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Approach to hydrophilic interaction chromatography column selection:

Application to neurotransmitters analysis

Article in Journal of Chromatography A · March 2010

DOI: 10.1016/j.chroma.2010.03.001 · Source: PubMed

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 Journal of Chromatography A, 1217 (2010) 3091–3104

Contents lists available at ScienceDirect

Journal of Chromatography A
journal homepage: www.elsevier.com/locate/chroma

Approach to hydrophilic interaction chromatography column selection:

Application to neurotransmitters analysis
Raluca-Ioana Chirita a,b , Caroline West a , Adriana-Luminita Finaru b , Claire Elfakir a,∗
a
Institut de Chimie Organique et Analytique CNRS UMR 6005, University of Orléans, 45067 Orléans, France
b
University of Bacau, Calea Marasesti 157, Bacau 600115, Romania

a r t i c l e i n f o a b s t r a c t

Article history: This paper presents the comparison between 12 hydrophilic interaction liquid chromatography columns
Received 11 August 2009 that are commercially available. The main factors influencing the retention and selectivity toward 12
Received in revised form 24 February 2010 neurotransmitters for these different HILIC systems have been studied. For additional information, the
Accepted 1 March 2010
retention data have been analyzed statistically by factor analysis. Principal component analyses (PCA)
Available online 6 March 2010
were calculated to evidence different separation behaviour between the stationary phases, based on the
retention data measured for three compound classes: anionic acidic compounds, cationic basic com-
Keywords:
pounds and zwitterionic amino acids. Finally, a generic procedure is suggested for optimization of HILIC
HILIC
Retention behaviour
analyses, depending on the ionization state of the analytes.
Stationary phases classification © 2010 Elsevier B.V. All rights reserved.
Neurotransmitters
Catecholamines
Fused core silica

1. Introduction nificantly increases solubility for polar and hydrophilic solutes.

In bioanalysis, most of the molecules of interest are very polar
and hydrophilic compounds. Their analysis can be difficult as they
are not or insufficiently retained under reversed-phase chromatog-
raphy (RPLC) conditions as well as not well soluble in non-polar
solvents constituting the mobile phase in normal-phase liquid
chromatography (NPLC). Hydrophilic interaction chromatography
(HILIC) appears to be an interesting alternative for applications
involving these types of polar compounds. The HILIC applications
have increased in number and diversity in the last decade. HILIC
has been applied for amino acids [1–4], peptides [3,5], sugars
[6], amadori compounds [7], urea [8], creatinine [9], nucleosides
[3,10,11], metal complexes [12], organophosphorus pesticides [13],
biogenic amines [14,15], acetylated histones [16], room temper-
ature ionic liquids [17], opiates and their glucuronides [18] and
a multitude of drugs and their metabolites [19–23] analysis. The
review by Hemström and Irgum [24] summarizes some of the
important issues that need to be taken into consideration when
optimizing a HILIC analysis. In HILIC, a hydrophilic stationary phase
and an aqueous–organic mobile phase with high organic content
(at least 60%) are used. This composition of mobile phase sig-
∗ Corresponding author at: Universite d’Orleans, Institut de Chimie Organique et
Analytique, CNRS UMR 6005, B.P. 6759, 45067 Orleans Cedex 2, France.
Tel.: +33 238494587; fax: +33 238417281.
E-mail address: claire.elfakir@univ-orleans.fr (C. Elfakir).
In HILIC, the retention is mainly caused by partitioning of the
analyte between a water-enriched layer immobilized at the sta-
tionary phase surface and the relatively hydrophobic mobile phase
[1]. A minimum of 2% of water is needed in order to ensure
the presence of the water layer. As in NPLC, under these chro-
matographic conditions, the more polar the analyte, the higher
the retention is. The mechanism of HILIC separation is somewhat
complicated since it consists not only of hydrophilic partitioning
interactions (the mobile phase—aqueous layer partitioning pro-
cess) but also secondary electrostatic (attraction or repulsion)
[3,13,25] and hydrogen-bonding [5,11] interactions. These sec-
ondary interactions can vary substantially with the variation in the
mobile phase additives such as acids or salts.
The number of HILIC—compatible columns has increased in the
last few years, as manufacturers have understood the potential of
this type of chromatography. Generally stationary phases are silica-
based and can often be charged in some pH region. They can roughly
be divided into bare silica, polar neutral, diol-bonded, amide-
bonded, positively charged amine-bonded, negatively charged
cation-exchange and zwitterionic phases. So the choice of an appro-
priate column can be really difficult considering that no column
classification is available as it is for the reversed-phase columns.
Considering the number of published applications the most popular
HILIC stationary phases are amide-bonded and bare silica columns
[24] but, as a great number of HILIC columns with different station-
ary phase chemistries are now available on the market, a detailed
study is necessary.

0021-9673/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.chroma.2010.03.001
3092 R.-I. Chirita et al. / J. Chromatogr. A 1217 (2010) 3091–3104
Brain neurotransmitters are polar compounds of high biological
significance. Among them catecholamines and indolamines play
an important role as anomalies in their concentrations lead to a
significant number of diseases such as Parkinson’s, Alzheimer’s or
Down’s disease, depression, schizophrenia, epilepsy, etc. In spite
of the fact that catecholamines and related compounds are among
the most important compounds from a biological point of view,
few papers [15,26] have so far described their determination under
HILIC conditions. Reversed-phase chromatography (RPLC) either on
ODS [27] or porous graphitic carbon [28] columns were used for
catecholamine analysis with highly aqueous mobile phases using
formic or acetic acid as additives; however, due to their ionisable
nature, most of the work concerning catecholamine separation con-
sists in using ion-pairing reversed-phase liquid chromatography.
The use of non-volatile ion-pairing reagents such as alkylsulfonic
acids [29–31] is one the most employed chromatographic meth-
ods but it is not compatible with MS detection. Volatile ion-pairing
agents such as perfluorinated acids [32,33] have already been
demonstrated as suitable for the separation of catecholamines
but the number of catecholamines simultaneously analyzed under
these conditions was smaller than the one we were interested
in. Ion-exchange chromatography [34,35] was also used for cat-
echolamine analysis, but these methods are only suitable for the
analysis of compounds of the same charge.
In this paper our aim was to evaluate the retention behaviour of
12 model solutes (dopamine DA, adrenaline A, noradrenaline NA,
serotonin S and some of their precursors and metabolites: tyro-
sine Tyr, 3,4-dihydroxy-phenylalanine DOPA, homovanillic acid
HVA, 3-methoxytyramine 3-MT, 3,4-dihydroxy-phenylacetic acid
DOPAC, tryptophan Trp and 5-hydroxyindole-3-acetic acid 5HIAA)
on 12 different commercial HILIC packing materials. The main
factors influencing the retention were studied and for additional
information the retention data have been analyzed statistically by
principal component analysis. Improved knowledge of the differ-
ent interactions between HILIC stationary phases and this family of
compounds will allow us to make a reasoned choice of stationary
phases to obtain the best possible separation in view of a future
quantification of these polar solutes by HILIC-MS/MS in biological
fluids or tissues.
2. Experimental
2.1. Chemicals and reagents
HPLC-grade acetonitrile (MeCN), methanol (MeOH), ethanol
(EtOH) and acetone were purchased from J.T. Baker (Noisy-
le-Sec, France) and perchloric acid from VWR Prolabo
(Darmstadt, Germany). Ammonium acetate and ammonium
formate, acetic acid and formic acid, 3,4-dihydroxybenzylamine
(DHBA) were purchased from Fluka (St.-Quentin-Fallavier,
France). Dopamine (DA), noradrenaline (NA), adrenaline (A),
3-methoxytyramine (3-MT), serotonin (S), homovanillic acid
(HVA), 3,4-dihydroxy-phenylalanine (DOPA), tryptophan (Trp),
3,4-dihydroxyphenylacetic acid (DOPAC), 5-hydroxyindole-3-
acetic acid (5HIAA), tyrosine (Tyr), 3,4-dihydroxy-phenylalanine
(DOPA) and tryptophan (Trp) were purchased from Sigma–Aldrich
(Saint-Quentin-Fallavier, France).
Deionized (18 M) water, purified using an Elgastat UHQ II sys-
tem (Elga, Antony, France) was used for preparation of analyte and
mobile phase solution.
2.2. Standards
Stock standard solutions of each catecholamine, indolamine,
and metabolite prepared at a concentration of 1000 ␮g mL−1 were
obtained by dissolving the adequate weighed amount of each
compound with 0.2 M perchloric acid. The use of perchloric acid
is dictated by the fact that the neurotransmitter analysis was
inscribed in a larger study aiming at analyzing these molecules
in brain extracts, which are prepared in perchloric acid. Thus it
was found important to maintain identical conditions in the whole
method development process. All stock solutions were stored at
−80 ◦ C. The studied solutions were obtained by diluting the cor-
responding stock standard solutions in buffer/organic modifier
mixture in order to have an injection solvent as close as possi-
ble to the mobile phase and a final analyte concentration about
5–10 ␮g mL−1 . In the HILIC mode the injection solvent composition
is very important. Retention time shift can be caused by a slightest
difference in the organic phase/aqueous phase ratio between the
injection solvent and the mobile phase. Moreover, it is imperative
to have similar salt content in injection solvent and mobile phase
in order to obtain good peak symmetry.
The mobile phase was a mixture of organic solvent and ammo-
nium formate or acetate buffer pH 3. PhoEBus, an application
program help for buffer studies (Analis, Namur, Belgium) was used
for the preparation of aqueous salt solutions. It was prepared by
specifying the salt concentration and pH values. The pH value of
the ammonium formate or acetate solution was adjusted with 1 M
formic acid or acetic acid aqueous solution, respectively. Mobile
phase pH was then adjusted and measured using a pH-meter cal-
ibrated in aqueous buffers, before the addition of organic solvent.
It is thus noted as the aqueous w S
w pH. The apparent pH (w pH) was
not measured but would presumably positively deviate from the
aqueous pH.
2.3. Instrumentation
Two chromatographic systems were used: the first one con-
sisted of a Merck-Hitachi quaternary pump model Lachrom L-7100
(Darmstadt, Germany), a Rheodyne (Cotati, CA, USA) model 7725
injection valve fitted with a 5 or 20 ␮L loop (depending on the col-
umn dimensions), a column oven Jet Stream 2 Plus and a 785A
UV–vis HPLC Detector (Applied Biosystems, Courtaboeuf, France).
The second one was an Agilent 1100 series (Waldbronn, Ger-
many) system with: binary pump, autosampler, column oven and
DAD detector. The UV detection was carried out at 280 nm in
order to obtain maximal absorbance for all the compounds and
MS detection was carried out using a Perkin-Elmer Sciex (Foster
City, CA, USA) API 300 mass spectrometer triple-quadrupole with
Ionspray as ion source. The chromatographic data handling was
accomplished using EZChrom Server software (Merck, Darmstadt,
Germany) and Chemstation software version A.08.03 (Agilent,
Santa Clara, USA), respectively.
Physicochemical parameters of the 12 columns studied are
reported in Table 1. The flow rates were of 0.2 and 1 mL min−1
for the columns that had internal diameters of 2 and 4.6 mm,
respectively, with the exception of the ZIC-HILIC and Ascentis HILIC
columns for which the optimized flow rates recommended by the
manufacturers are of 0.5 mL min−1 .
2.4. Data analysis
Marvin 4.1.11 software (ChemAxon, Budapest, Hungary) was
used to calculate the pKa and log D (pH 3) values for each analyte.
Log D is the logarithm of the ratio of the equilibrium concentra-
tions of all species (unionized and ionized) of a molecule in octanol
to same species in the water phase at 25 ◦ C. It differs from log P
in that ionized species are considered as well as the neutral form
of the molecule and thus is more representative of hydrophobic
character in buffered conditions.
Table 1
Physicochemical parameters of the commercial HILIC packing materials studied.

Bounded group Column Dimension (mm) Particles Pore Size Functional Specific surface Manufacturer
charge Diameter (␮m) (Å) group/support (m2 g−1 )
nature

Negatively Uptisphere strategy HILIC 250 × 2 5 100 Ultra pure spherical silica 450 Interchim
charged Pursuit XRs Si 150 × 2 3 Ultra pure spherical silica – Varian
Ascentis Express HILIC 150 × 2.1 2.7 90 Fused core silica – Supelco

Neutral Luna DIOL 150 × 2 3 200 Diol bonded on silica 200 Phenomenex

Pursuit XRs Diol 150 × 2 3 Diol bonded on silica – Varian

R.-I. Chirita et al. / J. Chromatogr. A 1217 (2010) 3091–3104

TSKgel Amide 80 250 × 2 5 100 Carbamoyl bonded silica 300 Tosoh Bio-sciences

Uptisphere 5 CN 250 × 4.6 5 120 Cyanopropyl bonded silica 320 Interchim

Positively Cosmosil HILIC 150 × 2 5 120 Triazole bonded on silica 300 Nacalai tesque
charged

Astec apHera NH2 150 × 2.1 5 300 Polyamine covalently bonded to a PVA copolymer – Sigma Aldrich
Polaris NH2 150 × 2 3 180 Aminopropyl silica – Varian

Silice Uptisphere 12 AA NH2 250 × 2 5 120 Aminopropyl bonded silica 340 Interchim

Zwitterionic ZIC-HILIC 150 × 4.6 5 200 Zwitterionic bonded on silica 140 Merck SeQuant

3093
3094 R.-I. Chirita et al. / J. Chromatogr. A 1217 (2010) 3091–3104
Principal component analyses (PCA) were performed with
XLSTAT 2009.2.03 software (Addinsoft, New York, NY, USA). Prior
to the calculation, all data were normalized: they were centred
(the mean was subtracted) and reduced (the rest was divided by
the standard deviation). This ensures uniform means of zero and
variances of unity.
3. Results and discussion
3.1. Charge state of analytes and elution order in relation to the
nature of the stationary phase
The chromatography of a set of neurotransmitters and related
substances was evaluated by performing HILIC using a variety
of hydrophilic stationary phases. The 12 solutes selected for our
study were divided into three groups: six biogenic amines (DA,
NA, A, 3-MT, S and DHBA), three amino acids (Tyr, DOPA and
Trp) and three carboxylic acids (HVA, DOPAC and 5HIAA). There
are three catecholamines known to occur in vivo: adrenaline
(A), noradrenaline (NA) and dopamine (DA) [36]. Tyrosine (Tyr)
and 3,4-dihydroxy-phenylalanine (DOPA) are their physiological
precursors. Homovanillic acid (HVA), 3-methoxytyramine (3-MT)
and 3,4-dihydroxy-phenylacetic acid (DOPAC) are some of their
metabolites present in the organism. Serotonin is an indolamine
that is produced from tryptophan (Trp) and is mainly metabo-
lized into 5-hydroxyindole-3-acetic acid (5HIAA). Fig. 1 presents
the chemical structures of the selected analytes and their associ-
ated pKa and log D (pH 3) values. To prevent degradation of analytes,
the chromatographic separation was carried out under acidic con-
ditions. In a purely aqueous buffer at pH 3, the six biogenic amines
are protonated, thus bearing a net positive charge. HVA, DOPAC
and 5HIAA, containing carboxylic functions, with respective pKa
values of 3.9, 3.6 and 4.2, are only partially dissociated, thus bear-
ing a partial negative charge. For the three amino acids (Tyr, Trp and
DOPA), the amine functions are protonated whereas the carboxylic
functions, with respective pKa of 2.0, 2.5 and 1.6, are essentially
deprotonated resulting in the presence of zwitterionic compounds,
with a global nominal net charge equal to zero.
However, in the HILIC mobile phase, a large proportion of the
composition is an organic solvent. It is a well-known fact that pH
and pKa values are affected by the presence of organic solvents. As a
result, the ionization state of the above solutes might be somewhat
different from what we expect, based on purely aqueous solutions.
McCalley [37] indicates that an acetonitrile–ammonium formate
buffer 85:15 (v/v), where the buffer pH was w S
w pH = 3, had a w pH =
5.2. Besides, the pKa values in the HILIC mobile phase of the 12
compounds investigated here are unknown to us. As a matter of
fact, if the amines and amine functions of the amino acids are still
most probably protonated in our HILIC mobile phase, it may well be
that the acids and acidic functions of the amino acids would have an
increased pKa . However, whether this pKa value increase is more
or less significant than the pH increase remains unknown. In the
following, we will therefore assume that the acidic functions are
all partially ionized.
Previous HILIC studies have shown that different selectivities
were obtained using the same mobile phase on different stationary
phases [13,38,39], demonstrating that analyte partitioning is not
the only phenomenon occurring in HILIC separations. Thus the pos-
sible secondary interactions between the stationary phase and the
analytes have to be taken in consideration for the column choice.
Among the 12 columns we have tested (Table 1), different elu-
tion orders were observed, as can be seen in Fig. 2. With regards
to chromatographic results the tested columns can be divided into
two groups: those that gave better separation of catecholamines
with low salt concentration (lower than 50 mM in the aqueous
component of the mobile phase): TSK gel Amide 80, Uptisphere
CN, Cosmosil HILIC and ZIC-HILIC (Fig. 2a) and those that gave bet-
ter results at high salt concentration (higher than 100 mM in the
aqueous component of the mobile phase): Uptisphere HILIC Silica,
Ascentis Express HILIC, Luna Diol, Astec apHera NH2 , and Upti-
sphere NH2 (Fig. 2b). The effect of the concentration of salt will
be discussed in a following section.
As the primary retention mechanism in HILIC is presumably
hydrophilic partition between the water-rich layer at the surface
of the stationary phase and the bulk organic-rich mobile phase, we
can first compare the elution order of the solutes to their log D (pH
3) values. Naturally, as mentioned above, these log D values are
only indicative, considering that the charge state of the analytes
in the organic solvent—buffer mobile phase may be somewhat dif-
ferent from what is expected, based on a purely aqueous solution.
For all columns by plotting log k vs. log D (figures not shown), it
appears that they are correlated on the silica, diol, amide, cyano,
and sulfobetaine phases with correlation coefficients comprised
between 0.65 and 0.85. The slopes are negative, indicating that the
most retained compounds are those possessing the lower log D val-
ues, that is to say the most hydrophilic compounds. This indicates
that hydrophilic partitioning plays a significant role in the reten-
tion mechanism on those phases, although evidently not the only
one, otherwise correlation coefficients would be much larger; other
mechanisms than hydrophilic partitioning must be present.
On the contrary, for the amine and triazole phases, the correla-
tion is extremely poor, showing that other retention mechanisms
should be considered to understand the elution orders observed.
The other mechanisms generally considered in HILIC are hydrogen-
bonding, dipole–dipole and other electrostatic interactions. The
presence of charges on the stationary phase surface offers the pos-
sibility of electrostatic interactions (attraction or repulsion) with
the charged analytes. The commercially available HILIC stationary
phases that we tested (Table 1) can be divided from this point of
view into different groups, depending on the charge or chargeable
state of their functional group and/or support. Considering only the
acido-basic properties of silanol groups and bonded ligands, three
groups can be defined, in a pH 3 aqueous mobile phase: neutral
(silica, diol, amide and cyano), positively (triazole or amino), and
zwitterionic (sulfobetaine-bonded column) charged or chargeable
functional groups. However, in the same manner as occurs for the
solutes, the charge state of the silica support and bonded station-
ary phase may vary when a large proportion of organic solvent is
introduced in the buffered mobile phase.
First of all, the silanol groups present on the bare silica phases
and in all stationary phases bonded on silica may be partially
ionized. Indeed, while the average pKa of silanols is generally con-
sidered to be between 4 and 6 in purely aqueous solutions, it is
unknown in highly organic mobile phases. It is mostly unclear
whether, as generally observed for weak acids, the pKa value should
increase or whether it should remain unaffected, considering a
water-rich layer is at the surface of the stationary phase [37]. We
will therefore assume that the silanol groups are partially ionized,
thus negative charges may be present at the material surface but
are difficult to control due to poor appreciation of the pH and pKa
in the presence of high proportions of organic solvent.
Secondly, as described for the amine solutes, it is most probable
that the amine-type stationary phases (Astec apHera NH2 , Polaris
NH2 and Uptisphere NH2 ) and the triazole stationary phase (Cos-
mosil HILIC) would remain positively charged in the solvent–buffer
mixture. For those phases, which are bonded on silica (Polaris NH2 ,
Uptisphere NH2 and Cosmosil HILIC), internal masking of resid-
ual silanol groups through interaction with the cationic ligand is
possible, thus negative charges may not be accessible to the solutes.
Finally, the sulfobetaine ligand of the ZIC-HILIC phase should
remain unaffected by mobile phase pH, thus would always have a
 R.-I. Chirita et al. / J. Chromatogr. A 1217 (2010) 3091–3104 3095

Fig. 1. Structures of the studied neurotransmitters. *Marvin 4.1.11 software was used to calculate the pKa and log D (pH 3).

nominal net charge of zero, as the sulfobetaine group carries both
a positive and a negative charge. However, whether the number of
positive and negative charges truly is equal on this stationary phase
is unsure. Accessibility to the positive charge of the quaternary
ammonium function, closer to the silica surface, is also questionable
as will be further discussed in a following section.
Considering all these factors, the columns we have tested can
be divided as follows: columns with neutral active functional
groups and possibly negatively charged support (silica, diol, amide
and cyano), cationic phases (amino and triazole) and zwitterionic
phases (sulfobetaine). Electrostatic interactions could be a valuable
explanation for the elution orders observed on the cationic station-
ary phases. Indeed, the cationic analytes (S, 3-MT, A, DA and NA)
were weakly retained and poorly separated on those phases, par-
ticularly on the triazole one (Cosmosil HILIC). Besides, the different
separations observed between the amino phases can be attributed
to different factors. First of all, the surface coverage of amino groups
might be different between the silica and polymeric columns, but
3096 R.-I. Chirita et al. / J. Chromatogr. A 1217 (2010) 3091–3104

Fig. 2. Analysis of test compounds on different HILIC systems. (a) Column temperature 25 ◦ C; mobile phase: MeCN–20 mM ammonium acetate aqueous solution, w w pH 3
(80:20, v/v); flow rate: 0.2 mL min−1 for TSK gel Amide 80 and Cosmosil HILIC; 1 mL min−1 for ZIC-HILIC and for Uptisphere CN. (b) Column temperature 25 ◦ C; mobile phase:
MeCN–150 mM ammonium formate aqueous solution w −1
w pH 3 (85:15, v/v); flow rate: 0.2 mL min . Detection UV 280 nm. Peak identities: (1) HVA; (2) 5HIAA; (3) DOPAC; (4)
3-MT; (5) S; (6). DA; (7) Trp; (8) A; (9) DHBA; (10) NA; (11) Tyr; (12) DOPA.

this information is unavailable. Secondly, non-specific hydrophobic
interactions on the polymer material might be another reason for
some of the observed differences. Finally, electrostatic interactions
may also play a part. On the silica-based column (Uptisphere NH2 )
the anionic compounds: DOPAC, HVA and 5HIAA are less retained
than on the polymer-based one (Astec apHera NH2 ) (Fig. 2b), proba-
bly because the partially deprotonated residual silanols are titrating
some of the amine groups in the coating, lowering the density of
available cationic ligands compared with the polymeric apHera col-
umn. Moreover, generally better results (data not shown), in terms
of peak shape and resolution, were obtained on the polymer-based
column than on the silica-based ones, although the Uptisphere col-
 R.-I. Chirita et al. / J. Chromatogr. A 1217 (2010) 3091–3104
Fig. 3. Influence of the organic solvent percentage on the retention factor. TSK
gel Amide 80 column; mobile phase: MeCN–20 mM ammonium acetate aqueous
solution, w −1
w pH 3 ((x:100 − x), v/v). Flow rate: 0.2 mL min .
umn contains more stationary phase than the apHera column. It
can indeed be seen in Table 1 that the former was 25 cm length,
while the latter was 15 cm length, with an identical internal diam-
eter (2 mm). All in all these observations indicate that the support,
and not only the functional groups, play a significant role in the
separation mechanism.
In the case of bare silica stationary phases, it had been shown
that the silica characteristics plays an important role on the col-
umn selectivity [37,40,41]. The Ascentis Express HILIC column uses
superficially porous particles (also called fused core). They are made
of high-purity silica particles that measure 2.7 ␮m in diameter
overall with a 0.5-␮m-thick porous shell. Relative to columns using
conventional sub-2 ␮m particles, Ascentis Express columns are
supposed to generate comparable efficiencies with less backpres-
sure [40]. However, in our study, the salt concentrations required in
the mobile phase to obtain sharp, symmetrical peaks for the basic
compounds were as high as on the standard silica columns made
of wholly porous particles.
Other types of interaction, namely hydrogen-bonding and
dipole–dipole interactions may be responsible for the alterna-
tive selectivities observed between other stationary phases. For
instance, slight differences can be observed between the amide
and diol phases, while significant differences are observed between
the cyano phase and the other “neutral” phases. In these cases, the
nature of the bonded ligand must be responsible for different inter-
action capabilities with individual solutes. Indeed, the carbamoyl
groups on the amide phase can participate in hydrogen-bonding
as hydrogen-bond donors, while the hydroxyl groups of the diol
phases can be proton-donors in hydrogen-bonding interactions;
the cyanopropyl ligand most probably participates in dipole–dipole
interactions.
An adequate separation of our 12 compounds was obtained with
the Pursuit Si column (Fig. 2b). This achieves our aim of separating
the catecholamines for analysis with MS detection. The coelution
of HVA, 5HIAA and DOPAC does not cause problems as these com-
pounds have different molecular masses.
3.2. Influence of the organic solvent nature and percentage
The principle of HILIC is verified for all the analytes and all the
columns: increasing the percentage of organic solvent in the mobile
phase leads to increasing retention factors (Fig. 3). The retention
difference can be very significant for a modification as small as 5%
in the organic solvent percentage, as can be seen in Fig. 3 for the
TSK gel Amide column: the retention factors for almost all the ana-
lytes double when increasing from 85 to 90% of MeCN. The increase
rate is not the same for all the compounds, inducing elution order
inversions. For example on the ZIC-HILIC column the selectivity is
different for a mobile phase containing either 80 or 60% of ace-
3097
Fig. 4. Effect of the organic solvent nature. Astec apHera NH2 column; mobile phase:
organic solvent/150 mM ammonium formate aqueous solution, w w pH 2.7 (85:15, v/v).
Flow rate: 0.2 mL min−1 . Detection MS. Peak identities: (1) HVA; (2) 5HIAA; (3)
DOPAC; (4) 3-MT; (5) S; (6) DA; (7) Trp; (8) A; (9) DHBA; (10) NA; (11) Tyr; (12)
DOPA.
tonitrile. This is possibly due also to the fact that the overall salt
concentration in the mobile phase changes, as the salt was only
added in the aqueous component of the mobile phase. Effect of salt
concentration will be further discussed below.
Not only the organic solvent percentage but also its nature can
influence the analytes retention. The elution strength of the most
commonly used organic solvents (MeOH, EtOH, MeCN and acetone)
had been tested on the different supports and had been found to be
in agreement with the data reported by Li and Huang [42] and Liu
et al. [38], meaning the alcohols have the greatest elution strength,
followed by acetone and MeCN.
A distinctive behaviour is observed with the aphera NH2 column,
on which not only the elution time but also the elution order of the
analytes, is affected by the organic modifier nature. The retention
order obtained with MeOH is different from those obtained with
acetone and MeCN, respectively (Fig. 4).
3.3. Influence of the salt type and concentration
When ionic compounds are analyzed, salt presence in the mobile
phase is necessary for the analyte elution and good peak shape.
In our case the effect of the salt concentration is different on the
different compounds and supports. The salts studied were lim-
ited to ones that were soluble in high amount of organic modifier
and to the volatile ones that could be further used when coupling
the chromatographic system to the mass spectrometric detection.
That is why only ammonium acetate and ammonium formate were
tested. For the catecholamines, on most of the columns (Uptisphere
HILIC Silica, Luna Diol, Uptisphere CN, Cosmosil HILIC, or Astec
apHera NH2 ), no significant differences were registered when using
either one of these two salts. For Ascentis Express HILIC signifi-
cant differences were noticed between the two salts: important
increases in the retention factors and deterioration of the peak
shapes were observed when ammonium formate was replaced by
ammonium acetate (data not shown). Small selectivity differences
were observed on the TSK gel Amide 80 and ZIC-HILIC columns. For
example, the NA/Tyr and Trp/DA pairs have elution order reversed
on the TSK column when ammonium acetate was replaced by the
formate salt.
3098 R.-I. Chirita et al. / J. Chromatogr. A 1217 (2010) 3091–3104
Fig. 5. Effect of the salt concentration. (a) Luna Diol column; (b) TSK gel Amide 80
column; (c) Astec apHera NH2 column; mobile phase: MeCN–ammonium formate
aqueous solution w −1
w pH 3 (80:20, v/v); flow rate: 0.2 mL min . Detection UV 280 nm.
Furthermore the salt concentration in the mobile phase has
more important effects on the analytes retention. As mentioned
in Section 3.1, some of the columns needed a significant amount
of salt (150 mM) in order to generate acceptable separations (Upti-
sphere HILIC silica, Ascentis Express HILIC and Luna Diol) or better
peak shape (Astec apHera NH2 and Uptisphere NH2 ). Yet even so,
large, poorly defined peaks were obtained, for example DOPA on
the Ascentis Express HILIC column, even at salt concentration of
150 mM. This highlights the importance of the electrostatic interac-
tions. The typical HILIC behaviour involves a retention increase with
the increase of the salt concentration in the mobile phase [25,43],
that is true for salt concentrations in the mobile phase that are infe-
rior to 30 mM [39]. However, for ionized solutes, as is the case for
most solutes in this study, it is important to also consider the pos-
sible contribution of the electrostatic interactions to the retention
changes, whenever charges are present on the stationary phase.
Among the columns tested for salt concentration effect (Upti-
sphere Silica, Ascentis Express HILIC, Luna HILIC, TSKgel Amide,
Uptisphere CN, Astec apHera NH2 , Cosmosil HILIC and ZIC-HILIC)
only the Luna Diol column showed a pure HILIC behaviour, meaning
retention slightly increases with the increase of the ammonium for-
mate concentration in the mobile phase, whatever the charge state
of the analyte (Fig. 5a). This increasing retention trend has also been
noticed when using a mobile phase that generates higher retentions
(MeCN–ammonium formate aqueous solution w w pH 3, 90:10, v/v),
for example Tyr retention factor increased from 3.9 to 5.4 when the
salt concentration in the aqueous component of the mobile phase
increased from 10 to 150 mM. This would suggest that the resid-
ual silanols are far less accessible than on the other bonded silica
phases. This is consistent with the known structure of this station-
ary phase, which is a cross-linked propane-diol phase bonded on
silica. Therefore, the residual silanol groups should be less easily
reached by the solutes.
On all other tested columns, the electrostatic attractions
between the charged chromatographic support and the oppositely
charged analytes play an important role in the compound reten-
tion and an ion-exchange chromatography behaviour is observed:
increased concentrations of salt lead to decreased retention. This
involves a retention decrease for the cationic analytes (NA, A, DA,
DHBA, 3-MT and S) on the stationary phases and supports possi-
bly possessing negative charges (Uptisphere HILIC Silica, Ascentis
Express HILIC, TSK gel Amide 80 and ZIC-HILIC) (Fig. 5b) when
the salt concentration in the mobile phase increases. The same
phenomenon is observed for the anionic and zwitterionic ana-
lytes (DOPA, DOPAC, 5HIAA, Tyr, Trp and HVA) on the positively
charged columns (Cosmosil HILIC, Astec apHera NH2 and Upti-
sphere NH2 ) (Fig. 5c). On both positively and negatively charged
columns, when our analytes have the same charge as the support
they were analyzed on, the retention increased with the increase
of the salt concentration. This phenomenon is in accordance with
that described by Guo and Gaiki [25] for the analysis of cyto-
sine, aspirin and salicylic acid on positively and negatively charged
columns. Guo and Gaiki suggested that this could be related to
both hydrophilic partitioning and decreased electrostatic repul-
sion between compounds and stationary phase functions bearing
identical charges.
Our work confirms for the TSK gel Amide column a behaviour
similar to that of the possibly negatively charged stationary phases
As previously observed by Alpert [3] the retention of the positively
charged basic amino acids decreased with the salt concentration
increase in the mobile phase on the TSK gel Amide column. This
implies the presence of negative charges on the stationary phase
that could be caused by deprotonated residual silanols of the silica
support. The hypothesis of deprotonated silanols has been issued
by Quiming et al. [43] to explain the electrostatic repulsions they
observed, between their analytes (methyl uric acids) and the “neu-
tral” diol-bonded silica column. The comparison by Guo and Gaiki
[25] of the TSK gel Amide column with ZIC-HILIC and bare silica for
the analysis of salicylic acid and its homologues, in pH conditions
closer to the ones used in our study, revealed, for the TSK column a
behaviour closer to that of the bonded ZIC-HILIC column. All these
results confirm that, in the addition to the functional groups, the
support nature plays a role in the separation mechanism under
HILIC conditions.
3.4. pH effect
Mobile phase pH can influence the analytes retention and/or
selectivity in HILIC due to the variation in ionization state of ana-
lytes according to the pH value. To investigate pH influence on
analytes retention, the pH of the ammonium formate aqueous solu-
tion was adjusted between 3 and 6.5 (formate ion would only buffer
well in the pH range 3–5) with formic acid before its combination
with MeCN.
In the HILIC mobile phase prepared from pH 6.5 buffer, the ion-
ization state of the solutes should be as follows: considering their
pKa values, amines functions should remain mostly protonated,
while acidic functions should now be fully ionized. For the station-
ary phases and silica supports, possible changes can be observed:
the amine and triazole phases probably remain mostly protonated
as well, while the silanol groups would now be essentially ionized.
 R.-I. Chirita et al. / J. Chromatogr. A 1217 (2010) 3091–3104
The diol functions should remain protonated, while the cyano and
amide functions, having no acido-basic properties, are unchanged.
Only two of the bare silica columns were tested in this case:
Uptisphere Silica and Ascentis Express HILIC. For these columns,
retention increased with the pH increase essentially for the acidic
compounds. The increased retention of the acidic solutes can be
attributed to increased hydrophilic character, as these compounds
are now fully ionized, so stronger hydrophilic interactions are now
possible. This hypothesis is confirmed by the fact that retention
generally increased for these solutes up to pH 5.0, and then lev-
elled off. At pH 5.0, it is expected that the acidic functions would
be fully ionized, and that no further changes should occur. Besides,
on Ascentis Express HILIC the peak shape of the basic compounds
deteriorated with pH increase. It is probable that, as silanol groups
become more and more deprotonated, a mixed-mode retention
mechanism occurs for the protonated solutes, comprising both
hydrophilic partitioning and electrostatic interactions with anionic
residual silanol groups, causing longer retention together with
peak shape deterioration. The retention increase with the pH was
also observed on the TSK gel Amide 80 column, which once more
behaves as a negatively charged column. The two neutral columns
used for the pH influence study (Luna Diol and Uptisphere CN) had
different behaviours. On Luna Diol retention remains constant for
all analytes when pH is varied. This is in accordance with the above
remarks on the least accessibility to residual silanols on this col-
umn. On the cyano-bonded column, retention remains constant
only for the amino acids (DOPA, Tyr and Trp). As the pH increased,
the basic solutes (3-MT, A, NA, DA, S and DHBA) and the acidic ones
(HVA, 5HIAA and DOPAC) had opposite behaviours: the retention
increased for the first group and decreased for the second on Upti-
sphere CN column. This implies that there is an increasing density of
negative charge with increasing pH on the CN support, most proba-
bly due to increasing deprotonation of the residual silanols with the
pH. These would cause increased electrostatic interactions: attrac-
tive to the cationic solutes inducing increased retention, repulsive
to the anionic solutes inducing decreased retention.
For the positively charged supports (Cosmosil HILIC and Upti-
sphere NH2 ) we could observe a retention increase, particularly
significant for the acidic compounds (HVA, 5HIAA and DOPAC).
This is consistent with increasing deprotonation of the acidic func-
tions, causing both increased hydrophilic character and increased
electrostatic interactions with the protonated triazole and amine
functions of the stationary phase.
Finally, for the zwitterionic column, ZIC-HILIC, the retention
increases (mainly for the positively and negatively charged com-
pounds) with the pH as it did on the negatively charged columns,
meaning that the analytes mainly interacted with the negatively
charged sulfonate group present at the sulfobetaine ligand end.
This is in accordance with past studies, indicating that sufficiently
large solutes cannot reach the quaternary ammonium function that
is placed close to the silica surface, as they might face electro-
static repulsion from the sulfonate end groups [15]. The resulting
behaviour of the sulfobetaine stationary phase is then comparable
to a negatively charged stationary phase.
3.5. Temperature influence
All the studies in the literature showed that selectivity changes
with temperature depending mainly on the nature of analytes and
differences in analyte–stationary phase interactions [44]. We could
observe that the temperature effect depends both on the analyte
and stationary phase nature.
On the two silica columns used for this study (Uptisphere HILIC
Silica and Ascentis Express HILIC) the temperature increase had
different effects. On the first column for all the compounds reten-
tion decrease was registered with the temperature increase. On the
3099
Fig. 6. Temperature effect. Ascentis Express HILIC column; mobile phase:
MeCN–150 mM ammonium formate aqueous solution w w pH 3 (90:10, v/v); flow rate:
0.5 mL min−1 . Detection UV 280 nm. (1) HVA; (2) 5HIAA; (3) DOPAC; (4) 3-MT; (5)
S; (6) DA; (7) Trp; (8) A; (9) DHBA; (10) NA.
fused core column different effects were observed for the different
analytes when temperature was increased as follows: some solutes
had a “normal behaviour” with decreased retention (S, 3-MT and
Trp), others were unaffected (DOPAC and HVA), while others had
an increased retention (DA, NA, A, DA and DHBA). This leads to dif-
ferent band spacing when the temperature is varied (Fig. 6). The
major differences occur between 10 and 20 ◦ C, leading to reversal
of elution order for Trp and NA.
Different behaviours were also observed for the two neutral
columns used for this study: TSK gel Amide and Luna Diol. For the
first column, retention variations were not very significant, while
on the second retention decreased for all the compounds when
temperature increased.
The temperature effect was studied on three of the positively
charged columns: Astec apHera NH2 and Cosmosil HILIC. For Astec
apHera NH2 and Cosmosil HILIC we could observe an increase of
the retention factor with the temperature increase for the acidic
compounds, while for the others analytes, the retention decreased
or remained constant.
Only for the ZIC-HILIC column, retention increased with the
temperature for all the analytes.
3.6. Comparison of stationary phases in all conditions
Lämmerhofer et al. [45] used principal component analysis
to compare HILIC chromatographic systems to mixed-mode ion-
exchange and reversed-phase systems, and also more specifically
on HILIC elution data, to investigate retention behaviours on sta-
tionary phases of varied nature. The intention in our data analyses
was to help in the initial choice of a stationary phase, but also to
investigate the similarities and differences in elution behaviour to
assess the usefulness of changing one column for another in the
method development process.
PCA calculations were performed on logarithms of the retention
factors after auto-scaling (all columns were centered and reduced)
to give all variables the same importance. Besides, this operation
ensures that the position of the columns in the PCA plot is related to
separation properties, while a PCA plot based on non-auto-scaled
data shows differences in retention behaviour.
Initially, a PCA was carried out combining all retention data
available for the three acidic solutes, the six basic solutes, and
3100 R.-I. Chirita et al. / J. Chromatogr. A 1217 (2010) 3091–3104

Fig. 7. Principal component analysis based on the retention data (log k) measured for A, DA, S, HVA, DOPAC, 5HIAA, DOPA, Tyr and TRP on the 12 stationary phases investigated,
in all operating conditions: (a) PC1–PC2 score plot and (b) PC1–PC2 loading plot.

Fig. 8. Principal component analysis based on the retention data (log k) measured on the 12 stationary phases investigated, in all operating conditions: (a) PC1–PC2 score
plot based on retention data of anionic acidic compounds (HVA, 5HIAA and DOPAC); (b) PC1–PC2 score plot based on retention data of cationic basic compounds (A, DA, S,
3-MT, NA and DHBA); (c) PC1–PC2 score plot based on retention data of zwitterionic amino acids (DOPA, Tyr and Trp).
 R.-I. Chirita et al. / J. Chromatogr. A 1217 (2010) 3091–3104 3101

Fig. 9. Principal component analysis based on the retention data (log k) measured on TSKgel Amide for the 12 solutes investigated, in all operating conditions: (a) PC1–PC2
score plot based on non-auto-scaled retention data; (b) PC1–PC2 loading plot based on non-auto-scaled retention data; (c) PC1–PC2 score plot based on auto-scaled retention
data; (d) PC1–PC2 score plot based on non-auto-scaled retention data. Black circles are related to the variation in acetonitrile percentage, from 70 to 90%. Pink squares stand
for the variation in column temperature (varied between 10 and 50 ◦ C). Blue diamonds are related to the variation in salt concentration in the mobile phase (from 10 to
75 mM). Green triangles stand for the data acquired at different pH (from 3 to 5). (For interpretation of the references to color in this figure legend, the reader is referred to
the web version of the article.)

the three amino acids. However, we feared that the cationic basic
solutes, being more numerous than the others, would excessively
weight this plot. Thus we calculated another PCA taking into
account the three anionic acidic solutes (HVA, 5HIAA and DOPAC),
three cationic basic solutes (A, DA and S), and the three zwitterionic
amino acids (DOPA, Tyr and Trp). Taking into account all stationary
phases and operating conditions tested, whenever data were avail-
able for all nine compounds, the calculation includes data for 87
observations. The PC1–PC2 score plot is presented in Fig. 7a, while
loading plot is presented in Fig. 7b.
PC1 and PC2 together account for about 85% of the variance, thus
the PC1–PC2 plots are amenable to interpretation. The three groups
of solutes are clearly distinguished on the loading plot, revealing
that they behave in a different manner on the stationary phases
tested. On the left-hand side of the score plot, the cationic-type
stationary phases (Cosmosil HILIC, Astec NH2 , Uptisphere NH2 and
Polaris NH2 ) are found, naturally essentially weighted by the reten-
tion of the anionic acidic compounds. On the right-hand side of the
score plot, the anionic-type (Ascentis Express HILIC, Pursuit SI and
Uptisphere SI), neutral (Luna DIOL, Pursuit DIOL, TSK gel Amide and
Uptisphere CN) and zwitterionic (ZIC-HILIC) stationary phases are
found, essentially weighted by the retention of cationic basic com-
pounds. ZIC-HILIC and Uptisphere CN are seen to be clearly different
from the others, based on their position relative to the PC2 axis, but
also based on the fact that they are the farthest to the centre points
on the PC2–PC3 plot (not shown). We can also point out that Luna
DIOL, which was seen to have less accessible silanol groups than the
other neutral phases, is positioned furthest to the top left of this col-
umn group, so it is clearly the least weighted by the position of the
cationic bases. This is all indicative of a global retention behaviour
separating compound families according to the following scheme:
(i) On anionic and neutral phases (apart from Uptisphere CN),
anionic acids are less retained than cationic bases, less retained
than zwitterionic amino acids.
(ii) On cationic phases, cationic bases are less or equally retained as
the anionic acids, less retained than zwitterionic amino acids.
(iii) On the zwitterionic phase (ZIC-HILIC), which appears to be dis-
criminated from the others on the PC2 axis, anions are less
retained than amino acids, less or equally retained as cations.
(iv) On the Uptisphere CN, anions are less retained than amino
acids, less retained than cations.
3102 R.-I. Chirita et al. / J. Chromatogr. A 1217 (2010) 3091–3104
Among each family of stationary phases providing generally
similar elution orders, some diversity is found. First of all, among
cationic stationary phases, a wide dispersion is found along the
PC2 axis, mainly weight by the retention of the zwitterionic amino
acids. It is clear that the salt concentration has a great effect on
the general retention behaviour of these phases, as the points are
scattered to a large extent (for Cosmosil HILIC and Astec NH2 , for
instance). On the ZIC-HILIC phase, the pH is seen to have a great
effect on separation behaviour, as the observations at pH ≥ 5 are
distant from observations at pH 4. This is most probably due to the
variation in ionization state of the solutes when increasing the pH,
affecting their retention and separation behaviour.
To achieve a clearer view of separation behaviour, we then cal-
culated three more PCAs: the first one based solely on retention of
the three anionic compounds (HVA, 5HIAA and DOPAC), PC1–PC2
score plot shown in Fig. 8a; the second one based solely on the
retention of the six cationic compounds (A, DA, S, 3-MT, NA and
DHBA), PC1–PC2 score plot shown in Fig. 8b; and the third one
based on the retention of the three zwitterionic amino acids (Trp,
Tyr and DOPA), PC1–PC2 score plot shown in Fig. 8c.
First of all, we can note that, as the retention behaviour of
compounds bearing the same charge is similar and as compound
number is small, the PC1 and PC2 axes together already carry
all information (except for Fig. 8b, because of the Uptisphere CN
eccentricity), thus the PC1–PC2 plots are readily amenable to inter-
pretation, without any need for attention to further components.
On all three figures, we can note that separation behaviour in
one compound family is not always as would have been expected
based on the observation of Fig. 7:
(i) The Uptisphere CN singles out for the separation of anionic and
cationic compounds, but nor for the separation of amino acids.
(ii) DIOL phases and silica gel phases are always separated, indi-
cating that they provide different elution orders of the solutes
of one compound family, or, when they provide identical elu-
tion orders, different selectivities are observed. For instance,
DIOL phases provide poor separation of HVA and 5HIAA, while
silica phases can separate these compounds. On the contrary,
silica phases provide poor separation for TYR and DOPA while
they are generally better separated on diol phases. Hydrogen-
bonding and dipole–dipole interactions probably play a part in
these differences.
(iii) ZIC-HILIC is generally in between diol and silica phases, closer
to the former.
(iv) For anions and zwitterionic species, diversity can also be found
among the cationic stationary phases. Polaris NH2 provided
poor separation of the anions, while Uptisphere NH2 was able
to separate them but with a clearly lower retention than other
amino phases. Astec NH2 is particularly singled out for separa-
tion of amino acids.
On the separation of amino acids, the salt concentration seems
to have a great influence on the separation behaviour, as ZIC-HILIC,
Astec NH2 and Uptisphere SI, on which the effect of salt concentra-
tion was investigated, are scattered to a large extent on the score
plot.
3.7. Relative importance of the adjusting parameters for each
stationary phase
For each column, when sufficiently varied data were available,
a principal component analysis was carried out to estimate the
relative importance of each optimization parameter (acetonitrile
percentage, salt concentration, pH and temperature) on both reten-
tion and separation.
In this case, PCA plots were carried out both before and after data
auto-scaling of retention data (logarithms of the retention factors),
thus two PCA plots were available in each case, one related to reten-
tion (non-auto-scaled data), and the other based on elution orders
of the solutes (auto-scaled data).
The PCA plots obtained for TSKgel Amide are presented in Fig. 9,
as an example, but conclusions are roughly the same for all columns.
In Fig. 9a and b, the PCA plots (score plot and loading plot, respec-
tively) based on non-auto-scaled data are presented. First of all,
we can point out the large percentage of variance explained by
the PC1–PC2 plots, and most particularly by the PC1 plot that car-
ries alone 80% of the variance, thus the figures presented are fully
amenable to interpretation. Secondly, the following points can be
deduced from the observation of both figures:
(i) The influence of pH is essentially associated to those three
solutes (HVA, 5HIAA and DOPAC), which face the strongest
variation in their ionization state in the pH range investigated
(the points related to pH variation are placed in the same direc-
tion as the acids).
(ii) The influence of the percentage of acetonitrile is the most
important one (the points are placed on a parallel line to PC1
axis), strongly affecting retention of all compounds.
(iii) Salt concentration and temperature only have limited influ-
ence on retention, and this influence is not identical to the
change in acetonitrile percentage (the points related to salt
concentration variation and temperature variation are placed
in an orthogonal fashion as compared to those related to ace-
tonitrile change).
In Fig. 9c and d, the PCA plots (score plot and loading plot,
respectively) based on auto-scaled data are presented. Again, the
percentage of variance explained by the PC1–PC2 plots is large,
but in this case the second component has a quite large signifi-
cance as compared to the first component (30% vs. 45% variance
explained). The conclusions regarding the influence of the opti-
mization parameters on the separation are quite different from the
above comments:
(i) The acidic compounds are no more separated from the others
on the loading plot, and no particular ordering depending on
compound family appears, indicating that all compounds can
be diversely affected by the varied optimization parameters, as
far as separation is concerned.
(ii) pH remains a significant parameter, thus would deserve being
investigated in the method development process, whenever
some solutes ionization may be affected, or stationary phase
ionization may be affected.
(iii) MeCN percentage is not as significant for separation as it is for
retention, indicating that most solutes tend to be affected in
the same manner by a change in MeCN proportion, while band
spacing is not so much affected.
(iv) Salt concentration is significant to modify band spacing.
(v) Temperature is still the least significant parameter to affect
separation quality.
These observations are quite consistent with the observations of
Guo et al. [39], who used experimental design to evaluate the rela-
tive significance of optimizing parameters on a variety of stationary
phases and concluded that MeCN proportion and salt concentration
were most significant than temperature. These results can then be
used as guidelines in HILIC method development (Fig. 10).
 R.-I. Chirita et al. / J. Chromatogr. A 1217 (2010) 3091–3104
Fig. 10. Decision tree for HILIC separations optimization.
4. Conclusions
On a practical note, these results can be used as follows:
(i) When analyzing anionic compounds, a cationic-type column
(amino or triazole) or zwitterionic (ZIC-HILIC) phase should
be selected first to provide sufficient retention. In the opti-
mization process, changing one column for another can be
advantageous to modify selectivities, and even sometimes elu-
tion order.
(ii) When analyzing cationic compounds, a neutral (cyano, diol
or amide), anionic-type (silica gel) or zwitterionic phase (ZIC-
HILIC) should be selected to achieve sufficient retention. In the
optimization process, changing one column for another, espe-
cially changing a neutral phase for an anionic phase (or vice
versa) can provide different selectivities and/or elution orders.
(iii) When analyzing globally neutral compounds such as zwitte-
rionic amino acids, any HILIC phase is potentially capable of
providing sufficient retention. In the optimization process, it
is more advantageous to replace a particular stationary phase
chemistry by a very different one, to provide different selec-
tivities and elution orders.
Based on these conclusions we propose the following procedure
for HILIC separation optimization at least for compounds with prop-
erties somewhat comparable to those studied here. Start with a
mobile phase composed of MeCN and 10 mM ammonium formate
aqueous solution (75:25, v/v) and then according to your results
follow steps of the decision tree presented in Fig. 10. If retention
3103
is too high, then the replacement of MeCN by MeOH should be
considered.
This method development scheme should be useful when polar
compounds amenable to HILIC chromatography need to be sepa-
rated.
Acknowledgments
This work was partially supported by a financial grant (PHC
Brancusi ECO-NET 18883PM) from the French Ministry of Foreign
and European Affairs that insured Raluca Chirita’s scholarship. The
authors would also like to thank Anna Ziemianin for her technical
support and the manufacturers Interchim, Supelco and Varian for
the gift of additional columns.
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