interactions among the solute, mobile phase, and stationary phase (column) The relative impor-tance of different solute–column interactions—and column selectivity—depends on the composition of the stationary phase and the molecular structure of the solute Figure 5.20 illustrates eight different interactions that can affect column selectivity:

(a) hydrophobic interaction

(b) steric exclusion of larger solute molecules from the stationary phase

(here referred to as ‘‘steric interaction’’)

(c) hydrogen bonding of an acceptor (basic) solute group by a donor (acidic)

group within the stationary phase (usually a silanol –SiOH)

(d) hydrogen bonding of a donor (acidic) solute group by an acceptor (basic)

group within the stationary phase (represented here by a group ‘‘X’’)

(e) cation-exchange or electrostatic interaction between a cationic solute and

an ionized silanol (–SiO−) within the stationary phase; also repulsion of

an ionized acid (e.g., R–COO−)

(f) dipole–dipole interaction between a dipolar solute group (a nitro group

in this example) and a dipolar group in the stationary phase (a nitrile group for a cyano column)

(g, h) π –π interaction between an aromatic solute and either a phenyl group

(phenyl column) (g), or a nitrile group (cyano column) (h)

(a)

Hydrophobic interaction

COCH3

Hydrogen bonding (acidic solute)

(d)

O

C

OH

(e)

Cation exchange

NH +

(g)

π-π interaction

(phenyl column)

O

O2N

π-π interaction (cyano column) O

C=N

O2N

NO2

(c)

Hydrogen bonding

(basic solute)

N

(b)

Steric exclusion

(f)

Dipole-dipole interaction

O

C= N

+ −

O 2 N

−

Figure5.20 Solute-column interactions that determine column selectivity (figures omit the connecting silane group [–Si(CH ) –])

O M ++ O

N N

Complexation of chelating solutes

(i )

Figure5.20 (Continued)

(i) complexation between a chelating solute and metal contaminants on the

particle surface

Interactions (a–e) can be significant for every column; dipole interactions (f ) are

only important in the case of cyano columns, andπ –π interactions (g, h) occur only

for phenyl and cyano columns [57] Both dipole andπ –π interactions are inhibited

by the use of acetonitrile as B-solvent, which further minimizes their importance

for separations with acetonitrile Complexation with surface metals (i) can result

from the use of a less pure, type-A silica, leading to broad, tailing peaks (very undesirable); the chelating soluteα,α-bipyridyl has been used to test columns for

metal complexation Because phenyl and cyano columns are used less often, and

type-A columns are not recommended, we will emphasize interactions (a–e) in this

chapter (but see [57, 58])

5.4.1.1 Solute–Column Interactions

The various solute–column interactions of Figure 5.20, which determine column selectivity, have been understood in general terms since the 1980s; see [59] for a good discussion of recent attempts at characterizing column selectivity However, only after 2000 did it become possible to reliably characterize RPC column selectivity

in terms of these interactions This was accomplished by the development and

application of the hydrophobic-subtraction model [60–62], which recognizes that

hydrophobic interactions are by far the most important contribution to RPC retention If only hydrophobic interactions were significant, a plot of values of log

k for one column against another would give a straight line with no scatter of

data around the line As seen in Figure 5.21, this is approximately the case for these two C18 columns (Inertsil ODS-3 and Stablebond C18)—however, values

of log k for aliphatic amides () and protonated strong bases () fall below

the best fit to these data These latter deviations are due to interactions of these solute molecules with silanol groups (silanol interactions are more significant for the StableBond C18 column) These and other smaller deviationsδ log k from this

plot (see the expanded inset of Fig 5.21) represent contributions to retention from

nonhydrophobic interactions b–e of Figure 5.20 It is possible to analyze values of

δ log k for the combination of different solutes and columns so as to separately

evaluate the five interactions of Figure 5.20a–e For columns other than phenyl or cyano (i.e., those for which only interactions a–e of Fig 5.20 are significant), values

δlog k

2.0

1.5

1.0

0.5

0.0

−0.5

−1.0

log k (StableBond C18)

y = 0.21 + 1.01 x

r 2 = 0.995 amides protonated bases

Figure5.21 Comparison of retention on two different C18columns Data for 90 different organic compounds Conditions: 15× 4.6-mm columns; 50% acetonitrile-water, pH-2.8

phosphate buffer; 2.0 mL/min; 35◦C Adapted from [61]

of k can be related to the interactive properties of the solute and the column:

log



k

k EB



=ηH − σS∗+ βA + αB + κC (5.3)

(i) (ii) (iii) (iv) (v)

Here k and k EB are values of the retention factor for a given solute and the

reference compound ethylbenzene (EB), respectively Terms i–v of Equation (5.3) correspond, respectively, to the interactions of Figure 5.20a–e Quantities η, σ,

β, α, and κ refer to properties of the solute molecule: hydrophobicity (η),

‘‘bulkiness’’ (σ), hydrogen-bond (H-B) basicity (β), H-B acidity (α), and effective ionic charge (κ) Corresponding column parameters are of primary practical interest:

H, hydrophobicity; S*, steric interaction, or resistance by the stationary phase to penetration by bulky solutes; A, H-B acidity; B, H-B basicity; and C, ion-exchange capacity or electrostatic (coulombic) interaction Columns with similar values of H, S*, etc., will possess similar selectivity and provide a similar retention order for peaks within the chromatogram Columns with different values of H, S*, etc., will differ in

selectivity and provide changes in relative retention The hydrophobic-subtraction model and Equation (5.3) best summarize our present understanding of RPC retention and column selectivity [59]

We will next relate terms i–v of Equation (5.3) to the interactions of Figure 5.20a–e Hydrophobic interaction is illustrated in Figure 5.20a, by the

interaction of the solute 2-n-octanone (CH3COC6H13) with the ligand groups of

a C8 column Values of column hydrophobicity H increase for longer ligands

(e.g., C18 vs C8), a higher concentration of ligand groups on the silica surface (μmoles/m2), smaller pore diameters (e.g., 8- vs 30-nm pores), and the presence of

column end-capping An increase in H increases the retention of more hydrophobic

molecules, those with larger values ofη

Steric exclusion or ‘‘steric interaction’’ is illustrated in Figure 5.20b by the

retention of two polycyclic aromatic hydrocarbon (PAH) isomers: the narrow, long naphthacene and the more ‘‘bulky’’ triphenylene Naphthacene is better able to squeeze between adjacent ligands, but if the spacing of column ligands is increased (lower ligand concentration), it becomes easier for the bulky triphenylene to enter

the stationary phase The column parameter S* measures the ‘‘tightness’’ of the

stationary phase or the difficulty that bulky solute molecules experience in squeezing

between the ligands; larger values of S* mean a ‘‘tighter’’ stationary phase and relatively less retention of bulky solute molecules Values of S* increase for longer

ligands, a higher concentration of the ligand (ligands closer together), and smaller pore diameters Solute bulkiness is measured by its value ofσ Steric exclusion is a somewhat complex phenomenon; see Section 5.4.1.2 below for further insights

Hydrogen bonding of a non-ionized basic solute (e.g., pyridine) by a column

silanol is illustrated in Figure 5.20c The hydrogen-bond acidity A of the column

is due to the presence of surface silanols, and therefore decreases when the column

is end-capped (due to the removal of some silanols and blocking of others; see the

example of Fig 5.16d) The silanols of type-A columns are usually more acidic than

those present in type-B columns; therefore values of A tend to be larger for type-A

columns The H-B basicity of the solute is measured by its value ofα; unprotonated amines and amides are more basic and have larger values ofα, while nitriles and nitro compounds are much less basic and have smaller values ofα Most other polar compounds have intermediate H-B basicities and values ofα

Hydrogen bonding of a solute that is a H-B acid (e.g., butyric acid) is illustrated

in Figure 5.20d The H-B basic group ‘‘X:’’ in the stationary phase is not specified because the nature of X differs for different kinds of RPC columns For type-B

alkylsilica columns, there is so far no compelling explanation for what groups

‘‘X:’’ consist of It has been suggested that water dissolved into the stationary phase corresponds to these groups ‘‘X:’’, on the basis of an inverse correlation

of values of A and H —as well as other arguments In the case of some type-A alkylsilica columns with large values of B, contaminating metals in the silica appear

to comprise the ‘‘X:’’ groups Alternatively, for embedded-polar-group columns, the polar group (which is usually a H-B base) very likely corresponds to the ‘‘X:’’ group.

An increase in B leads to increased retention of carboxylic acids, which have large

values ofα; the retention of other H-B acids, such as alcohols and phenols, is less

dependent on B (smaller values of α)—although phenols are preferentially retained

on embedded-polar-group columns (i.e., phenols appear as stronger H-B acids when these columns are used)

Column ion-exchange capacity C is a measure of the ionization of the silica

and the accessibility of ionized silanols Silanol ionization and/or accessibility (and

values of C) increase (1) as mobile-phase pH increases, (2) for non–end-capped

columns, and (3) for type-A versus type-B columns While the main effect of larger

values of C is to increase the retention of protonated bases, it also results in a

decrease in retention for ionized acids (because of electrostatic repulsion); the value

ofκfor a solute is approximately equal to its molecular charge (e.g., +1 for fully protonated bases,−1 for fully ionized acids) The main difference in selectivity for

type-A versus type-B columns is determined by their low-pH values of C; type-B columns have values of C < 0.25 at pH 2.8, while type-A columns have C > 0.25.

For columns with values of C < 0.00 at low pH, it is believed that these columns

carry a net positive charge [63], presumably the result of protonated amine groups

that are introduced during the manufacturing process for some columns Values of

H, S*, A, and B are assumed not to change with the pH of the mobile phase Values of the column-selectivity parameters H, S*, etc., have been measured

for over 400 different columns; see [64] for a partial listing, or for a current list of values contact one of the authors (or http://www.USP.org/USPNF/columnsDB.html) Average values of these column parameters are summarized at the top of Table 5.8

for several different kinds of RPC column Within a given column type, there is

also a significant variation in values of H, S*, etc., as illustrated at the bottom of

Table 5.8 for several type-B C18columns Consequently not all columns of a given

kind can be regarded as equivalent in terms of selectivity Apart from values of H and S*, for example, average retention as measured by values of k for ethylbenzene

(last column of Table 5.8) increases with the surface area of the particle

5.4.1.2 Shape Selectivity

The following, minor digression examines two distinct forms of steric exclusion; for now, the reader may prefer to skip to Section 5.4.2.

Two separate manifestations of steric exclusion have been described: steric

interaction, as measured by term ii of Equation (5.3), and shape selectivity [65].

Differences between these two phenomena are illustrated in Figure 5.22 for the

separation of two isomeric hydrocarbons on a polymeric column (Fig 5.22a) and

a monomeric column (Fig 5.22b) The basis of shape selectivity is illustrated in Figure 5.22a for a ‘‘narrow’’ molecule i, a ‘‘wide’’ molecule j, and a polymeric alkylsilica column The ‘‘wide’’ molecule j is excluded from part of the stationary

phase because its minimum cross-section (double-headed arrow) exceeds the spacing

between ligands (molecule j cannot ‘‘squeeze’’ between the ligands) In a monomeric column (Fig 5.22b), the ligands are further apart, so as to allow access of both narrow and wide molecules (i and j) to the stationary phase When a molecule

has access to the stationary phase, steric exclusion affects retention in a different way; now the hydrodynamic diameter of the molecule becomes important, rather than its minimum cross-section When the hydrodynamic diameter of the solute

molecule is comparable in size to the spacing between ligands (as for molecule i),

the retained molecule is restricted in its possible orientations within the stationary phase This restriction of the solute molecule reduces its retention, in a similar way as for size-exclusion chromatography (Section 13.8.1) Note that shape selectivity and steric interaction lead to dissimilar effects on retention as a function of molecular shape; thus they clearly represent two different contributions to retention [61] Because of the ‘‘either-or’’ nature of shape selectivity, it can result in relatively large changes in relative retention—whereas steric interaction has a smaller effect on RPC selectivity

Figures 5.22c, d illustrates the potential advantage of shape selectivity for

the separation of polycyclic aromatic hydrocarbons (PAHs) The polymeric column

Table 5.8

Characterization of Column Selectivity by means of the Hydrophobic-Subtraction Model

(Eq 5.3)

Different column types

C18(type-B, wide-pore) 0.95 0.01 −0.05 0.01 0.22 0.31 3.2

C18(type-B, monolith) 1.01 0.02 0.12 −0.02 0.11 0.31 3.2

C18(type-B, hybrid) 0.98 0.01 −0.14 −0.01 0.13 0.05 6.3

C18(polar end-capped) 0.90 −0.04 −0.02 0.02 −0.02 0.40 7.4

Different narrow-pore, type-B C18columnsa

Zorbax StableBond C18c 1.00 −0.03 0.26 0.00 0.14 1.04 7.6 Zorbax Eclipse XDB-C18c 1.08 0.02 −0.06 −0.03 0.05 0.09 9.1 Kromasil 100–5C18d 1.05 0.04 −0.07 −0.02 0.04 −0.06 12.5 ProntoSIL 120–5 C18 SHe 1.03 0.02 −0.11 −0.02 0.11 0.40 8.7

Nucleodur C18 Gravityh 1.06 0.04 −0.10 −0.02 −0.08 0.32 11.0

aAll columns 5- μm particles; data of [73].

bAdvanced Materials Technology;cAgilent;dAkzo Nobel;eBischoff;fGL Science;gGrace-Alltech;

hMacherey Nagel;iACT;jMerck;kPhenomenex;lSupelco;mThermo/Hypersil;nWaters;oTosoh Bioscience.

(a) (b)

(min)

(c)

(d)

Solute minimum cross-section

(width)

O

OH

Si

OH

Si

Si-OH

i

j

Solute hydrodynamic diameter

i

j

shape selectivity

Polymeric column with

closely spaced ligands

steric interaction

Monomeric column with widely spaced ligands

steric interaction

column

shape selectivity

column

Figure5.22 Different manifestations of steric exclusion Shape selectivity (a) compared with steric interaction (b) (c) Separation of a mixture of 13 polycyclic aromatic hydrocarbons on

a polymeric column (d) Separation of same sample with same conditions on a monomeric column (c) and (d) are adapted from [65].

for the separation of Figure 5.22c exhibits greater shape selectivity and therefore

provides a much greater differentiation (and better separation) of these different isomeric C22 PAHs, versus the corresponding separation in Figure 5.22d with a

monomeric column (where shape selectivity is minimal) Long, narrow molecules (compared to those that are short and wide) are preferentially retained when shape selectivity is more important, while short, wide solute molecules (of similar molecular weight) are more retained when steric interaction is dominant As a rule, we can say that shape selectivity is more important when C30 or polymeric columns are used, and sample molecules are both large and have very different ratios of length to width Most RPC separations are carried out with monomeric columns other than

C30, in which case steric interaction and values of S* largely define the effect of steric

exclusion on column selectivity For further details on the practical utility of shape selectivity, see Section 6.3.5.2

5.4.2 Column Reproducibility and ‘‘Equivalent’’ Columns

Column manufacturers try to ensure that each column (e.g., Waters Symmetry C18) has similar properties and will perform satisfactorily and reproducibly in a routine

RPC assay Consequently the plate number N and column pressure drop for each

column usually is measured prior to its sale (Section 5.7); columns whose values of

N fall below some minimum value are discarded Similarly other tests are carried out

by the manufacturer (Section 5.7) to ensure that column selectivity stays the same from one batch to the next of the column packing (similar to the measurements of

values of H, S*, etc.) An example is shown in Figure 5.23 for several successive

batches of ZorbaxR Rx-C18, where the retention times for dimethylaniline and

toluene are plotted against the batch number Values of k for the two solutes vary

by±4% (1 SD), mainly as a result of small, unimportant differences in the surface

areas of the silica particles The ratio of these two k-values ( α) is a more direct

measure of column selectivity (primarily the important column-selectivity parameter

C of Eq 5.3); values of α vary by only ±0.5% Consequently it appears that the

selectivity of different batches of this column packing should be similar, especially for separations that involve protonated basic compounds

During the 1970s and 1980s HPLC column manufacturing had not yet devel-oped to its present advanced state, and column selectivity often varied significantly among different batches of a particular column More recently column repro-ducibility has improved (e.g., see [67]), but it is still possible that one column batch will differ enough from another in terms of selectivity to result in a failed separation—especially for demanding separations Various means exist for dealing with the latter problem (Section 6.3.6.1), one of which is to select an ‘‘equivalent’’

column from a different source Using values of H, S*, etc., as in Table 5.8b, it is possible to identify one or more columns with similar values of H, S*, etc., by means

5

4

3

2

1 α 0

k

Lot number

CH3 N(CH3)2

Figure5.23 Monitoring different batches of column packing for possible changes in selectiv-ity Sample: dimethylaniline and toluene Conditions: 150× 4.6-mm Zorbax Rx-C18columns; 50% acetonitrile-water plus pH-7 phosphate buffer; 1.6 mL/min; 22◦C Adapted from [66]