Analytes Prone to Hydrolysis

NPC is ideally suited for the analysis of compounds prone to hydrolysis

because it employs nonaqueous solvents for the modulation of retention. An
example of the use of NPC in the analysis of a hydrolysable analyte was
demonstrated by Chevalier et al. [28] for quality control of the production of
benorylate, an ester of aspirin. A major issue in benorylate production is the
potential formation of impurities suspected of causing allergic side effects;
therefore monitoring of this step is critical to quality control. The presence of
acetylsalicylic anhydride prohibited the use of RPLC since it can be easily
hydrolyzed in the water-containing mobile phase. However, an analytical
method based on the use of normal-phase chromatography with alkylnitrile-
bonded silica as the stationary phase provided an ideal solution to the analy-
sis. Optimal selectivity was achieved with a ternary solvent system:
hexane–dichloromethane–methanol, containing 0.2 v/v% of acetic acid to
prevent the ionization of acidic function and to deactivate the residual silanols.
The method was validated and determined to be reproducible based on pre-
cision, selectivity, and repeatability.
Another application that demonstrates the advantages of using NPC for the
separation of analytes prone to hydrolysis is the reaction monitoring for the
formation of 9,10-anthraquinone [29]. Anthraquinone is an important inter-
mediate in the manufacturing of various dye products but also is used as a cat-
alyst in the isomerization of vegetable oils. It is produced in large amount by
Friedel–Crafts reaction of phthalic anhydride with benzene in the presence of
AlCl3 catalyst.

The development of a normal-phase HPLC method was warranted due to the

presence of phthalic anhydride, which is unstable in water. Analysis in organo-
aqueous solvent systems that are used in RPLC would lead to an on-column
reaction forming the respective carboxylic acid degradation product. Figure
5-5 shows the chromatogram obtained for the separation of 9,10-anthraquinone
from the reactants and impurities on a silica column. The method was suc-
cessfully applied to monitor the reaction conversion and also to determine the
stability of 9,10-anthraquinone at the specified storage conditions.
In addition, sometimes a normal-phase HPLC method at subambient tem-
perature must be applied for analytes that are extremely prone to hydrolysis.
In the synthesis of leukotriene D4 antagonist, accurate quantitation of mesy-
late intermediate is essential for process optimization. Owing to its inherent
instability, analysis of mesylate intermediate must be carried out under
normal-phase conditions with nonprotic solvents; however, significant cycliza-
tion of mesylation was still observed in such condition at room temperature.
The authors concluded that the on-column reaction of the mesylate was silica-
catalyzed cyclization. By conducting the normal-phase HPLC analysis
at −30°C, it was demonstrated that on-column cyclization was adequately
inhibited [30].
Extremely Hydrophobic Compounds
NPC has been used in the analysis hydrophobic compounds such as polyaro-
matic hydrocarbons [31–33]. An interesting example of an application of NPC
involving extremely hydrophobic compounds was recently offered by Liu and
NORMAL-PHASE

Typical chromatogram of a reaction mixture collected during the course of

reaction of phthalic anhydride with benzene in the presence of AlCl3, as catalyst.
Peaks:
1, benzene; 2, anthraquinone; 3, phthalic anhydride; 4, maleic anhydride; 5,
unknown.
Chromatographic conditions: Column: Spherisob silica, 250 4.6mm, 10 m;
mobile
phase, n-heptane–ethanol–chloroform–acetic acid (89 : 5 : 5 : 1, v/v/v/v); flow
rate, 1mL/
min; detection, UV at 254 nm; temperature, 27°C. (Reprinted from reference 29,
with
permission.)
Warmuth [34] when they adopted NPC for the analysis of supermolecules,
such as hemicarcerplexes. Hemicarcerplexes are complexes formed with hemi-
carcerand host and guest molecules. As shown in Figure 5-6, hemicarcerands
possess a very hydrophobic structure with molecular weight over 2000 and is
insoluble in protic solvents. A normal-phase HPLC method was developed
using a silica column with dichloromethane and diethylether as the mobile-
phase system.The authors demonstrate that the chromatographic retention of
hemicarcerplexes is mainly dominated by its size. Furthermore, a linear rela-
tionship between the logarithmic retention factor and the size of the hemi-
carcerplexes was observed for linear guest molecules independent of their
polarity.
Separation of Isomers
With more and more complex molecules being investigated as drug candi-
dates, isomer separation has become increasingly challenging. Despite being a
workhorse analytical tool, reversed-phase chromatography is limited in its
ability to distinguish between isomers [35–39]. On the other hand, NPC has
established itself as the technique of choice for the separation of positional
isomers as well as stereoisomers due to the specific nature of interactions.The
separation of positional isomers of alkyl-substituted polyaromatic hydrocar-
bons (PAHs) in the petroleum industry is an example where NPC has been
employed successfully. Alkyl substitution significantly increases PAH reten-
tion in RPC so that alkyl-substituted PAHs with low ring number have reten-
tion times close to some of the nonsubstituted higher-ring-numbered PAHs,
resulting in co-elution.Wise et al. [40] reported that aminopropyl-bonded silica

A schematic of hemicarcerplex. (Reprinted from reference 34, with

permission.)
phase yields a separation sequence of PAHs solely based on the number of
conjugated rings independent of the type of alkyl substitution.
Separations involving cis/trans isomers can also be accomplished by
employing NPC. An example of this application is the separation of tricyclic
antidepressant doxepin, which is marketed as a mixture of geometric isomers
in a cis/trans ratio of 15 : 85 [41].When a spherisorb silica column is used with
a hexane–methanol–nonylamine mobile-phase system, the cis isomer of
doxepin elutes first. The structures of the two isomers and the chromato-
graphic separation are shown in Figure 5-7. NPC has also been successfully
employed in the separation of cis/trans isomers of steroids. Four diastereomers
NORMAL-PHASE

(a) Structures of cis and trans isomers of Doxepin and (b) normal-phase
chromatographic separation of isomers of doxepin. I, cis-doxepin; II, trans-
doxepin; III,
nortriptyline; IV, cis-N-desmethyldoxepin; V, trans-N-desmethyldoxepin.
Chromato-
graphic conditions: Column: Spherisob silica, 150 4.5mm, 3 m. Hexane:
methanol :
nonylamine, 95 : 5 : 0.3 (v/v/v); flow rate, 1.0 mL/min; detection, 254 nm;
temperature,
23°C. (Reprinted from reference 41, with permission.)
consisting of two pairs of cis/trans isomers (see Figure 5-8 for structures) were
separated using a silica column and hexane-dichloromethane-2-propanol
mobile-phase system as shown in Figure 5-9 [42].
Other interesting examples of positional isomer separation involving NPC
are (a) the separation of dihydrodipyridopyridopyrazines, a new family of
antitumor agents, on a silica Nucleosil 50 A-10 m column [43] and (b) the
separation of celecoxib isomers by Chiralpak AD column [44]. NPC was also
employed successfully for the resolution of (a) four configurational isomers of
a steroidal calyx pyrrole [45], (b) regio- and stereoisomers of eicosanoids [46],
(c) retinal and retinol isomers [47], and (d) several E/Z isomers pairs of
vitamin A [48].
In certain cases, such as the separation of PAHs obtained from a coal
liquefaction process, using reversed-phase HPLC is complicated as sample
preparation is elaborate.This is due in large part to the fact that most complex
fuel-related materials contain compounds that are not usually soluble in
acetonitrile, the solvent of choice in reversed-phase HPLC. Here, NPC, which
employs a variety of solvents, offers an alternative to the analysis of such
samples. Separation of five well-studied coal liquefaction process stream
samples was achieved and 19 isomers were resolved when NPC was used [33].
The method employed a tetrachlorophthalimidopropyl-modified silica column
(TCPP) with a charge-transfer mechanism.
One of the most challenging tasks in isolating secondary metabolites from
fermentation broths is the removal of numerous structural analogs of the
desired product formed by the host organism. Pneumocandin B0 is a potent
antifungal agent produced as a recently discovered secondary metabolite by
the fermentation of Zalerion arboricola [49]. Pneumocandin B0 is the product
of interest, with a molecular weight of 1069Da.Pneumocandin C0, which differs
from B0 only by a single carbon shift of hydroxyl group, is a key impurity co-
produced by the fermentation. This impurity is proved to be intractable by
reversed-phase chromatography or crystallization.The isomer was successfully

Structures of cis and trans isomers of steroids. (Reprinted from reference

42, with permission.)
separated in NPC mode by employing LiChrospher Silica stationary phase and
an ethyl acetate/methanol/water mixture (86/7/7) mobile-phase system.
Carbohydrates
NPC has also found some applications in the field of carbohydrate analysis.
Typical stationary phases used for this application are alkyl amine-, diol-, or
polyol-bonded silicas [50–53]. Alkyl amino-bonded silicas are commonly used
for the separation of saccharides and oligosaccharides in various matrixs, such
as food or biological fluids. Although water is used as part of mobile phase,
the retention behavior of carbohydrate follows the NPC retention behavior.
NORMAL-PHASE HPLC

Chromatograms of isomers 3–6. Chromatographic conditions: Column:

APEX silica, 250 4.6mm, 5 m (Jones chromatography); mobile phase, hexane-
dichloromethane-2-propanol. (a) 82 : 10 : 8 (v/v/v), (b) 84 : 10 : 6 (v/v/v); flow
rate,
1 mL/min; detection, 280 nm; temperature, ambient. (Reprinted from reference
42, with
permission.)
Carbohydrates are eluted in the order of increasing polarity, and retention
decreases when water content increases.With an aminopropyl silica column,
Koizumi et al. [54] showed the resolution of d-glycooligosaccharides up to a
degree of polymerization of 30–35 under isocratic conditions, with a binary
mixture of acetonitrile/water. Similarly, with the use of a polyamine polymer
resin-bonded silica or an amino-bonded silica, separation of maltooligosac-
charides up to a degree of polymerization of 28 was achieved [55].
Separation of Saturated/Unsaturated Compounds
The surface of the silica may be dynamically coated with transition metals, and
the selectivities observed can be attributed to the complexes between the
metal ions and the analyte species [56]. The use of silver-impregnated silica
(adsorption of salts of transition metals on the silica surface) has been used
for the analysis of saturated and unsaturated fatty acid methyl esters (FAME)
and triacylglycerols (TAG) [57]. The retention of the unsaturated FAME and
TAG can be attributed to the stability of the complex that is formed between
the electrons of the carbon–carbon double bonds and the silver ions. The
predominant interaction for saturated analytes is with the polar silanol groups.
The secondary interactions are those of the silver ions with the unpaired elec-
trons of the carbonyl oxygens of the analytes. The amount of silver adsorbed
onto the silica and the pH (employment of acidic or basic modifiers) have been
determined to have an effect on the retention and resolution of certain acidic
and basic compounds and fatty acids [58].
CONCLUSIONS
In normal-phase chromatography, polar stationary phases are employed and
solutes become less retained as the polarity of the mobile-phase system
increases. Retention in normal-phase chromatography is predominately based
upon an adsorption mechanism. Planar surface interactions determine suc-
cessful use of NPC in separation of isomers. The nonaqueous mobile-phase
system used in NPC has found numerous applications for extremely
hydrophobic molecules, analytes prone to hydrolysis, carbohydrates, and sat-
urated/unsaturated compounds. In the future, with the advent of new station-
ary phases being developed, one should expect to see increasingly more
interesting applications in the pharmaceutical industry.