HPLC COLUMN .pdf

HPLC process development

1. THE COLUMN

2. Different particle configurations.
Totally porous silica particles
are the most common
because of their greater
column capacity and
availability in a wider variety
of options (stationary phase,
particle and pore size,
column dimensions, etc.).
The most popular particles
have diameters in the 1.5-to
5-μm range.

3. Poresizeand surfaceareaare usually related
reciprocally.
For compounds have >500 Da, the average pore
diameter are about 1.2 nm. For compounds with
molecular weights <500 Da, the average pore diameter
should preferably be about 9 nm or larger. Larger
molecules require larger pores; for example, proteins
are usually separated with 30-nm-pore particles.
Particle surface-area, average pore-diameter, and
pore-diameter distribution typically are measured by
the adsorption of nitrogen or argon, using the
Brunauer–Emmett–Teller (BET) procedure.

4. Visual appearance of several silica particles for RPC;
magnification in (b)is7×greater than in(a).

5. Column efficiency as a function of particle size and type. Sample, naphthalene.
Conditions: 50 ×4.6-mm, C18 columns; mobile phase is 60%acetonitrile-watermobile
phase; 23
◦
C.

6. Narrower particle-size-
distribution for the superficially
porous (Halo™)pack-
ing of Figure 5.3, compared with
that of a commercial totally
porous packing.
Cross-section of superficially porous (Halo™)
particles with 9-nm pores (electron
micrograph).

7. Silica surface showing different types of silanols.

8. Separation of protonated basic compounds on type-A (a) compared with type-B
(b) columns. Sample: four tricyclic antidepressants. Conditions: 150 ×4.6-mm C18
columns; mobile phase is 30%acetonitrile-water with pH-2.5 phosphate buffer.

9. Aggregation of microparticles to form totally porous particles.

10. Cross section of representative monolith packings (electron micrographs). (a)
Silica-based; (b) polymeric.

11. RPC packings usually are made by covalently reacting (‘‘bonding’’) an
organosilane with the silanols on the surface of a silica particle to form the
stationary phase or ligand R:
Synthesis of various bonded-phase column packings by the reaction of a silane
with silica. (a, d),Monomeric packings; (b, c), potentially polymeric packings.

12. Some alternative bonded phases based on different reaction conditions.

13. Some alternative bonded phases based on
different reaction conditions.

14. Options for increasing the stability of alkylsilica columns. (a, b), protection of
the—Si–O–bond by a steric-protected bonded phase (for low-pH conditions only); (c,
d)protection of the bonded phase by end-capping.

16. Synthesis of organic/inorganic hybrid particle. Courtesy ofWaters Corporation.

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

18. Basis of RPC 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)
(i) complexation between a chelating solute and metal contaminants on the
particle surface

19. Comparison of retention on two different C18 columns. 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

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

21. Solute-column interactions that determine column selectivity (figures omit the
connecting silane group [–Si(CH3)2–]).

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 monomericcolumn. (c)and(d

23. Monitoring different batches of column packing for possible changes in selectivity.
Sample: dimethylaniline and toluene. Conditions: 150 ×4.6-mmZorbax Rx-C18
columns; 50%acetonitrile-water plus pH-7 phosphate buffer; 1.6 mL/min; 22◦
C.

24. Schematic of equipment for packing
columns by the slurry procedure.

25. REVERSED-PHASE
CHROMATOGRAPHY
FOR NEUTRAL(naturally or using mobile
phase pH) SAMPLES

27. Retention in RPC as a function of temperature and the polarity of the solute, mobile phase and
column. Sample: as indicated in figure. Conditions: (a-c) 150 ×4.0-mm 5-μm) Symmetry C18
column, and (d) 150 ×4.6-mm (5-μm) ZorbaxStable Bond cyanocolumn; 2.0 mL/min; mobile
phaseis acetonitrile/water, with mobile-phase composition (%B) and temperature indicated in
figure(bolded values represent changes from [a]).

28. Where k
wrefers to the (extrapolated) value of k for 0% B (water as mobile phase), S
is a constant for a given solute when only %B is varied, and φ is the volume-fraction
of organic solvent B in the mobile phase (φ ≡ 0.01% B).
Variation of log k with%B.
Sample is 4-nitrotoluene.
Conditions: 250 ×4.6-mm
(5-μm) ZorbaxC8 column;
mobile phase consists of
organic/watermixtures; 35◦
C; 2mL/min.

29. Separation of a mixture of four nitro-substituted benzenes as a function of solvent
strength (%B). Sample: 1, nitrobenzene; 2, 4-nitrotoluene; 3, 3-nitrotoluene; 4,
2-nitro-1,3-xylene. Conditions: 100 ×4.6-mm (3-μm) ZorbaxC8 column; mobile phase
consists of acetonitrile-water mixtures (varying %B); 35◦C; 2 mL/min.

30. Different possibilities for the retention of a solute molecule in reversed-phase
chromatography. (a) Solvophobicinteraction; (b)adsorption ; (c) partition; (d)
comparison of RPC retention (k) with octanol-water partition P; sample; eight amino
acids; column: C8; mobile phase: aqueous buffer (pH-6.7); 70
◦
C.
it was observed that RPC
retention(values of k)
correlates with partition
coefficients P for the
distribution of the solute
between octanol and
water.

31. •Relation of K (related to t
R) and P (related to
polarity of sample) suggests that a partition
processbest describes RPC retention.
•However, later studies showed that correlations of
log P versus log k, as in last Figure (for amino acids)
are less pronouncedwhen the sample consists of
molecules with more diverse structures, which
makes the latter argument on behalf of partition
less compelling.

32. A surprising observation was made for the RPC retention of various
homologous series (CH3–[CH2]n − 1 –X), where X represents a functional
group such as –OH or –CO2CH3.
The plot of log k versus n for a homologous series and a C8 column; a
discontinuityin the expected linear plot (dashed line) is observed (arrow)
when n equals 8 for the solute (CH3 –[CH2]7 –X). It was concluded from
this observation that the contribution to retention for successive –CH2-
groups in the solute becomes slightly smaller when the length of the
solute molecule just exceeds the length of the alkyl ligand.
Presumably there is a decreased interaction with the column for solute
molecules that are too long to penetratefully into the stationary phase (or
attach to a single column ligand), with a corresponding decrease in the
retention of –CH2-groups that ‘‘stick out of’’ the stationary phase.

33. (a) Illustrative plot of log k versus number of –CH2-groups n for a homologous series CH3 –
(CH2)n−1 –X;C8 column; (b) illustration of the ‘‘overlapping’’ of alkyl chains in the solute and
column; (c–f ) plots of experimental methyleneselectivity αCH2 versus carbon number n
cfor
indicated columns of differing ligandlength. Average data for several homologous series;
90%methanol-water as mobile phase; 25
◦
C.
This figure supports the
solvophobic-interaction
model.

34. •Increasing amounts of the B-solvent (e.g.,
acetonitrile) are taken up by the stationary
phase as %B increases. Likewise some solutes
may interact with underivatized silanols
present on the particle surface.
•Thus there is not a clear distinction between
partition and adsorption in RPC

35. Role Stationary Phase
The use of mobile phases that are
predominantly aqueous (φ ≈ 0) can lead to
greatly reduced sample retention—the
opposite of that predicted.
When firstobserved, this reduced retention was
attributed to ‘‘phase collapse,’’ whereby alkyl
ligandsclump together and tend toward a
horizontal rather than vertical alignment with
the particle surface.

36. Selectivity
The most effective way to improve the resolution (or speed) of a
chromatographic separation is to initiate a change in relative
retention (selectivity).

37. Separation of a moderately irregular sample (mixture of eight nitro-aromatic compounds) as
a function of solvent strength (%B). Sample: 1, nitrobenzene; 2, 2,6-dinitrobenzene; 3,
benzene (shaded peak); 4, 2-nitrotoluene; 5, 3-nitrotoluene; 6, toluene; 7, 2-nitro-1,3-
xylene; 8, 1,3-xylene. Conditions: 100 ×4.6-mm (3-μm) ZorbaxC8 column; mobile phase
consists of acetonitrile/water mixtures; 35
◦
C; 2 mL/min.
Regular samples are often
composed of structurally
similar molecules; for
example, in the last
separation Figure that the
sample is a mixture of
mono-nitro alkylbenzenes.
Here we have irregular
separation because there are
different compounds.

38. Separation of a mixture of substituted benzenes as a function of solvent strength (%B). Sample: 1, p-cresol; 2,
benzonitrile; 3, 2-chloroaniline; 4, 2-ethylaniline; 5,3,4-dichloroaniline; 6, 2-nitrotoluene; 7, 3-nitrotoluene; 8, toluene; 9,3-
nitro-o-xylene; 10,4-nitro-m-xylene..
Solvent-type selectivity:Separation of a mixture of substituted benzenes with methanol or mixtures of methanol-
acetonitrileas mobile phase.
Solvent-Type Selectivity

39. Solvent-type selectivity: fine-tuning the B-solvent. Same sample and conditions as
in Figures upper (peaks 1–4 only), plus added figure (d); (b) is 30%MeOH +
23%ACN, and (d) is 35%MeOH + 20%ACN.

40. Solvent-strength nomographfor reversed-phase HPLC (adapted from [28]). Two
mobile phases of equal strength (46%ACN and 57%MeOH) marked by •,asanexample

41. Temperature
Retention of polycyclic aromatic
hydrocarbons as a function of separation
temperature.
(a)Sample (fused-ring aromatic
hydrocarbons): 1, anthracene; 2,
fluoranthene; 3,triphenylene; 4,
chrysene; 5, 3,4-benzofluoranthene; 6,
1,2,5,6-dibenzoanthracene.
(b)(b) Sample same as (a), plus added
poly-aryls: A,1,1 ,-dinaphthyl; B, 1,3,5-
triphenylbenzene; C, 9,10-
diphenylanthracene.

42. Separation of a mixture of
10 organic compounds of
diverse structure on four
different columns. Sample:
1, 4-nitrophenol; 2, 5,5-
diphenylhydantoin; 3,
acetophenone; 4,
benzonitrile; 5, 5-
phenylpentanol; 6,anisole;
7, toluene; 8, cis-chalcone;
9, ethylbenzene; 10, trans-
chalcone.
Column Selectivity

43. Separation of isomers with a cyclodextrin-bonded column. Conditions: 250 ×4.6-mm (5-μm) Cyclobond I
column; 30% acetonitrile–pH-4.5 buffer; 35◦C; 2.0 mL/min.
While it is possible to achieve the baseline separation of some isomersby RPC
with alkylsilica columns, the use of a cyclodextrin columnmay be a better
choice
Silver-ion complexation of olefins has been found to be a useful means for
enhancing the RPC separation of cis-from trans-olefin isomers.

44. Column selectivity changes:
(1)a change in column source (i.e., part number
(2)a change in separation conditions (‘‘method
adjustment’’).

45. A change in column source
Example of the use of values of Fs to
select columns of similar selectivity
for possible replacement in a routine
HPLC assay. Gradient separations
where only the column is changed for
the separations of a–d. Asterisks mark
peaks of interest, values of Fs
calculated from Equation (5.5) (ionic
[not neutral] sample).

46. Example of method adjustment
for a seven-component mixture of
neutral compounds. Sample: 1,
oxazepam; 2, flunitrazepam; 3,
nitrobenzene; 4, 4 nitrotoluene;
5,benzophenone; 6, cis-4-
nitrochalcone; 7, naphthalene.
Conditions: 150 ×4.6-mm C18
column (B differs from A only in a
10% lower ligandcoverage); 2.0
mL/min; acetonitrile-water
mobile phases; other conditions
shown in figure.
Method adjustment

47. Comparison of separation by an original versus ‘‘orthogonal’’ method. Gradient
separations where the column and organic solvent are changed (mobile-phase pH =
6.5 for both a and b). Asterisks mark gradient artifacts (not solute peaks).
Orthogonal Separation

48. General method-development approach for use in this and following
chapters.

49. Multiple-Variable Optimization
Multiple-variable optimization in each case
relies on an experimental design: a plan for
the required experiments, as illustrated for
certain combinations of conditions that affect
selectivity for neutral samples.

50. Experimental designs for the simultaneous optimization of various separation conditions for
optimum selectivity. (a) Solvent strength (%B) and temperature (T); (b) solvent strength and
solvent type (MeOHand ACN); (c) solvent type (MeOH, ACN, and THF).

51. Preferred solvents for maximum change in solvent-type selectivity. Tetrahydrofuran
(THF) is used less often because of its higher UV cutoff, susceptibility to oxidation,
slower column equilibrationwhen changing the mobile phase (e.g., from THF/water
to ACN/water), and incompatibility with PEEKtubing .
Mixtures of Different Organic Solvents

52. Use of seven solvent-type-selectivity experiments for the separation of a mixture of nine
substituted naphthalenes. Sample substituentsare: 1,1-NHCOCH3; 2,2-SO2CH3; 3,2-OH; 4,1-
COCH3; 5,1-NO2;6,2-OCH3; 7, -H (naphthalene); 8,1-SCH3; 9, 1-Cl. Mobile phases (circled):
1, ACN;
2,MeOH; 2 exchange:
1, ACN; 2,MeOH;
3, 39%tetrahydrofuran/water;
4,1:1mixture of 1 and 2;
5, 1:1 mixture of 2 and 3;
6, 1:1 mixture of 1 and 3;
7, 1:1:1 mixture of 1, 2, and 3.

53. Separation of six
steroids by changes
in solvent strength
(%B) and type.
Sample: 1,
prednisone; 2,
hydrocortisone; 3,
cortisone; 4,
dexamethasone; 5,
corticosterone; 6,
cortexolone.

54. Separation of a mixture of 6 organic compounds of diverse structure by changes
in solvent strength (%B) and temperature. Sample: 1, methylbenzoate; 2, benzophenone;
3, toluene; 4, naphthalene; 5, phenothiazine; 6, 1,4-dichlorobenzene. Conditions: 125 ×
3.0-mm C18 column; mobile phase acetonitrile/watermixtures; 1.0 mL/min.

55. Optimized separation of a mixture of 10 organic compounds of diverse structure
on four different columns by varying solvent strength (%B) and temperature. Sample
and conditions as in Figure 6.14, except as indicated in figure.

56. Illustrations of a change in column conditions to either improve resolution or decrease run time.
Sample components (non-ionized for these conditions; pH-2.6): 1,phthalic acid; 2, 2-
nitrobenzoic acid; 3, 2-fluorobenzoic acid; 4, 3-nitrobenzoic acid; 5;2-chlorobenzic acid; 6, 4-
chloroaniline; 7, 3-fluorobenzic acid; 8, 2,6-dimethylbenzoic acid;9, 2-chloroaniline; 10, 3,4-
dichloroaniline. Conditions: 4.6-mm C18 columns (5-μm) withindicatedlengths L; mobile phase
is 30%ACN-buffer for (a)and(b); 40%ACN-buffer for (c)and (d); 40◦Cin(a)and(b), 30◦Cin(c)and(d);
flowrates indicated in figure

57. NONAQUEOUS REVERSED-PHASE
CHROMATOGRAPHY (NARP)
The mobile phase for NARP separations will therefore consist of a mixture of
more polar (A-solvent) and less polar (B-solvent) organic solvents. Often
the A-solvent will be ACN or MeOH, while the B-solvent can be THF,
methylenechloride, chloroform, methyl-t-butylether(MTBE), or other
less polar organic solvents.

58. Non-aqueous reversed-phase (NARP) separations of carotenes. Conditions:
250 ×4.6-mm C18 column; 8%chloroform-ACN mobile phase; 2.0 mL/min;
ambient temperature.

59. SPECIAL PROBLEMS
•(1) poor retention for very polar samples (k ≥
1)
SOLVING: a. changing mobile phase pH, b) ion pair reagents, c)
using higher surface area (smaller pore diameter), d) Using
normal phase chromatography
•(2) peak tailing (asymmetry factors As >2)
SOLVING: a) Using TFH or TEA for acid and base samples
interaction with sp, b) Using another column or guard column
(new one)

60. NORMAL-PHASE
CHROMATOGRAPHY

61. NPC used for
(1) analytical separations by thin-layer
chromatography (TLC, Section 1.3.2),
(2) the purification of crude samples (preparative
chromatography and sample preparation),
(3) the separation of very polar samples
(4) the resolution of achiral isomers.

62. Packing NPC
•Inorganic: alumina, magnesia, magnesium silicate (Florisil), and
diatomaceous earth (Celite, kieselguhr),
•Synthetic (unbonded) silica: a more neutral, less active
surface, with less likelihood of undesirable sample reactions during
separation strong particles of controlled size and porosity that can
withstand the high pressures.

63. Three polar-bonded-phase packings
(1)cyano columns, where –(CH2)3–C ≡N groups
are bonded to silica particles,
(2)diol columns bonded with –(CH2)3 –O–CH2 –
CHOH–CH2OH groups, and
(3)amino columns with –(CH2)3 –NH2 ligands.

64. mono-substituted benzenes (substituents indicated for each peak; e.g., –H is benzene, –Cl is
chlorobenzene). Conditions: 150 ×4.6-mm silica (5-μm particles); 20%CHCl3-hexane mobile
phase; ambient temperature; 2.0 mL/min. (a) Chromatogram is recreated from data of [1]; (b)
retention of (a) compared with RPC retention from Figure 2.7c for benzenes substituted by the
same functional group (50%acetonitrile-water as RPC mobile phase).

65. •Because the column in NPC is more polar than the mobile phase, more-
polar solutes will be preferentially retained or adsorbed—the opposite of
RPC.
•Butthe correlation of Figure 8.1b is moderately strong (r
2
= 0.76), there is
also significant scatter of the data. That is, NPC separation cannotbe
regarded as the exact opposite of RPC retention.

66. Differences NPC vs RPC
(1) Different Polarity of mp and sp
(2) Different behaviour for the number n of alkyl
carbons in the solute molecule (its carbon
number C
n),
(3) Difference conditions for isomeric solutes
separation.

67. Comparison of NPC separation (a) with RPC separation (b–d) for a mixture of alkyl-substituted anilines. Conditions: 150 ×
4.6-mm C8 column (5-μm particles) in (a), 150 ×4.6-mm cyano column (5-μm particles) in (b–d); mobile phase is 60%
methanol–pH-7.0 buffer in (a), and 0.2%isopropanol-hexane in (b); ambient temperature and 2.0 mL/min in (a) and (b).
Sample (peak numbers): 1–3, 2-, 3-and 4-methylaniline; 4, 2,6-dimethylaniline; 5, 2-ethylaniline; 6, 2,5-dimethylaniline; 7,
2,3-dimethylaniline; 8, 2,4-dimethylaniline; 9, 3-ethylaniline; 10, 4-ethylaniline; 11, 3,4-dimethylanilne; 12, 2,4,6-
trimethylaniline; 13,2-i-propylaniline; 14,4-i-propylaniline.

68. That is, NPCcan separate solutes of differing functionality,but
differences in solute carbon number have much less effect on
retention.
NPC permits the group-separation of petroleum samples into
saturated hydrocarbons, olefins, benzenes, and various
polycyclic aromatic hydrocarbons—according to the number
of double bonds in the molecule, but with little effect of
differences in alkyl substitution or solute molecular weight.
Similarly lipid samples can be resolved into mono-, di-, and tri-
glycerides (as well as other compound classes).

69. Theory
Retention in NPC is best described by a displacementprocess,
based on the fact that the silica surface is covered by a
monolayer of solvent molecules that are adsorbed from the
mobile phase. Consequently, for a solute molecule to be
retained in NPC, one or more previously adsorbed solvent
molecules must be displaced from (leave) the silica surface in
order to make room for the adsorbing solute.

70. Hypothetical examples of solute retention on silica for chlorobenzene (a,b non-localized) and
phenol (c,d localized). Mobile phase in (a,b) is a less-polar solvent (CH
2Cl
2); mobile phase in (c,d)
is a more-polar solvent (tetrahydrofuran, THF).

71. Retention differs for a more-polar mobile-phase solvent
such as THF and a more polar solute such as phenol.
Here the interaction of solvent and solute molecules
with surface silanols will be stronger, as indicated by
the arrows that connect the two interacting
species—in contrast to the weaker and less specific
interactions shown for phenol sample in CH2Cl2 (as
mp).
As a result there is a ratio 1 :1 interaction of a surface
silanol with a polar group in a molecule of either
solute or mobile phase—called localizedadsorption.
Under these conditions adsorbed molecules can
assume a vertical rather than flat configuration.

72. Solvent nomograph for normal-phase chromatography and silica columns.

73. Solvent-strength selectivity in normal-phase chromatography. Sample: 1,2-
aminonaphthalene; 2, 2,6-dimethylquinoline; 3, 2,4-dimethylquinoline; 4, 4-
nitrophenol; 5, quinoline; 6, isoquinoline. Conditions: 150 ×4.6-mm silica column
(5-μm particles); ethylacetate (B)-cyclohexane (A) mixtures as mobile phase;
ambient temperature; 2.0 mL/min. Peaks 1 and 4 are shaded to emphasize their
change in relative retention as %B is varied.

74. Corresponding separations by TLC and column chromatography
(involving the same sample, mobile phase, temperature, and
especially the same silica as stationary phase) should yield
similar values of k for each compound in the sample.
The RF value of a solute in TLC is defined as its fractional
migration from the original sample spot (point at which the
sample is applied) toward the solvent front (end of solvent
migration during TLC).
Use of TLC Data for Predicting NPC Retention

75. Solvent-strength selectivity in normal-phase chromatography. Sample: 1,2-aminonaphthalene; 2, 2,6-
dimethylquinoline; 3, 2,4-dimethylquinoline; 4, 4-nitrophenol; 5, quinoline; 6, isoquinoline. Conditions: 150 ×4.6-
mm silica column (5-μm particles); ethy-lacetate (B)-cyclohexane (A) mixtures as mobile phase; ambient
temperature; 2.0 mL/min. Peaks 1 and 4 are shaded to emphasize their change in relative retention as%B is
varied.

76. •Experimental studies have shown that solvent-
type selectivity in NPC depends mainly on the
strength of the B-solvent (ε
0
B).
•As ε
0
Bincreases, the B-solvent becomes more
strongly attached to a specific silanol, resulting
in localized adsorption of the B-solvent.

77. Example of solvent-type selectivityfor normal-phase chromatography. Sample: 1, 2-methoxynapthalene; 2, 1-nitronapthalene; 3, 1,2-
dimethoxynapthalene; 4,1,5-dinitronapthalene; 5, 1-naphthaldehyde; 6, methyl-1-naphthoate; 7, 2-naphthaldehyde; 8, 1-naphthylnitrile; 9, 1-
hydroxynaphthalene; 10, 1-acetylnapthalene; 11, 2-acetylnapthalene; 12, 2-hydroxynaphthalene. Conditions: 150 ×4.6-mm silica column (5-μm
particles); mobile phases (%v) indicated in figure (50% water-saturated), except that (c) contains 6% added CH2Cl2 to achieve miscibility of ACN
(hexane is the A-solvent in each case) 35
◦
C; 2 mL/min.(a–c) Separations with indicated mobile phases; (d–f ) correlations of retention data from
(a–c).

78. Comparison of NPC separation (a) with RPC separation (b–d)for a mixture of alkyl-substituted anilines. Conditions: 150 ×
4.6-mm C8 column (5-μm particles) in (a), 150 ×4.6-mm cyano column (5-μm particles) in (b–d); mobile phase is 60%
methanol–pH-7.0 buffer in (a), and 0.2%isopropanol-hexane in (b); ambient temperature and2.0mL/min in (a)and(b).
Sample (peak numbers): 1–3, 2-, 3-and 4-methylaniline; 4, 2,6-dimethylaniline; 5, 2-ethylaniline; 6, 2,5-dimethylaniline;
7, 2,3-dimethylaniline; 8, 2,4-dimethylaniline; 9, 3-ethylaniline; 10, 4-ethylaniline; 11, 3,4-dimethylanilne; 12,2,4,6-
trimethylaniline; 13,2-i-propylaniline; 14,4-i-propylaniline.

79. •Isomeric solutes of identical alkyl-carbon
number (e.g., C1, consisting of o-, m-, and p-
methylanliline) are seen to be bunched
together, while solutes of differing carbon
number (e.g., C1 vs. C2) are well separated.

80. Column Selectivity
Comparison of retention and
selectivity among different NPC
columns. Sample: 1, chrysene; 2,
perylene; 3, 1-nitronaphthalene;
4, 1-cyanonaphthalene; 5,2-
acetonaphthalene; 6,
naphthalene-2,7-
dimethylcarboxylate; 7, benzyl
alcohol. Conditions: 150 ×4.6-mm
columns (column type indicated in
figure); hexane mobile phase; 35◦
C; 2.0 mL/min. Chromatograms (a
− c) reconstructed from data of
[26]; (d) estimated from data of
[1] (note extreme change in
retention range for silica column d
vs. polar-bonded columns a–c).

81. Factors that contribute to isomer selectivity for NPC separation on silica columns. (a, b)
Steric hindrance; (c, d) electron donation; (e, f ) relative positions of polar groups
within the solute molecule; (g) intramolecular hydrogen bonding of two polar groups.

82. In the case of polar-bonded-phase NPC columns, sterichindrance effects
(Figs. 8.13a,b) will be lessimportant because the silica surface is further
removed from the polar cyano, diol, or amino group of the stationary
phase—hence contributing less to steric hindrance between the solute
and the stationary phase.
Similarly the matchingof polar groups in the solute molecule with polar
groups in the stationary phase (Figs. 8.13e,f ) will be easier for a polar-
bonded-phase column (with less effect on isomer selectivity) because the
cyano, diol, or amino groups are not rigidly positioned on the surface but
are connected to the silica surface by a flexible –CH2 –CH2 –CH2 –linkage.
Finally, the attractionof polar groups in the solute molecule to the polar
stationary phase is weaker for polar-bonded-phase columns than for silica,
which in turn reduces the effect of each of the contributions to isomer
separation in Figure 8.13.

83. METHOD-DEVELOPMENT SUMMARY

84. HYDROPHILIC INTERACTION CHROMATOGRAPHY
(HILIC)
Hydrophilic interaction chromatography (HILIC) can be regarded
as normal-phase chromatography with an aqueous-organic
mobile phase; for this reason it is sometimes referred to as
‘‘aqueous normal-phase chromatography.’’

85. Separation of a mixture of derivatized oligosaccharides by HILIC
with mobile phases of varying%-water. Conditions: 200 ×4.6-mm
PolyHydroxyethyl A column (5-μm particles); mobile phases are
water-acetonitrile as indicated in the figure; 2 mL/min.

86. Columns
Subsequently a variety of different bonded-silica packings have
been employed for HILIC, which can be categorized as follows:
bare silica, polar neutral (e.g., cyanopropyl), diol-bonded,
amide-bonded, polypeptide-bonded, positively charged
amine-bonded (anion-exchange), negatively charged (cation-
exchange), and zwitterionic phases.

87. HILIC Problems
•Peak shape (both fronting and tailing)
•Column bleed
•Irreversible sorption
•Slow
•equilibration of the column