Journal of Chromatography A 1677 (2022) 463324

Contents lists available at ScienceDirect

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

The impact of low adsorption surfaces for the analysis of DNA and
RNA oligonucleotides
Honorine Lardeux a,b, Alexandre Goyon c, Kelly Zhang c, Jennifer M Nguyen d,
Matthew A Lauber d, Davy Guillarme a,b, Valentina D’Atri a,b,∗
a

Institute of Pharmaceutical Sciences of Western Switzerland (ISPSO), University of Geneva, CMU-Rue Michel Servet 1, Geneva 4 1211, Switzerland
School of Pharmaceutical Sciences, University of Geneva, CMU-Rue Michel Servet 1, Geneva 4 1211, Switzerland
Small Molecule Pharmaceutical Sciences, Genentech Inc., DNA Way, South San Francisco, CA 94080, USA
d
Waters Corporation, 34 Maple Street, Milford, MA 01757, USA
b
c

a r t i c l e

i n f o

Article history:
Received 18 March 2022
Revised 7 July 2022
Accepted 8 July 2022
Available online 9 July 2022
Keywords:

Oligonucleotides
Ion-pairing reversed-phase chromatography
(IP-RPLC)
Hydrophilic interaction chromatography
(HILIC)
Bioinert surfaces
Low adsorption surfaces

a b s t r a c t
As interest in oligonucleotide (ON) therapeutics is increasing, there is a need to develop suitable analytical methods able to properly analyze those molecules. However, an issue exists in the adsorption of
ONs on different parts of the instrumentation during their analysis. The goal of the present paper was
to comprehensively evaluate various types of bioinert materials used in ion-pairing reversed-phase (IPRPLC) and hydrophilic interaction chromatography (HILIC) to mitigate this issue for 15- to 100-mer DNA
and RNA oligonucleotides. The whole sample flow path was considered under both conditions, including
chromatographic columns, ultra-high-performance liquid chromatography (UHPLC) system, and ultraviolet (UV) flow cell. It was found that a negligible amount of non-specific adsorption might be attributable
to the chromatographic instrumentation. However, the flow cell of a detector should be carefully subjected to sample-based conditioning, as the material used in the UV flow cell was found to significantly
impact the peak shapes of the largest ONs (60- to 100-mer). Most importantly, we found that the choice
of column hardware had the most significant impact on the extent of non-specific adsorption. Depending on the material used for the column walls and frits, adsorption can be more or less pronounced. It
was proved that any type of bioinert RPLC/HILIC column hardware offered some clear benefits in terms
of adsorption in comparison to their stainless-steel counterparts. Finally, the evaluation of a large set of
ONs was performed, including a DNA duplex and DNA or RNA ONs having different base composition,
furanose sugar, and modifications occurring at the phosphate linkage or at the sugar moiety. This work
represents an important advance in understanding the overall ON adsorption, and it helps to define the
best combination of materials when analyzing a wide range of unmodified and modified 20-mer DNA
and RNA ONs.
© 2022 The Authors. Published by Elsevier B.V.
This is an open access article under the CC BY license ( />
1. Introduction
Therapeutic oligonucleotides have gained increasing attention
thanks to their high potential to treat a large variety of diseases
[1,2]. By the end of 2021, 16 oligonucleotide-drug therapies have

been approved by Food and Drug Administration (FDA) or European Medicines Agency (EMA), with twelve of them having received approval since 2016 [3]. Milasen, a personalized oligonucleotide specifically developed for a single patient suffering from
∗
Corresponding author at: Institute of Pharmaceutical Sciences of Western
Switzerland (ISPSO), University of Geneva, CMU-Rue Michel Servet 1, Geneva 4 1211,
Switzerland.
E-mail address: (V. D’Atri).

Batten disease, is a promising example of oligonucleotide-based
customized medicine [4,5].
To support the development of these complex drugs, robust and
sensitive analytical methods are required. Ion-pairing reversedphase liquid chromatography (IP-RPLC), also known as ion-pair
chromatography, is recognized as the gold standard method for
the characterization of oligonucleotide products and related impurities [6,7]. Being complex amphiphilic molecules, ONs present
hydrophilic and negatively-charged backbone. Therefore, they are
not sufficiently retained on hydrophobic RPLC stationary phases.
For this reason, ion-pairing (IP) agents such as N-alkyl amines are
added to the mobile phase, forming oligonucleotide ion-pairs that
may be separated based on their related hydrophobicity. At elevated temperatures applied to these separations, oligonucleotides

/>0021-9673/© 2022 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license ( />

H. Lardeux, A. Goyon, K. Zhang et al.

Journal of Chromatography A 1677 (2022) 463324

adopt a linear form such that a length-based separation is primarily observed [8–10]. Even if the ion-pair formation in solution is
a commonly depicted mechanism, the chromatographic separation
may also be explained by the initial adsorption of the IP agent on
the hydrophobic stationary phase via its alkyl chains, followed by
an ion-exchange process between the charged surface and the analyte [11]. It is generally accepted that both mechanisms coexist to

explain the retention model of the so-called “ion-pair chromatography” [12,13].
IP-RPLC of oligonucleotides has been widely explored over the
years. First achieved by Fritz et al. in 1978, IP-RPLC separation
of oligonucleotides historically used triethylamine (TEA) as the IP
agent [8,14,15]. To overcome some limitations in terms of ESI-MS
sensitivity, Apffel et al. suggested in 1997 the addition of hexafluoroisopropanol (HFIP) to facilitate the ESI process as well as the IP
efficiency [11,16–21]. While alternative IP agents have been widely
evaluated [22–31], TEA-HFIP mobile phase remains widely used for
oligonucleotide characterization [6,7,16,32].
The highly polar nature of ONs makes it possible to also consider hydrophilic interaction chromatography (HILIC). In a HILIC
separation, charged oligonucleotides can be separated on a polar
stationary phase, usually bonded with polar groups such as amide
or diol moieties, using a highly-organic mobile phase that contains
salts to enhance retention capabilities and selectivity. The separation mechanism involves the partitioning of analytes between the
bulk mobile phase and a water-rich layer immobilized on the stationary phase surface. Retention is further achieved through ionic
and hydrogen bonding interactions [33].
HILIC was first introduced by Alpert in 1990, but HILIC for ON
analysis has grown exponentially in the last few years [34]. The
popularization of MS-friendly, ion-pairing free buffers such as ammonium acetate or formate that substitute the previous use of triethylammonium acetate in HILIC mode further encourages the development of HILIC [35–44].
It has been widely reported that oligonucleotides, because of
their electron-rich backbone, suffer from undesired, and often adsorptive, interactions with materials traditionally used in chromatographic analyses. The main construction material of chromatographic systems and columns is stainless-steel to ensure pressure resistance. Despite its mechanical strength, easy manufacturability and compatibility with most eluents, stainless-steel was
found to be susceptible to corrosion with many diverse chromatographic eluents [45,46]. The resulting positively-charged metal oxide layer at the surface of the metallic components may cause
problems of metal leaching, impacting the chromatographic and
MS performance, as well as leading to irreversible adsorption of
analytes [47]. This non-specific adsorption is even more critical
when working at low to neutral pH, being that these are conditions under which metals are more electropositive and most likely
to cause ionic interactions with negatively-charged species such
as oligonucleotides [42,48–51]. These unwanted ionic interactions
with the oligonucleotides are further increased as the stainlesssteel surface becomes more corroded and as the number of phosphate groups increases [51,52]. Non-specific adsorption may also
be a result of polarity-based interactions between hydrophobic

ion-pairs and hydrophobic materials from the flow path.
This phenomenon negatively impacts chromatographic performance by reducing recovery and altering peak shapes (tailing,
asymmetry). Consequently, sensitive detection as well as accurate
quantitation are hindered, and reliability and reproducibility become compromised [53,54]. Several approaches have traditionally
been used in an attempt to minimize oligonucleotide adsorption.
In one case, a strong acid or a sacrificial sample can be used to
mask active sites of metallic surfaces and thereby passivate a chromatographic system or column [55,56]. Chelators such as ethylenediaminetetraacetic acid (EDTA) may also be used to trap metal ions

and prevent adsorption. However, their use can come with certain
drawbacks, such as ion suppression and persistence in the system.
In addition, these techniques are time-consuming and not longstanding [57–60].
In the last few years, chromatographic instrument manufacturers have focused their developments on strategies to permanently
mitigate adsorption of problematic analytes. Low adsorption systems and columns have been offered and are based on the use
of novel surface technologies. Made of bioinert and/or biocompatible materials, they provide a solution to suppress interactions of
oligonucleotides with surfaces [53–55,60–63]. In general, the term
“bioinert” refers to a surface that hampers adsorption, while the
term “biocompatible” is used to define a corrosion-resistant material [61,64]. Therefore, oligonucleotide analyses require the use of
bioinert materials, which are also biocompatible.
In this work, we present a comprehensive evaluation of bioinert
strategies to prevent non-specific adsorption of oligonucleotides in
IP-RPLC and HILIC. In IP-RPLC mode, three columns made of different bioinert hardware (i.e. titanium-lined, PEEK-lined and hybrid organic/inorganic surface columns) were compared to their
stainless-steel counterparts using model oligonucleotide samples
(DNA and RNA oligonucleotides ranging from 15- to 100-mer). Similarly, bioinert HILIC columns were compared to their stainlesssteel counterparts. As bioinert HILIC columns are just recently
emerging, only PEEK-lined and hybrid organic/inorganic surface
columns are available. To our knowledge, HILIC columns in a
titanium-lined hardware are not yet offered. Finally, the impact of
instrumentation hardware was also investigated. Three chromatographic systems with different fluidic path material (i.e. stainlesssteel, MP35N and titanium, and hybrid organic/inorganic surface)
were considered and the impact of the UV flow cell was also highlighted. The last part of the study deals with the extension of our
observations with the analysis of a wide range of unmodified and
modified 20-mer oligonucleotides.

To our knowledge, a systematic comparison of the impact of
bioinert columns consisting of different column hardware has
never been reported before. More importantly, the evaluation of
non-specific adsorption of DNA and RNA oligonucleotides in HILIC
mode has been here comprehensively investigated for the first
time by using bioinert column hardware.
This work was essential to understand the contribution of each
hardware parameter on the overall oligonucleotide adsorption and
conclude on a combination of materials to preferentially use in future studies.
2. Experimental
2.1. Chemicals and reagents
Oligonucleotides were purchased from Eurogentec (Seraing, Belgium) and Integrated DNA Technologies (IDT, Leuven, Belgium).
Type 1 water was obtained from a Milli-Q purification system
from Millipore (Bedford, MA, USA). LC-MS grade methanol (art.
M/4062/17) and acetonitrile (art. A/0638/17) were purchased from
Thermo Fisher Scientific (Reinach, Switzerland). Ammonium acetate (≥98%, art. 32301), 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP,
≥99%, art. 105228), triethylamine (TEA, ≥99.5%, art. 90340), and
RNase-free water (art. 95289) were purchased from Sigma-Aldrich
(Buchs, Switzerland).
2.2. Sample preparation
Eppendorf DNA LoBind® tubes, Eppendorf Dualfilter T.I.P.S® and
polypropylene vials were systematically used during this work to
eliminate any risk of additional adsorption that would bias our results. 100-μM oligonucleotide aliquots were initially prepared by
2


H. Lardeux, A. Goyon, K. Zhang et al.

Journal of Chromatography A 1677 (2022) 463324


Table 1
Sequences, molecular masses and modification types of investigated oligonucleotides. Phosphorothioate (PS) linkages are indicated by a ∗ , 2’-O-methoxyethyl modifications
(MOE) by a X, 2’-O-methyl (OMe) modifications by a Y, and locked nucleic acids (LNA) by a Z.
Compound Name

Length (mer)

DNA/ RNA

Sequence (5’-3’)

Molecular weight (g.mol−1 )

Modification

dT15-35:
Equimolar mixture of
dT15, dT20, dT25, dT30,
dT35

15
20
25

DNA

TTT (TTT)3 TTT
TTT (TTT)5 TT
TTT (TTT)7 T


4500.9
6021.9

Unmodified

dT40-100:
Equimolar mixture of
dT40, dT60, dT80, dT100
rU15-30:
Equimolar mixture of
rU15, rU20, rU30
dT20
dA20
dG20
dC20
rU20
dT20-PS
rU20-PS
rU20-MOE
rU20-OMe
dT20-LNA

30
35
40
60
80
100
15
20

30
20
20
20
20
20
20
20
20
20
20

DNA

DNA

DNA
DNA
DNA
DNA
RNA
DNA
RNA
RNA
RNA
DNA

TTT (TTT)8 TTT
TTT (TTT)10 TT
TTT (TTT)12 T

TTT (TTT)18 TTT
TTT (TTT)25 TT
TTT (TTT)32 TT
UUU (UUU)3 UUU
UUU (UUU)5 UU
UUU (UUU)8 UUU
TTT TTT TTT TTT TTT TTT TT
AAA AAA AAA AAA AAA AAA AA
GGG GGG GGG GGG GGG GGG GG
CCC CCC CCC CCC CCC CCC CC
UUU UUU UUU UUU UUU UUU UU
T∗ T∗ T∗ TTT TTT TTT TTT TT∗ T ∗ T∗ T
U∗ U∗ U∗ UUU UUU UUU UUU UU∗ U ∗ U∗ U
XXX UUU UUU UUU UUU UUX XX
YYY UUU UUU UUU UUU UUY YY
ZZZ TTT TTT TTT TTT TTZ ZZ

reconstituting lyophilized material in the appropriate volume of
RNase-free water and stored at – 20°C (DNA oligonucleotides) or
– 80°C (RNA oligonucleotides). Oligonucleotides samples were prepared by diluting the oligonucleotide material to 2 μM in RNasefree water or 10:90 H2 O/ACN prior to RPLC or HILIC analysis, respectively. Equimolar oligonucleotide mixtures were prepared by
mixing aliquots and diluting the oligonucleotide product to 2 μM.
Table 1 lists investigated oligonucleotides and their characteristics.
A 20-mer double-stranded DNA oligonucleotide was also studied in HILIC mode. A single-stranded ON, having 5’-TTC GCC
TCG CAG TGC GCC TT-3’ sequence and molecular weight of
12 237 g.mol−1 , and its complementary sequence were annealed
to form the duplex by using the following protocol. 100-μM singlestranded oligonucleotides were mixed in an annealing buffer consisting of 100 mM ammonium acetate in H2 O, heated at 85°C for
5 min, then allowed to slowly cool down to room temperature and
aliquoted. The final concentration of the duplex was 50 μM. Duplex sample was then prepared by diluting the material to 2 μM in
10:90 H2 O/ACN.


7542.9
9063.8
10584.8
12105.6
18189.8
24273.7
30357.6
4530.6
6061.4
309123.1
6021.9
6202.2
6522.2
5721.6
6061.4
6118.4
6157.8
6494.2
6145.6
6190.0

Unmodified

Unmodified

Unmodified
Unmodified
Unmodified
Unmodified
Unmodified

Phosphorothioate (6)
Phosphorothioate (6)
2’-O-methoxyethyl (6)
2’-O-methyl (6)
Locked Nucleic Acid (6)

flow-through-needle injector. The TUV detector was equipped with
a 500-nL analytical flow cell (10 mm path length). The flow path
of this instrument contains hybrid surface technology (HST), which
is described as MaxPeakTM High Performance Surfaces by Waters.
It is an ethylene bridge hybrid siloxane surface that is applied to
materials by vapor deposition.
For sake of consistency and proper data comparison, the same
TUV detector was used on the three instruments while being
equipped with different flow cells. In all cases, absorbance data
were acquired at 260 nm. Data acquisition and instrument control
were performed by Empower 3 software (Waters).
2.3.4. Columns
RPLC and HILIC columns used for this study, including bioinert columns and their stainless-steel analogs, have been listed in
Table 2. The complete history of RPLC and HILIC column injections
is reported in Table S1 and Table S2, respectively.
2.4. Chromatographic conditions

2.3. Instrumentation and columns
Mobile phases for IP-RPLC analyses were composed of 15 mM
TEA, 400 mM HFIP in water, pH 7.9 (mobile phase A) and a mixture of 50:50 mobile phase A and methanol (mobile phase B).
The flow rate was set at 0.4 mL/min and the column temperature at 70°C. A gradient of 40–50%B in 15 min was used for dT40–
100, while a gradient change of 20%B in 20 min was used for the
other oligonucleotides. Gradients of 30–50%B were used for dT1535, dT20, dA20, dG20, dC20, dT20-PS, rU20-MOE, dT20-LNA; and
20-40%B for rU15-30, rU20, rU20-PS, rU20-OMe.

Mobile phases for HILIC analyses were composed of 50 mM ammonium acetate in water, pH 6.9 (mobile phase A, no adjustment
of pH) and acetonitrile (mobile phase B). The flow rate was set at
0.3 mL/min and the column temperature at 40°C. Gradient conditions were the same for all oligonucleotides. A gradient of 55–25%B
in 30 min was used for Waters columns, while a gradient of 75–
45%B in 30 min was used for YMC columns.
It should be noted that the goal of this work was to evaluate the contribution of each hardware parameter on the overall oligonucleotide adsorption, therefore possible effects of mobile
phase compositions and pH were not investigated.

2.3.1. H-Class
The Acquity UPLCTM H-Class system (Waters, Milford, MA, USA)
was equipped with a quaternary solvent delivery pump, an autosampler including a 10-μL flow-through-needle injector and a
tunable ultraviolet (TUV) detector with a 500-nL analytical flow
cell (10 mm path length). The flow path of the instrument was
made of stainless-steel.
2.3.2. H-Class Bio
The Waters Acquity UPLCTM H-Class Bio system was equipped
with a biocompatible quaternary solvent delivery pump, an autosampler including a 10-μL flow-through-needle injector. The TUV
detector was equipped with a 1500-nL titanium flow cell (5 mm
path length). The flow path of this instrument primarily consists
of an MP35N alloy.
2.3.3. Premier system
The Waters Acquity PremierTM system was equipped with a binary solvent delivery pump and an autosampler including a 10-μL
3


Diol
Diol
Ethylene-bridged hybrid organic-inorganic particles
Ethylene-bridged hybrid organic-inorganic particles


1.7
1.7

1.9
1.9

150 × 2.1
150 × 2.1

150 × 2.1
150 × 2.1

130
130

2.6
150 × 2.1

100

3. Results and discussions
3.1. Impact of column material
Column hardware represents more than 70% of the sample accessible surfaces during an analysis [60]. Generally composed of
a stainless-steel tube and stainless-steel frits, it is often assumed
that the LC column will introduce the most significant source of
adsorption problems. Several strategies exist to minimize sample
losses and distorted peaks. While a “sample conditioning” protocol is often used prior to analysis, bioinert columns have recently
become available, and they are meant to be a permanent solution
to non-specific adsorption on column hardware. Columns featuring titanium, PEEK and hybrid organic/inorganic surface technologies are now commercially available as alternatives to conventional
stainless-steel columns.

To examine currently available techniques, we compared three
different bioinert RPLC columns (one of each technology) to their
stainless-steel analog. The first technology was a hybrid surface
technology (HST) column described as MaxPeakTM High Performance Surfaces by Waters (Wpr), which is the bioinert version of
the BEH C18 column (Wss). The second one is a PEEK-lined column
from YMC (Ypk), which is the bioinert version of the YMC-Triart
C18 column (Yss). Last one is a titanium-lined column described
as bioZenTM by Phenomenex (Pti), which is the bioinert version of
the Kinetex® EVO C18 (Pss).
Besides, bioinert HILIC alternatives are still emerging. To the
best of our knowledge, commercially available bioinert HILIC
columns are currently limited to hybrid organic/inorganic surface
and PEEK technologies. Therefore, we compared the two existing
bioinert HILIC column hardware types with their stainless-steel
analogs. The first one is a HST column from Waters (Wpr_H) that
is the bioinert version of a BEH Amide column (Wss_H). The second one is a PEEK-lined column from YMC (Ypk_H), which is the
bioinert version of the YMC-Triart Diol (Yss_H).
A Premier system comprised of hybrid surface flow path components was systematically used for this column comparison study.

YMC-Triart DIOL-HILIC
YMC-Triart DIOL-HILIC
Metal-free
Yss_H
Ypk_H

3.1.1. Sample conditioning of columns
A sample conditioning step was incorporated into the comparison of these columns. The dT15-35 sample (mixture of polydeoxythymidylic acids of 15-, 20-, 25-, 30- and 35-mer), which has
many times been used for column and system performance testing, was chosen as the “conditioning” sample [53,60]. Consecutive
injections of a solution containing 4 pmol of each oligodeoxyribonucleotide (ODN) were made on each brand-new column until
consistent peak areas were achieved.

For the IP-RPLC mode, results have been reported in Fig. 1 for
Waters (Wss vs. Wpr, Fig. 1A, B), YMC (Yss vs. Ypk, Fig. 1C, D) and
Phenomenex (Pss vs. Pti, Fig. 1E, F) columns. For each column, the
first injection was taken as the reference value (100%) and relative
peak areas of each ODN were expressed as % to plot the evolution of peak areas over injections. Fig. 1 also presents the overlaid
chromatograms of the first and last injections of the sample conditioning protocol.
With stainless-steel columns, very low peak areas were obtained for the first injection on the columns, with early eluting
peaks particularly affected, as shown in Fig. 1A and C for the Wss
and Yss columns. This was even worse for the Pss column (Fig. 1E)
which resulted in nearly complete sample loss regardless of the
ODN. Because of non-specific adsorption, the use of a brand-new

YMC
YMC

Acquity UPLC BEH Amide
Acquity Premier BEH Amide
Wss_H
Wpr_H

Waters
Waters

Phenomenex
bioZenTM Oligo
Pti

Biocompatible titanium
(BioTi)
Stainless-steel

MaxPeakTM High
Performance Surfaces
Stainless-steel
PEEK-lined stainless-steel

1.9
1.9
2.6
150 × 2.1
150 × 2.1
150 × 2.1
YMC
YMC
Phenomenex
Yss
Ypk
Pss

Wpr

Waters

Acquity UPLC Oligonucleotide
BEH C18
Acquity Premier
Oligonucleotide BEH C18
YMC-Triart C18
YMC-Triart C18 Metal-free
Kinetex EVO C18
Wss


Waters

All oligonucleotides were concentrated at 2 μM and injection
volume was 2 μL. Gradient conditions were optimized during preliminary studies. All gradients were systematically followed by an
8-min re-equilibration to the initial conditions.

120
120

Amide
Amide

C18

C18
C18
C18

Ethylene-bridged hybrid organic-inorganic particles
Ethylene-bridged hybrid organic-inorganic particles
Organo-silica core-shell particles with ethane
cross-linking
Organo-silica core-shell particles with ethane
cross-linking
Ethylene-bridged hybrid organic-inorganic particles
Ethylene-bridged hybrid organic-inorganic particles

1.7
150 × 2.1


MaxPeakTM High
Performance Surfaces
Stainless-steel
PEEK-lined stainless-steel
Stainless-steel

120
120
100

C18
Ethylene-bridged hybrid organic-inorganic particles

1.7
150 × 2.1
Stainless-steel

130

C18
Ethylene-bridged hybrid organic-inorganic particles

Particle
size (μm)
Column
dimensions (mm)
Name

Manufacturer


Column Hardware (tube
and frits material)

130

Journal of Chromatography A 1677 (2022) 463324

Acronym

Table 2
List of investigated chromatographic columns and their properties.

Pore size
˚
(A)

Particle type

Ligand
type

H. Lardeux, A. Goyon, K. Zhang et al.

4


H. Lardeux, A. Goyon, K. Zhang et al.

Journal of Chromatography A 1677 (2022) 463324


Fig. 1. Monitoring of peak area increases during sample conditioning in IP-RPLC mode using the Premier system. Overlaid chromatograms from the first injection (before
conditioning) and the last injection (after conditioning) of the mixture dT15-35 when using (A) stainless-steel Waters (Wss), (B) bioinert Waters (Wpr), (C) stainless-steel
YMC (Yss), (D) PEEK-lined YMC (Ypk), (E) stainless-steel Phenomenex (Pss), or (F) titanium-lined Phenomenex (Pti) columns. 15 injections (300 pmol) and 4 injections (80
pmol) were required for sample-based conditioning of the stainless-steel and bioinert columns, respectively. 100% peak area corresponds to first injection (inj. 1).

column without conditioning cannot give reliable results. In the
subsequent injections, the peak areas gradually increased with a
plateau reached after 15 injections. This corresponds to an actual mass load of 300 pmol, required to mask the active sites of
stainless-steel material from the columns. We can also notice differences in terms of adsorption behavior between ODNs. The shortest oligodeoxythymidines (dT15 and dT20) systematically showed
the greatest increase in peak areas over conditioning time, with
relative peak areas up to 4200% in the 15th injection. This may be
explained by their elution order rather than length. Early eluting
compounds will sacrificially saturate adsorption sites as they go
through the column, leading to later eluting oligonucleotides being less adsorbed to the column hardware [54]. As a result, dT35
relative peak area showed a 175 to 580% value in the last injection, which is moderately high compared to dT15 or dT20. Despite
differences in adsorption behavior, these results demonstrate the
need to carefully condition stainless-steel columns.
Contrary to their stainless-steel analogs, nearly full recovery for
all oligonucleotides was achieved upon the first injection on bioinert RPLC columns. To verify this observation, four successive injections of the ODN mixture were performed, corresponding to 80
pmol loaded on column. Fig. 1B, D and 1F showed that relative
peak areas over the four injections varied from 98 to 104% regardless of the ON length and type of bioinert column. Adsorption was
reduced upon the first injection, and reliable results were obtainable without sacrificing time or samples. In addition, resolution of
minor peaks from failure sequences can be seen to be afforded
with the bioinert columns upon the first injection. However, this

was not achieved with brand-new stainless-steel columns, where
sample conditioning was needed to detect such minor impurities.
The same sample conditioning protocol was applied to both
stainless-steel HILIC (Fig. 2A and C) and bioinert HILIC (Fig. 2B

and D) columns. Different than with IP-RPLC, each HILIC column
showed different behavior and did not require the same amount
of sample to be effectively passivated. As shown in Fig. 2C, Yss_H
columns showed the greatest number of injections required to
reach a plateau in terms of peak area, with 20 injections corresponding to 400 pmol of oligonucleotide in total. In any case
the utility of sample conditioning the stainless-steel columns to
limit undesired interactions with the metallic column hardware
was again demonstrated.
However, despite the conditioning protocol, the increase in peak
areas over conditioning is not as pronounced as in IP-RPLC mode
(up to 440% vs. up to 4200%, respectively). Surprisingly, bioinert HILIC columns also required some conditioning injections to
achieve consistent peak areas for the dT15-35 sample (Fig. 2B and
D) while it was barely necessary in IP-RPLC. Indeed, 10 and 14 injections (200/280 pmol) were performed to reach the plateau of
peak area on Wpr_H and Ypk_H columns, respectively, demonstrating that this sample conditioning protocol was quite slow with a
very gradual increase in peak areas.
Finally, these findings highlight the benefits of using a bioinert
column for the analysis of metal-sensitive analytes from a “sample conditioning” point of view. Sample conditioning was found
to be required with standard RPLC columns, while bioinert RPLC
columns seem to show ready to use performance upon the first

5


H. Lardeux, A. Goyon, K. Zhang et al.

Journal of Chromatography A 1677 (2022) 463324

Fig. 2. Monitoring of peak area increases during sample conditioning in HILIC mode using the Premier system. Overlaid chromatograms from the first injection (before
conditioning) and the last injection (after conditioning) of the mixture dT15-35 when using (A) stainless-steel Waters (Wss_H), (B) bioinert Waters (Wpr_H) column, (C)
stainless-steel YMC (Yss_H), (D) PEEK-lined YMC (Ypk_H) columns. 15 injections (300 pmol) and 10 injections (200 pmol) were required for sample-based conditioning of

the Wss_H and Wpr_H columns, respectively, while 20 injections (400 pmol) and 14 injections (280 pmol) were required for sample-based conditioning of the Wss_H and
Ypk_H, respectively. 100% peak area corresponds to the first injection (inj. 1).

injection. Besides, the use of bioinert HILIC columns did not completely suppress non-specific adsorption, but required a reduced
number of conditioning injections in comparison with stainlesssteel HILIC columns.

the two remaining stainless-steel columns (Yss and Pss) produced
a peak area equal to only 60–82%, with increasing values for the
larger ODNs such as dT35 (similar behavior to what was already
explained in Section 3.2.1.). This confirms that adsorption of ONs
(equal to 20–40%) is still taking place on these two columns, despite the column conditioning procedure. This could be due to differences in metal surface areas, microsite corrosion across the various materials or column manufacturing procedures, but ultimately
it seems to be an indication of the number of active sites where
ONs can adsorb. Batch-to-batch testing of different hardware lots
was not possible here, and it could be equally possible that the
behavior of stainless-steel columns in this regard is highly variable.
As reported in Fig. 3B, more pronounced differences were observed
between the RPLC columns when analyzing the dT40–100 sample,
most likely due to the increasing sizes of the ONs. Some differences were observed between the reference bioinert hybrid surface
(Wpr) column and its stainless-steel counterpart (Wss). Indeed,
relative areas varied from 90% for dT40 to only 40% for dT100,
and the first eluted peak was not the one to show the lowest recovery. In addition, the PEEK column (Ypk) behaves very well for
dT40 (relative peak area of 101%), but adsorption was significantly
more pronounced when increasing the ON size (relative peak area
of 46% for dT100). On the contrary, the stainless-steel column from
the same provider (Yss) has a relatively constant behavior independent of the ON size, with relative peak area comprised between 70
and 82%. Finally, the titanium column (Pti) has the exact opposite
behavior to the PEEK column (Ypk), with a significant reduction
of adsorption from dT40 (relative peak area of 44%) to dT100 (relative peak area of 97%). The stainless-steel column from the same
provider (Pss) showed similar behavior, but adsorption was slightly
less pronounced, in particular for the smaller ONs of this sample

(dT40 and dT60). Interpretation of these results is quite difficult
since there is likely to be an interplay between several different
factors (mostly related to the larger size of these ONs and their
molecular masses ranging between 10 and 30 kDa). However, it
is also important to keep in mind that the columns were initially

3.1.2. Analysis of unmodified oligonucleotides
In addition to the initial column conditioning of the different
columns employed in this work, the behavior of these columns
was evaluated for the analysis of three different types of model
oligonucleotide (ON) samples. These included the previously analyzed dT15-35 sample, but also a mixture of larger ODNs (dT40100, a mixture of poly-deoxythymidylic acids of 40-, 60-, 80and 100-mer), and finally a mixture of small oligoribonucleotides
(ORNs, rU15-30, a mixture of poly-uridylic acids of 15-, 20- and
30-mer).
To obtain a consistent comparison and draw reliable conclusions, the six RPLC columns and the four HILIC columns shared
the same history in terms of usage before the injections of the
three model ON samples were carried out (as reported in Table S1
and Table S2). In Fig. 3, the relative peak areas of the different ON
products are provided, while the chromatograms related to these
data for bioinert columns are reported in Fig. S1. For each individual ON and in both IP-RPLC and HILIC modes, the Waters Premier
column (Wpr and Wpr_H, respectively) was taken as the reference
value (100%) for the calculations. The columns were already conditioned, and a plateau was reached as described in Section 3.1.1 and
reported in Figs. 1 and 2.
Concerning the results obtained in IP-RPLC mode (Fig. 3A–C),
no significant differences were observed between the stainlesssteel and bioinert (Wss and Wpr) Waters columns when analyzing the dT15-35 sample (Fig. 3A). The behavior of the two other
bioinert columns (Ypk and Pti) was also in line with our expectations, and values of 105–110% (not significantly different from
100%) were experimentally observed, which means that all three
bioinert type columns produced the same peak areas for the dT1535 mixture. On the other hand, despite the column conditioning,
6



H. Lardeux, A. Goyon, K. Zhang et al.

Journal of Chromatography A 1677 (2022) 463324

Fig. 3. RPLC bioinert columns composed of hybrid surfaces, PEEK- or titanium-lined hardware (Wpr, Ypk and Pti) in comparison with their stainless-steel analogs (Wss, Yss,
Pss), and HILIC bioinert columns composed of hybrid surfaces and PEEK-lined hardware (Wpr_H, Ypk_H) in comparison with their stainless-steel analogs (Wss_H, Yss_H).One
injection of each mixture, namely dT15-35 (A, D), dT40-100 (B, E), and rU15-30 (C, F), was performed on each previously conditioned column using the Premier system.
Relative peak areas for each oligonucleotide are reported. 100% corresponds to peak area using the Wpr and Wpr_H column for the IP-RPLC and HILIC mode, respectively.

conditioned with a dT15–35 sample (see Section 3.1.1.). Since the
chemical nature of the dT40-100 sample is different from the material used for conditioning, the column adsorption sites may not
have been perfectly masked and lead to partial adsorption of larger
ONs (Fig. 3B). This itself highlights an inherent drawback to having
to rely on sample-based column conditioning. Fig. 3C shows the
adsorption data experimentally obtained for small ORNs, namely
the rU15–30 sample. In this case, the differences observed between
the RPLC columns were more pronounced than for the dT15-35
sample (Fig. 3A), but less than for the dT40-100 sample (Fig. 3B). It
is important to mention that two bioinert columns (Wpr and Ypk)
showed comparable behavior, with no issue related to adsorption
for the small RNA products. The third bioinert column (Pti) offered
very good performance in terms of adsorption, with relative peak
area values ranging from 75 to 90%, and there was no problem
with peak shape for this class of molecules (see Fig. S1). For the
three stainless-steel columns (Wss, Yss and Pss), undesired adsorption became systematically more pronounced versus their bioinert
counterparts, with an average increase of 20 to 40%. As expected,
losses appeared to be increasingly less pronounced for the larger
ON species.
Concerning the results obtained in HILIC mode (Fig. 3D–F), sample conditioning was not sufficient to permanently mitigate adsorption. For the dT15-35 sample (Fig. 3D), the relative peak areas
from the stainless-steel columns (Wss_H and Yss_H) varied from

20 to 90% and from 30 to 40% respectively. That means that nonspecific adsorption was still significant on the HILIC stainless-steel
columns, even after a conditioning procedure (see previous section). However, these two columns behave quite differently. Indeed,

the Yss_H column offers consistent analyte recovery whatever the
ON size, while sample losses become more and more significant
with increasing ON size when using the Wss_H.
Some significant differences were also observed between the
bioinert columns (HST or PEEK) and those made from stainlesssteel. On average, the HST material (Wpr_H) offered 20–30% better
recovery than the PEEK-coated column (Ypk_H) that was found to
result in almost stable relative peak areas whatever the ON length.
Despite some differences in terms of peak areas, peak shapes were
excellent on the two bioinert columns for the dT15-35 sample, as
illustrated in Fig. S1.
Similar results were obtained for the mixture of small ORNs
(rU15-30, relative peak areas presented in Fig. 3F) vs. small ODNs
(Fig. 3D), but the recoveries experimentally obtained were improved compared to what was observed for all the columns for
the small ODNs. ORN relative peak areas were measured to be
50–80% for the Wss_H, 50–60% for the Yss_H and 80–95% for
the Ypk_H column. Values for the small ODNs were 20–90% for
the Wss_H, 30–40% for the Yss_H and 70–80% for the Ypk_H column. These results demonstrate that ORNs are less prone to adsorption than ODNs under HILIC conditions. Besides some changes
in peak areas, it is important to notice that peak shapes were
again excellent on the two bioinert columns (Fig. S1). Finally,
some larger ODNs (dT40–100) were also analyzed on the four
HILIC columns, and these results are reported in Fig. 3E. Here,
the performance of the columns ranked the same as with small
ONs. The Wpr_H always showed better results compared to the
other ones in terms of adsorption (reference column, relative peak
area values of 100%) followed by the other bioinert HILIC col7



H. Lardeux, A. Goyon, K. Zhang et al.

Journal of Chromatography A 1677 (2022) 463324

umn (Ypk_H, values of 70–85%), the Waters stainless-steel column (Wss_H, values of 60–70%) and the YMC one (Yss_H, values of 40–50%). Non-specific adsorption did not vary according
to the length of oligonucleotides across any of the tested HILIC
columns. At most, there was 10–15% variation of relative peak
area with a given column. This behavior is in line with the previously obtained results, except on the Wss_H column where
the decrease of relative peak areas with oligonucleotide size was
not any longer observed with large oligonucleotides (dT40-100).
These results suggest that sample losses on the Wss_H column
are very dependent on the size of the oligonucleotide between
a 15- and 40-mer length, but that differences in size beyond 40
residues might have a diminishing effect. As illustrated in Fig. S1,
all peaks remained symmetrical on the two bioinert columns for
the mixture of large ODNs. Broader peaks were observed on the
Wpr_H vs. the Ypk_H column. Nevertheless, selectivity and resolution were always greater on the Wpr_H column, which might
in part be tied to its stationary phase and its corresponding
retentivity.
In summary, it appears that the smaller DNA and RNA samples
(15- to 35-mer) behaved quite similarly in terms of non-specific
adsorption in both modes, with adsorption being generally more
pronounced under HILIC conditions. The larger oligodeoxyribonucleotides (40- to 100-mer) showed different behavior on the different columns investigated in this work. Most of the observed chromatographic differences are related to the size of these ON species,
which are clearly more difficult to characterize.

nL. PEEK is used as the tubing material on the inside of the flow
cell apparatus that is itself mostly made of Teflon. The second one
is commonly used with the Waters Acquity H-Class Bio system to
limit adsorption of biopharmaceutical products during their analysis. It is a titanium flow cell having a path length of only 5 mm
and a volume of 1500 nL.

Fig. 4 shows the results obtained for the dT15-35, dT40-100 and
dT15-35 samples with each combination of UHPLC instrument and
UV flow cell material when analyzing the mixtures in HILIC mode.
Results concerning IP-RPLC mode have been instead reported in
the Supplementary Information (Fig. S2).
3.2.1. Impact of UV flow cell conditioning
Some preliminary HILIC experiments were performed with the
UV analytical flow cell mounted on a Waters Premier instrument
before and after oligonucleotide sample conditioning. Fig. 5 shows
HILIC chromatograms of the small (dT15-35) and large (dT40–100)
ODNs samples as obtained with the original and then the conditioned UV flow cell. The experiments with the original UV flow cell
were performed on a brand-new Premier instrument, as received
from manufacturing. This means that the UV flow cell had not
seen any ON sample before the experiment reported in Fig. 5 (blue
trace). On the other hand, the conditioned UV flow cell corresponds to a part that had been used for about one month and
exposed to numerous injections of ONs. This corresponds to the
black trace in Fig. 5.
As shown, some clear differences were observed between the
black and the blue traces and were seen upon zooming in on
the baseline (bottom chromatograms in Fig. 5). Differences were
not drastic for small ODNs of around 15–20 nucleotides (nt), even
though some minor species (probably corresponding to shortmers
and longmers) were less resolved and/or hardly visible on the UV
flow cell that was not conditioned (blue trace). Differences between the UV flow cells were amplified for the larger ODNs of the
dT15-35 sample corresponding to dT25 to dT35, where there was
a significant loss of resolution between minor species and a significant drift in baseline. The situation was at its worst with the
mixture of large ODNs (dT40-100). Here, weakly retained components in the sample were hardly detected when using the unconditioned, original UV cell. In addition, the four main peaks (dT40,
dT60, dT80 and dT100) were poorly resolved and a strong baseline drift was observed. On the contrary, the sample-conditioned
UV flow cell provided better signal intensity, improved peak symmetry, higher resolution and less baseline drift. These observations clearly demonstrate the need to properly condition the UV
flow cell before its first use. This also proves that non-specific

adsorption within the UV flow cell was most pronounced with
large ONs.

3.2. Impact of instrumentation
The instrument could also be responsible for non-specific adsorption and its impact might very well be different under HILIC
vs. IP-RPLC conditions since mobile phase compositions are quite
different. For this part, a previously conditioned bioinert Waters
column (Wpr or Wpr_H) was systematically employed to minimize
as much as possible non-specific adsorption within the column and
to thereby more sensitively investigate the impact of the UV flow
cell and UHPLC instrumentation.
Indeed, in the last few years, all LC instrument manufacturers have released several different chromatographic systems, which
have been referred to as bioinert, biocompatible and iron-free
[55,61,64]. These systems have been designed to minimize nonspecific adsorption losses due to metal interactions and/or to offer
a better compatibility with mobile phases containing high amount
of salts. Historically, bioinert HPLC systems were made of PEEK,
but with the emergence of UHPLC conditions, advanced materials such as titanium and MP35N alloys have also been applied to
build instrumentation that can withstand elevated pressures. In addition, a new bioinert UHPLC system was recently released by Waters where the flow path is covered with a hybrid surface technology that is created through the vapor deposition of ethylene
bridged hybrid inorganic/organic surfaces. To have a clear view
of what can be done with the currently available UHPLC instruments for the analysis of ON products, three different systems from
the same manufacturer were compared. The first one was a regular stainless-steel UHPLC instrument (Waters Acquity H-Class). The
second one was a biocompatible UHPLC system (Waters Acquity HClass Bio), where the flow path is composed of corrosion resistant
MP35N alloy. Finally, the last one was a new UHPLC system (Waters Acquity Premier) constructed with hybrid surface flow path
components.
Besides the evaluation of three different UHPLC instruments,
two different UV flow cells were also tested. The first one is included on the commercial Waters Acquity H-Class and Waters Acquity Premier instruments. It is a regular light-guided analytical
flow cell with a path length of 10 mm and a volume of only 500

3.2.2. Impact of instrumentation material
The impact of UHPLC instrumentation on the non-specific adsorption of ONs was assessed using three different Waters chromatographic systems. The H-Class and Premier instrument are

originally equipped with an analytical UV flow cell (mostly made
of Teflon wetted parts) that was already conditioned with the
dT15-35 sample (see Section 3.2.1.), while the H-Class Bio is originally equipped with a titanium UV flow cell. Therefore, the original
instrument configurations were compared with the use of the analytical flow cell for the H-Class Bio instrument. Importantly, to have
adsorption data that can be reliably compared between the three
instruments, the same UV detector was used on the three instruments. Differences in sensitivity due to the detector itself and in
particular the UV lamp were therefore avoided.
HILIC experimental results have been summarized in Fig. 4 for
the three model mixtures of ONs. ON relative peak areas were plotted for the four different systems, and the instrument configura8


H. Lardeux, A. Goyon, K. Zhang et al.

Journal of Chromatography A 1677 (2022) 463324

Fig. 4. Comparison of instruments namely H-Class, H-Class Bio and Premier equipped with the same UV detector with an analytical flow cell, except for the H-Class Bio
where the use of a titanium flow cell was also discussed. Letter M indicates that the configuration is the one that is commercially available. Histograms corresponding to
HILIC-UV chromatograms of the three mixtures using the Waters bioinert column (Wpr_H).

Fig. 5. HILIC-UV chromatograms of DNA mixtures of oligonucleotides showing the impact of sample conditioning of the UV analytical flow cell on oligonucleotide adsorption.
a. The UV analytical flow cell was used for the first time. b. The same analytical flow cell was used after being sample conditioned. A previously conditioned Wpr_H column
was used on the Premier system.

9


H. Lardeux, A. Goyon, K. Zhang et al.

Journal of Chromatography A 1677 (2022) 463324


tion combining the Premier system with the conditioned analytical
flow cell was taken as the reference (100%). Regardless of the UHPLC instrument, the peak areas obtained with the titanium UV flow
cell were about 2-fold lower than with the analytical UV flow cell
(as a result of its 5 vs. 10 mm path length). Since the analytical UV
flow cell was already conditioned, no significant differences were
observed between ONs varying in size and type. As illustrated in
Fig. 4, there were almost no differences for large ODNs and small
ORNs between the three UHPLC instruments equipped with the
analytical flow cell (relative peak areas values ranged from 95 to
105%).
However, some slight differences were observed between the
Premier system and the two remaining UHPLC instruments when
analyzing small ODNs (dT15-35). In this particular case, relative
peak areas on the two other instruments were equal to 85-90%
vs. 100% on the Premier system. The Premier system having being used for the column conditioning studies, it therefore saw significantly higher quantities of samples and in particular the dT1535 sample. This might explain the slightly improved recoveries of
short ODNs with as much likelihood as the hybrid surfaces of the
instrument flow path inherently contributing such a sizable effect.
In the end, it appears that the non-specific adsorption of oligonucleotides on any type of UHPLC instrument can be negligible, at
least with the mass loads applied here for the sake of sample characterization. In this type of work, care should at least be taken to
condition the UV flow cell.
The impact of the instrumentation on non-specific adsorption
of ONs was also evaluated in IP-RPLC mode. Fig. S2 shows the results obtained for the dT15-35, dT40-100, and rU15-30 samples
with each combination of UHPLC instruments and UV flow cell
material (3 systems and 2 UV flow cells). As in HILIC mode, the
two-fold decrease of UV signal observed for each ON when modifying the analytical UV flow cell for a titanium flow cell can be
attributed to the shorter path length (5 mm vs. 10 mm). When
looking at chromatograms of 15- to 35-mer ONs (Fig. S2A and S2C),
peak shapes remain strictly identical whatever the type of instrument and UV flow cell material employed meaning that no significant adsorption issues were observed, even when using the Waters Acquity H-Class system which is composed of stainless-steel.
Fig. S2B shows the results obtained for the mixture of larger ODNs
(dT40-100 sample). Here, the instrument had a clear impact on adsorption and above all peak shapes of the largest ONs. Interestingly, the peak shapes of the largest ONs (60- to 100-mer) were

strongly degraded with severe tailing and broadening observed
when using the regular vs. titanium UV flow cell. To understand
this behavior, it is relevant to mention that there are some significant differences between the two UV flow cells in terms of their
surface exposed materials. Indeed, the analytical flow cell is mostly
composed of Teflon which is a hydrophobic material where the
complexes of ON and TEA (which are also hydrophobic) can adsorb,
despite their short residence time, and that can lead to peak shape
distortion.
Besides the UV flow cell, some additional (more limited) differences were also observed for dT80 and dT100 between the different UHPLC systems, especially when using the analytical flow cell.
Indeed, the H-Class system offered worse performance (asymmetry
at 10% for dT100 was 4.39) vs. the H-Class Bio (asymmetry at 10%
was 1.85) or the Premier instrument (asymmetry at 10% was 2.18).
This confirms that large ONs (60- to 100-mer) are more prone to
adsorption and that the latter two UHPLC systems should be preferentially used.
Based on these findings, it is clear that the analytical UV flow
cell of 10 mm is suitable for use with small ONs (15- to 40-mer) in
order to achieve maximum sensitivity (namely a 2-fold improvement). On the contrary, despite the more limited sensitivity, the
titanium flow cell, even with its 5 mm path length, should be

preferably used in IP-RPLC mode to achieve suitable peak shapes
for large ONs (60- to 100-mer).
3.3. Application to the analysis of unmodified and modified 20-mer
oligonucleotides
Differences in the general features of the oligonucleotide have
shown to impact non-specific adsorption, and therefore, chemical
modifications of the oligonucleotide structure may influence their
recovery.
Indeed, it should be noted that therapeutic ONs have to be
chemically-modified to ensure proper pharmacokinetic properties
and sufficient activity in vivo [65]. In this context, modifications

often involve the phosphate linkage and the furanose sugar moiety (deoxyribose in DNA and ribose in RNA). Among the modifications involving the phosphodiester backbone, the most widely
used is a phosphorothioate (PS) bond, in which a sulfur replaces
one of the non-bridging oxygen atoms of the phosphate linkage
(reported in blue in Fig. 6) [66]. In addition, modifications applied to the furanose sugar moiety (reported in red in Fig. 6) include substitutions in the 2’-position, with 2’-O-methyl (OMe), 2’O-methoxyethyl (MOE), and locked nucleic acid (LNA) [66]. Based
on the performed chemical modifications and base composition of
the ONs (U/T, C, A, G, reported in grey in Fig. 6), a change in the
general features of the ON occurs and potentially impacts nonspecific adsorption.
The influence of chemical modifications on the 20-mer ON adsorption in both IP-RPLC and HILIC modes was therefore investigated and the list of the evaluated ONs is reported in Table 1.
As a result of previous findings, the bioinert hybrid surface
columns (Wpr and Wpr_H) and their stainless-steel counterparts
(Wss and Wss_H) were used in combination with the best LC system configuration (consisting of the Premier instrument equipped
with an analytical flow cell) to evaluate the impact of ON chemistry on adsorption. Corresponding chromatograms are reported in
Figs. 7 and 8. The main chromatographic descriptors and percentage recoveries (calculated as the ratio between the stainless-steel
reference column and the bioinert column areas) are summarized
in Table 3.
First of all, and as reported in Table 3, a higher recovery
of all the ONs was obtained with the bioinert HST columns
(Wpr/Wpr_H) as compared to their stainless-steel counterparts
(Wss/Wss_H). It is worth mentioning that the electropositive metal
oxide layer on the surface of stainless-steel columns is generally
thought to result in ionic interactions with the negatively-charged
backbone of the ONs and therefore be the cause of non-specific adsorption [62,67]. This metal oxide is masked in the case of the hybrid surface (Wpr/Wpr_H) columns. Indeed, this hypothesis is supported by the chromatograms of all ONs shown in Figs. 7 and 8,
that correspond to the first and second set of 20-mer ON samples,
respectively. As reported in Table 3, a better recovery can be observed, indicating that ionic interactions between column surfaces
and the ONs are attenuated with the bioinert columns.
Concerning the detailed impact of modifications, differences
in the ON sugar (deoxyribose/ribose) and base composition (U/T,
C, A, G) were first evaluated. The IP-RPLC-UV and HILIC-UV
chromatograms of this set of samples were reported in Fig. 7A
and Fig. 7B, respectively. For this part of the work, 20-mer

homomolecular oligodeoxyribonucleotides (ODNs) were considered, namely an oligodeoxyadenosine (dA20), an oligodeoxycytidine (dC20), and an oligodeoxyguanosine (dG20) to be compared
with an oligodeoxythymidine (dT20). A change on the sugar composition was also applied and a 20-mer oligoribonucleotide (ORN),
namely oligouridine (rU20), was analyzed. By fixing the length of
the ONs at 20-mer, and therefore the number of the ON phosphate groups, it was possible to examine the extent of adsorption
10


H. Lardeux, A. Goyon, K. Zhang et al.

Journal of Chromatography A 1677 (2022) 463324

Fig. 6. Chemical structures of nucleic acids and chemical modifications of the investigated oligonucleotides. Abbreviations: 2’-O-MOE, 2’-O-methoxyethyl; 2’-O-Me, 2’-Omethyl; DNA, deoxyribonucleic acid; LNA, locked nucleic acid; RNA, ribonucleic acid (For interpretation of the references to color in this figure, the reader is referred to the
web version of this article.).

11


H. Lardeux, A. Goyon, K. Zhang et al.

Journal of Chromatography A 1677 (2022) 463324

Fig. 7. (A) IP-RPLC-UV and (B) HILIC-UV chromatograms of 20-mer non-modified oligonucleotides using the bioinert (Wpr/Wpr_H) vs. stainless-steel reference (Wss/Wss_H)
columns from Waters. % recovery calculated as the ratio between the stainless-steel and the bioinert columns areas are reported in red. Premier system was used. For
gradient conditions, please refer to Section 2.4 (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.).

12


H. Lardeux, A. Goyon, K. Zhang et al.


Journal of Chromatography A 1677 (2022) 463324

Fig. 8. (A) IP-RPLC-UV and (B) HILIC-UV chromatograms of 20-mer modified oligonucleotides using the bioinert (Wpr/Wpr_H) vs. stainless-steel reference (Wss/Wss_H)
columns from Waters. % recovery calculated as the ratio between the stainless-steel and the bioinert columns areas are reported in red. Premier system was used. For
gradient conditions, please refer to Section 2.4 (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.).

13


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Journal of Chromatography A 1677 (2022) 463324

Table 3
Chromatographic parameters of 20-mer oligonucleotides analyzed by IP-RPLC and HILIC using the bioinert column (Wpr/Wpr_H) vs. stainless-steel reference
column (Wss/Wss_H) from Waters. A Premier LC system was used for these measurements. Corresponding chromatograms are provided in Figs. 7 and 8. tR ;
retention time (min), As; asymmetry factor @10. % recovery calculated as the ratio between the stainless-steel reference column (Wss or Wss_H) and the
bioinert column (Wpr or Wpr_H) areas.
Wpr column
Oligos
dT20
dA20
dG20
dC20
rU20
dT20-PS
rU20-PS
rU20-MOE
rU20-OMe
dT20-LNA


tR
7.36
6.33
8.70
4.46
6.20
7.79
7.04
7.24
9.73
6.81

As
1.14
1.09
0.95
1.10
1.07
0.79
0.60
1.10
1.16
1.07

Oligos

∗

91449

71736
23513
48107
35697
69116
101987
60582
102905
69846

Height

tR

19707
16032
918
11417
8418
10850
7105
12575
22407
15944

7.90
7.00
9.62
5.18
6.80

7.72
7.04
7.94
9.94
7.65

As
1.10
1.10
0.56
1.06
0.99
0.99
0.60
1.11
1.12
1.13

Wpr_H column
tR

dT20
dA20
dG20
dC20
rU20
dT20-PS
rU20-PS
rU20-MOE
rU20-OMe

dT20-LNA
ds

Area

Wss column

9.27
8.19
9.93
10.73
14.89
7.46
13.76
10.04
12.54
10.07
13.71

As
1.18
1.47
1.68
1.83
1.08
1.14
0.92
1.12
1.14
1.10

0.89

Area
112015
114668
1900
37691
56310
91023
134301
93994
146944
93498
113938

Area
63371
52172
10429
26655
20571
54682
54906
42929
66108
60342

Height
18903
15396

270
7974
5081
10777
3972
11045
15989
14314

Wss_H column
Height

tR

20133
18747
64
5134
8893
13538
17173
15159
24542
16525
16516

9.53
8.46
10.10
11.00

15.10
7.71
13.98
10.28
12.76
10.31
13.95

As
1.38
4.59
1.43
3.78
1.48
1.12
1.15
1.24
1.23
1.09
1.12

Area
53026
5705
576
4605
24741
86244
114032
82634

126573
87066
69807

Height
8088
493
27
224
3602
12127
14890
12207
20163
14268
10014

% recovery
69.3%
72.7%
44.4%
55.4%
57.6%
79.1%
53.8%
70.9%
64.2%
86.4%
%
recovery

47.3%
5.0%
30.3%
12.2%
43.9%
94.7%
84.9%
87.9%
86.1%
93.1%
61.3%

The columns shared the same history in terms of usage before the injections of the three model oligonucleotide samples were carried out.

phenomena in correlation with the other ON functional groups,
namely the extracyclic functional groups on the different bases and
the sugar hydroxyl groups.
Interpreting the chromatographic behavior of the 20-mer ONs
analyzed in IP-RPLC mode (Fig. 7A), ORN rU20 was among the
species displaying low recovery. Specifically, ORNs are more hydrophilic than ODNs because it contains ribose and not deoxyribose. A ribose sugar has a hydrophilic hydroxyl that largely defines the RNA macromolecule and that might be responsible for
the enhanced adsorption of the ORN by interactions involving the
hydroxyl groups themselves [68]. Same conclusion was drawn in
HILIC mode (Fig. 7B) [43]. In addition, synergistic electrostatic
interactions might be taking place between the rU20 functional
groups (i.e. phosphate groups, sugar hydroxyl groups, and the extracyclic functional groups on the U) and the column oxide layer
surface. Decreased recovery (44%) was therefore also observed on
the Wss_H vs. the Wpr_H column.
Based on the HILIC chromatographic profiles (Fig. 7B), it was
also possible to correlate the extent of non-specific adsorption
with various ON functional groups (i.e. extracyclic functional

groups on the different bases and the sugar hydroxyl groups).
Specifically, differences were seen for dA20, dG20 and dC20, which
must be indicative of extracyclic nucleobases or the possible formation of intramolecular secondary structures. For dA20 and dC20
(both containing exocyclic amino groups), very low % recoveries
were observed on the Wss_H column in comparison to the Wpr_H
column (5% for dA20 and 12% for dC20, respectively). This could be
traced back to the involvement of the nucleobases in potential surface interactions. However, it should be considered that stainlesssteel columns (Wss_H) required longer initial conditioning times
than bioinert columns (Wpr_H) and it might be possible that a
like-for-like conditioning process might be needed for some samples. In this specific case, the conditioning was carried out with a
mixture of poly-dT (see Section 3.1.1). Thymidine (T), compared to
A, C, and G, is the nucleobase with the most limited capacity for

hydrogen bonding, and it contains two oxide groups and a methyl
as heterocycle functional groups [48,68]. Indeed, it could be possible that extended column conditioning might be required for ONs
containing exocyclic amino groups, like in the case of dA20 and
dC20, that are capable of even more extensive hydrogen bonding
and electron sharing. This might better mitigate the non-specific
adsorption encountered with the stainless-steel column in HILIC
mode. As shown in Fig. S3, a significant increase in the recovery
of dA20 was achieved after an additional column conditioning was
performed in the form of 15 consecutive injections of dA20. In
short, analysts must be careful if they are to rely on sample-based
conditioning of stainless-steel columns.
Interestingly, in both modes, dG20 showed a low recovery no
matter the column used (lower area and height values as compared to all other ONs, as reported in Table 3). Despite not being supported by the data, a possible explanation for this behavior might be the formation of long and stable supramolecular structures characterized by the cooperative binding of interlocked slipped strands forming stable G-quadruplexes [69,70].
These supramolecular structures can have a remarkably increased
size as compared to a linear 20-mer strand, while showing an extremely high thermal stability (utterly resistant above temperature
of 100°C) [70]. Therefore, when analyzing dG20, the overly high
peak adsorption (as compared to the other 20-mer ONs) might be
eventually traced back to the formation of these heterogeneous intramolecularly hydrogen-bonded complexes.

Concerning the DNA duplex that was analyzed in HILIC mode
(Fig. 7B), it should be considered twice as many phosphate groups
per length versus a single-stranded molecule. In addition, the extracyclic functional groups on the bases are involved in the hydrogen bonding between the two complementary strands and therefore not available for further interactions. The higher percentage
recovery of the duplex DNA (61%) in comparison with the singlestranded DNA sequences (dT20, dA20, dG20, and dC20) shows that
the duplex without the exposed functional groups on the nucle14


H. Lardeux, A. Goyon, K. Zhang et al.

Journal of Chromatography A 1677 (2022) 463324

obases results in improved recovery despite the greater number of
phosphate groups and a completely different secondary structure.
The DNA duplex in this study was predicted to have a melting temperature (Tm ) of 66°C (based on the provider specifications). Interestingly, under the applied HILIC conditions, no traces of single
strands were found, confirming that HILIC was exceptionally well
suited to the analysis of this duplex ON. When analyzed by high
temperature IP-RPLC, this ON was detected only in its melted state
(Fig. S4).
To this point, more pronounced adsorption behavior is observed
in HILIC vs IP-RPLC mode (Fig. 7, Table 3), and may be explained
by differences in pH conditions. It has been reported that the oxide layer on the metal surface of stainless-steel columns has an
isoelectric point (pI) of approximately 7 [62]. Therefore, at mobile phase pH values equal to this pI, as in the case of the HILIC
conditions used in this work, the surface oxide layer should be
50% positively-charged and therefore interfering with the HILIC
mechanism and actively participating in the binding of negativelycharged ONs. On the contrary, in IP-RPLC mode, the mobile phase
pH (7.9) being higher than pI, net positive surface charge is reduced and therefore negatively-charged analytes are slightly less
prone to adsorption. According to this hypothesis, these additional electrostatic interactions in HILIC mode explain the more
pronounced adsorption observed on stainless-steel HILIC columns
(Wss_H) compared to stainless-steel RPLC columns (Wss).
Additional modifications involving the phosphate backbone

(phosphorothioate PS linkages) and the sugar moiety (OMe, MOE,
and LNA residues) were also evaluated in IP-RPLC and HILIC modes.
Chromatographic parameters and chromatograms are reported in
Table 3 and Fig. 8. For these analyses, dT20 and rU20 were kept
as reference ONs and the modifications were applied to three nucleotides at both the 5’- and 3’-positions (as reported in Table 1).
As reported in Table 3, ONs containing PS linkages showed increased adsorption in comparison to their non-modified counterparts in IP-RPLC mode (Fig. 8A). This effect could have been predicted by the fact that the sulfur atom is less electronegative than
oxygen, leading to weaker ion-pairing with the components of the
mobile phase and therefore additional risk of undesired ionic interactions with the column surfaces.
Contrary to IP-RPLC, in HILIC mode the backbone of a PScontaining ON is not involved in additional interactions with an
ion-pairing agent (as in IP-RPLC). Differences in electronegativity
between PO and PS moieties therefore explain much better recovery and peak resolution of PS ONs under HILIC conditions as compared to IP-RPLC mode, especially concerning rU20-PS.
Regarding modifications involving the sugar moiety, it should
be noted that they might change the lipophilicity of the ONs
(which relates to favorable pharmacokinetic properties) [71].
Specifically, the absence of the hydrophilic hydroxyl that characterizes the RNA is responsible for diminishing ionic interactions
and therefore obtaining a better recovery. In agreement with this
hypothesis, sugar-modified ONs (rU20-MOE, rU20-OMe, and dT20LNA) showed a better recovery as compared to rU20 in both chromatographic modes, with remarkably higher area and height values, as reported in Table 3.
Interestingly, in HILIC mode (Fig. 8B, Table 3), a consistent and
even better recovery (in the range 85% – 95%) than in IP-RPLC was
observed for all the sugar-modified ONs on the Wss_H in comparison to the Wpr_H column. In addition to what was previously
described in IP-RPLC, rU20-MOE, rU20-OMe, and dT20-LNA each
contains a modification that masks the hydrophilic hydroxyl of the
RNA. This might impede hydrogen-bonding between the HILIC stationary phase and the hydroxyl groups of ORNs (substituted in
sugar-modified ONs). The combination of all these effects would
explain the improvements in recovery and resolution as compared
to rU20 in HILIC mode.

4. Conclusions
The goal of this study was to understand the contribution of
each step of the chromatographic process in the problematic of

non-specific adsorption of DNA and RNA oligonucleotides ranging
from 15- to 100-mer residues in length. Under the conditions used
in this paper (concentrations in the μM range), column hardware
is of significant impact because it is generally made of stainlesssteel and represents more than 70% of the flow path surface that
a sample will encounter throughout an analysis. Columns made
of different bioinert hardware (i.e. titanium-lined, PEEK-lined and
hybrid organic/inorganic surface columns) were compared to their
stainless-steel counterparts in both IP-RPLC and HILIC modes. In
this context, successive injections of a mixture of ONs (conditioning sample) were carried out to saturate any potential adsorption sites of the column hardware. For the IP-RPLC stainless-steel
columns, the peak areas gradually increased and a plateau was
reached after about 15 injections. It is noteworthy that full recovery of all oligonucleotides from the conditioning sample was
achieved upon first injection when using the IP-RPLC bioinert
columns. Nevertheless, the injections of DNA and RNA mixtures of
ONs that followed showed that the sample conditioning procedure
is not longstanding, and that IP-RPLC bioinert columns should be
preferred.
Standard stainless-steel HILIC columns showed some utility but
only after long sample conditioning. However, it was found that
this strategy does not completely solve the problem and specific
protocols might be required based on the nature of the samples
used for conditioning versus the samples to be analyzed. Bioinert
HILIC columns should also be preferentially used when possible. To
the best of our knowledge, commercially available bioinert HILIC
columns are currently limited to hybrid organic/inorganic surface
and PEEK technologies. Both these hardware surfaces were tested
in comparison to their stainless-steel counterparts and remarkable
improvements in peak recoveries were found, across a wide range
of different ONs including dT15-35 as short DNA, dT40-100 as long
DNA, and rU15-30 as short RNA.
Next, three different chromatographic systems were compared,

and it was found that non-specific adsorption of ONs on instrumentation materials was not significant. Recoveries from an LC
with bioinert flow paths were, at most, only improved by 10%.
However, the conditions applied here correspond to relatively high
mass loads that would be suited to characterizing a drug substance. The analysis of trace samples, like is encountered with
pharmacokinetic studies, might show more meaningful differences.
Particular care should instead be taken with the UV flow cell,
since strong adsorption might occur on UV flow cells that have not
been passivated through sample-based conditioning. Conditioning
of the UV flow cell is therefore highly recommended to ensure
consistent results. Analysts might also need to be equally scrupulous whenever a new flow path component is installed into their
LC. In addition, the material used in the UV flow cell (titanium
or Teflon) was found to significantly affect the peak shapes of the
largest ONs (60- to 100-mer) analyzed by IP-RPLC, for which titanium should be preferentially used.
Finally, the evaluation of a larger set of ONs was performed,
including a DNA duplex and ONs having different base compositions (U/T, C, A, G), furanose sugars (DNA/RNA), and modifications
occurring at the phosphate linkage (PS) or at the sugar moiety
(OMe, MOE, and LNA). Based on our data, the choice of the column hardware had the most relevant impact on the extent of nonspecific adsorption. In addition, HILIC separations have proven to
be extremely attractive for the analysis of RNA-based ONs, duplex,
PS- and sugar-modified ONs. However, bioinert HILIC columns are
mandatory for successful HILIC operation, especially with this particular type of oligonucleotide analysis.
15


H. Lardeux, A. Goyon, K. Zhang et al.

Journal of Chromatography A 1677 (2022) 463324

Declaration of Competing Interest
[10]


Jennifer Nguyen and Matthew Lauber are employees of Waters Corporation (Milford, MA, USA) that has provided the Waters
columns and the Acquity Premier system used in this work.

[11]

CRediT authorship contribution statement
[12]

Honorine Lardeux: Conceptualization, Investigation, Writing –
original draft, Visualization. Alexandre Goyon: Resources, Writing
– review & editing. Kelly Zhang: Resources, Writing – review &
editing. Jennifer M Nguyen: Resources, Writing – review & editing. Matthew A Lauber: Resources, Writing – review & editing.
Davy Guillarme: Conceptualization, Funding acquisition, Resources,
Project administration, Writing – original draft, Writing – review &
editing, Supervision. Valentina D’Atri: Conceptualization, Writing
– original draft, Writing – review & editing, Visualization, Supervision, Project administration.

[13]

[14]

[15]

[16]

Acknowledgements
[17]

The authors wish to thank Jean-Luc Veuthey from the University of Geneva for his fruitful comments and discussions, Daniel
Eßer (YMC Europe GmbH, Dinslaken, Germany) for providing the

YMC columns used in this work, Brian Rivera (Phenomenex Inc,
Torrance, CA, USA) for providing the Phenomenex columns, Szabolcs Fekete (Waters) for sharing insights on potential ON retention effects, and Sebastien Besner (Waters) for sharing considerations about flow cell materials. The authors also wish to thank
Waters Corporation (Milford, MA, USA) for the loan of the Acquity
Premier system and for the gift of the Waters columns used in this
work.

[18]

[19]

[20]

[21]

Supplementary materials

[22]

Supplementary material associated with this article can be
found, in the online version, at doi:10.1016/j.chroma.2022.463324.
[23]

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