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Miniaturization of liquid chromatography coupled to mass spectrometry
Journal Pre-proof Miniaturization of liquid chromatography coupled to mass spectrometry. 1. Current trends on miniaturized LC columns Karen Mejía-Carmona, Juliana Soares da Silva Burato, João Victor Basolli Borsatto, Ana Lúcia de Toffoli, Fernando Mauro Lanças PII:
S0165-9936(19)30120-7
DOI:
https://doi.org/10.1016/j.trac.2019.115735
Reference:
TRAC 115735
To appear in:
Trends in Analytical Chemistry
Received Date: 7 March 2019 Revised Date:
13 November 2019
Accepted Date: 14 November 2019
Please cite this article as: K. Mejía-Carmona, J. Soares da Silva Burato, J.V. Basolli Borsatto, A. Lúcia de Toffoli, F.M. Lanças, Miniaturization of liquid chromatography coupled to mass spectrometry. 1. Current trends on miniaturized LC columns Trends in Analytical Chemistry, https://doi.org/10.1016/ j.trac.2019.115735. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.
1
Miniaturization of liquid chromatography coupled to
2
mass spectrometry.
3
1. Current trends on miniaturized LC columns
4 5
Karen Mejía-Carmona, Juliana Soares da Silva Burato, João Victor Basolli Borsatto, Ana
6
Lúcia de Toffoli and Fernando Mauro Lanças*
7 8
University of São Paulo, São Carlos, Institute of Chemistry of São Carlos, SP, Brazil
9 10
*Corresponding author. Tel: +(55) 16 3373 9983; Fax: +(55) 16 3373 9984
11
E-mail address: [email protected] (F. M. Lanças)
12 13 14
Highlights
15
•
Recent advances in miniaturized liquid chromatography
16
•
New materials employed for capillary columns separations
17
•
Recent advances in the development of capillary columns for miniaturized liquid
18
chromatography
19 20
Contents
21
1. Introduction
22
2. Theoretical aspects
23
3. Packed columns
24
4. Monolithic columns
25
5. Porous Layer Open Tubular Columns (OT-PLOT)
26
6. Wall Coated Open Tubular (WCOT) Columns
27
7. Recent applications of miniaturized LC
28
8. Concluding remarks
1
29
Abstract
30
Over the last decade, the use of miniaturized columns in liquid chromatography (LC) has
31
seen intense growth in a wide range of research areas. This report brings an overview of
32
the current developments and applications of miniaturized columns in LC, focused on the
33
type of columns used. Following a short introductory section on some theoretical aspects,
34
the fundamental bases on their design and fabrication, new stationary phases and
35
incorporated materials, performance evaluation/characterization, main advantages, and
36
recent applications of filled capillary and open tubular (OT) capillary columns are
37
introduced and critically discussed. Prospects on the different types of miniaturized
38
columns and their hyphenation with mass spectrometry (MS) reported in the last years are
39
also introduced and discussed.
40 41
Keywords: miniaturized liquid chromatography, particle packed capillary column,
42
monolithic capillary columns, open-tubular columns, wall coated open-tubular columns,
43
porous layer open tubular columns, mass spectrometry.
44 45
Abbreviations
46 47 48 49 50 51 52 53 54 55 56 57 58 59
β-CD, β-cyclodextrin; capillary-LC, capillary liquid chromatography; CLSM, confocal laser scanning microscopy; CTCs, circulating tumor cells; dp, particle diameter; ESI, electrospray ionization; EI, electron ionization; FP, fully porous particles; GC, gas chromatography; h, reduced plate height; H, plate height; HILIC, hydrophilic interaction chromatography; HIC, hydrophobic interaction chromatography; HPLC, high-performance liquid chromatography;
2
60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79
i.d., inner diameter; KPL, kinetic performance limits plots; LC, liquid chromatography; MS, mass spectrometry; MS/MS, tandem mass spectrometry; nano-ESI, nano-electrospray ionization; nano-LC, nano liquid chromatography; NP, non-porous particles; OT, open tubular; PDMS, polydimethylsiloxane; PLOT, porous layer open tubular; SEM, scanning electron microscopy; SP, superficially porous particles; TMOS, tetramethoxysilane; UHPLC, ultra-high pressure liquid chromatography; υ, reduced linear velocity; WCOT, wall coated open tubular;
1. Introduction
80
Liquid chromatography (LC) has become a widespread analytical technique used in
81
many fields of natural and health sciences [1–3]. Following the current trends in analytical
82
chemistry, miniaturization is among the main LC innovation areas along the last ten years
83
[4].
84
Horvárth et al. [5] were one of the pioneers in LC miniaturization. In 1967 they
85
compared a stainless-steel uncoated capillary tubing of inner diameter (i.d.) of 0.3 mm with
86
a 50 µm pellicular particle packed capillary of 1.0 mm i.d. for the separation of
87
nucleotides. This study drove the authors to the conclusion that packed small-bore
88
capillaries were more efficient than uncoated capillary ones. One decade later, Tsuda and
89
Novotny [6,7] established the required equipment for working with capillary LC, by
90
miniaturizing the overall capillary LC system. They diminished the column i.d. to 50–200
91
µm, and the particle size down to ~30 -10 µm, as well as have implemented modifications
92
on conventional injection and detector systems aiming to reduce the band-broadening
3
93
effects. At the same time, Ishii et al. [8] worked in the development of a micro-high
94
performance liquid chromatography (MHPLC) system, successfully applied to both packed
95
[9] and open tubular (OT) capillary columns. These seminal works were an essential
96
contribution that allowed miniaturized LC to significantly advance in the 80s, along with
97
other
98
chromatography, waking up the interest of researchers from different fields [10]. Recently,
99
Novotny et al. [10] reported a comprehensive review of the historical aspects, advances,
100
techniques
as
capillary
electrophoresis
and
capillary
supercritical
fluid
and efforts of capillary LC developments from the beginning until the present.
101
Nowadays, the separation in miniaturized LC is performed in capillary columns
102
similar to those used in high-resolution gas chromatography. As a result of all the
103
developments made in miniaturized LC, it is possible now to find commercially available
104
capillary and nano liquid chromatography systems (capillary-LC and nano-LC), including
105
–and principally– using ESI-MS as a detector.
106
One of the most important advantages offered by miniaturized LC is the viability to
107
work in applications were just a small sample volume may be collected and analyzed,
108
covering areas such as forensic, clinical, and omics. Besides, the low flow rate employed
109
means lower solvent consumption, allowing the use of specialized solvents, and leading to
110
enhanced detection as a result of the lower chromatographic dilution [11,12].
111
Miniaturized LC is a general term employed for describing liquid chromatography
112
using i.d. columns below to 1.0 mm. In practice, to make an accurate description of this
113
approach, it is necessary to consider both the internal column diameter and the mobile
114
phase flow rate used [13]. In general terms, for practical purposes, capillary-LC employs
115
capillary columns with i.d. between 100–500 µm and flow rates of 1–10 µL/min, while
4
116
nano-LC employs capillary columns with i.d. < 100 µm with flow rates from 10 to 1000
117
nL/min [2].
118
The design of columns has been one of the main drivers on LC miniaturization, as
119
exposed by Desmet et al. [11]. There are two main types of capillary columns: filled
120
capillary columns and open tubular capillary columns (Fig. 1). The former can be filled
121
with a particle bed or a monolith, and the second has a thin layer of stationary phase
122
attached to the inner surface of the column, leaving an open tubular space.
123
124
Particle packed capillary columns commonly use the same particles employed in
125
conventional HPLC columns; silica-based particles are the most employed. Silica particles
126
can be prepared in different ways, producing unique characteristics, resulting in a wide
127
range of packed capillary columns commercially available [12]. In LC, it is known that
128
reducing the particle size and the internal diameter of columns improves the separation
129
performance; the use of sub-2 µm particles enables greater efficiency at faster flows,
130
reducing analysis time [14]. Nevertheless, this advantage is limited by the high back-
131
pressure generated, requiring specialized instrumentation similar to that used in ultra-high
132
pressure liquid chromatography (UHPLC) [15]. Additional information about particles and
133
support materials, as well as packing capillary columns methods, can be found in several
134
reviews [2,14–17].
135
Monolithic capillary columns - another way to prepare filled columns - since their
136
introduction in the early 90s, have gained particular interest because of their easy in-situ
137
preparation and high permeability that decrease the back-pressure when compared to
138
particle packed capillary columns. A monolithic column consists of a continuous
139
polymeric block structure made from either silica-based or organic-based polymers that fill
5
140
the whole capillary tube [1]. Capillaries with i.d. ranging from 20–500 µm are commonly
141
employed. The efficiency of monolithic columns depends on the stationary phase
142
morphology, macropore size, uniform skeleton, and kind of polymers employed.
143
Improvements in the efficiency –similarly by that obtained by the reduction particle sizes
144
in packed columns– can be achieved by controlling both the external porosity and pore size
145
[14]. Currently, the synthesis of a wide range of monolithic materials containing metallic
146
nanoparticles, metal-organic frameworks, carbon-based nanomaterials, as well as
147
monoliths functionalized with boronic acid, zwitterionic monomers and other
148
functionalities [18], extended the range of applications of capillary LC.
149
On the other hand, open tubular (OT) columns, firstly introduced for gas
150
chromatography (GC) in 1958 by Golay [19], became an unique type of column which
151
have a thin layer of stationary phase on the inner wall of the capillary. By the late 1970s,
152
Tsuda et al. [20] explored the OT concept in LC studies, including the design, theory, and
153
practical aspects of the technique. However, the research interest decayed quickly, mainly
154
due to its lower performance compared to particle packed columns, and the lack of suitable
155
miniaturized instrumentation and detectors at the time. Recently, with the commercial
156
availability of highly sensitive detectors, reduced volume injectors, and micro and
157
nanoflow LC instruments, the interest in applications and fundamental research in OT
158
columns emerged again.
159
In contrast to particle packed columns, the most critical advantages of OT columns
160
is the exclusion of eddy dispersion contribution –counting roughly to one-half of band-
161
broadening in packed columns– and their high permeability, which allows the use of longer
162
columns [11]. However, to achieve a high chromatographic efficiency are required
163
columns with downsized inner diameters (~5 µm), a fact that severely compromises the
6
164
backpressure and column length [21]. OT columns are classified into two main types: wall
165
coated open tubular (WCOT), and porous layer open tubular (PLOT).
166
Although the former is the most employed in GC, consisting of a thin cover film of
167
non-porous stationary phase, WCOT columns were poorly explored in LC because of their
168
lower sample capacity and efficiency, mainly due to their non-porous film. Later efforts
169
aiming to obtain thicker films and increase their sample capacity were made, employing
170
multilayer coatings and unique chemical bonding processes. Despite this, WCOT columns
171
were overshadowed by a new type of open tubular LC column termed PLOT columns,
172
once they showed to be superior to the LC purposes at that time [1]. In practice, LC-type
173
WCOT capillary columns have been to the moment employed more successfully in
174
capillary electrochromatography (CEC) than in micro and nano-LC [22].
175
In a PLOT column, also initially developed for GC, the stationary phase is a porous
176
material that offers a higher surface area and better retention capacity compared to WCOT
177
columns. Among the stationary phases employed, organic polymeric or silica polymeric-
178
based materials are the most utilized. Polystyrene (PS), polydivinylbenzene (DVB), and
179
polymethacrylate (PMMA) are the current phases applied to proteomic analyses. Thin
180
layer polymeric stationary phases are usually deposited by the free-radical polymerization
181
method, either by thermal or photo-initiation approaches, yielding several layer structures
182
and morphologies by modifying such as the energy supplied through the polymerization
183
time [3,23,24].
184
The last generation of silica-based PLOT columns was recently obtained by the sol-
185
gel method, as reported by Forster et al. [25]. They employed a solution of
186
tetramethoxysilane (TMOS), urea and polyethylene oxide (PEO) as progeny agent in
187
preparing fused silica capillaries of 10–100 µm i.d. The authors obtained PLOT columns
7
188
with a good mass loadability that provided high-efficiency separations (>170,000 plates),
189
successfully applied to normal phase separations [25,26]. Recently, Hara et al. [27]
190
demonstrated that capillary columns with 5 µm of i.d. coated with mesoporous silica, 550
191
nm layer width, and 2.6 m length, achieved ~950,000 plates.
192
In this first review of a series on coupling miniaturized LC columns to mass
193
spectrometry (MS), we provide an overview of the recent advances on capillary-LC and
194
nano-LC, focused on the capillary columns, stationary-phases synthesis and packing
195
methods, and their recent applications with particular emphasis on the coupling to mass
196
spectrometry (MS). In a second review, we will discuss the miniaturized instrumentation,
197
hyphenation with mass spectrometry, and the current status of chip-based systems.
198 199
2. Theoretical aspects
200
The behavior of the analyte's molecules inside a column and how it affects the
201
analysis performance have been one of the most important topics to be understood in all
202
chromatography techniques. The main fundaments were first developed for GC and further
203
transferred to LC. Among the researchers that have developed the theory of
204
chromatography in the last century, J. J. van Deemter and coworkers receive the main
205
attention. In their work, the authors described how the molecules of analytes are distributed
206
along the column during the separation based on three terms [28]. The first term (A-term),
207
also called eddy diffusion, demonstrates how the molecules of analytes spread by flowing
208
through the column. It is independent of the flow but depends on the column morphology
209
(particle diameter (dp) for packed columns and i.d. for OT columns, as an example). The
210
second term (B-term) is related to the longitudinal diffusion and depends on the diffusion
211
coefficient of the mobile phase, but also on the flow rate. The last term, (C-term) called the
212
mass transfer resistance, depends on the transference of the analytes between the mobile 8
213
and stationary phase and presents a linear dependence on the flow rate. Those terms were
214
used to correlate the plate height and the flow rate of analysis generating Equation 1 (Table
215
1). The plate height (H) (Equation 2, Table 1), an important metric to be considered in the
216
evaluation of the separation performance, is defined as the rate of increase of the peak
217
variance per unit of length of the column. The lower the H, the better the separation
218
performance. The van Deemter equations not only brought a deeper view of the
219
chromatography fundamentals but also resulted in a powerful tool to evaluate the
220
performance of the columns. The so-called van Deemter plots have been applied for
221
generations of scientists to evaluate the performance of the chromatographic separation in
222
combination with other graphical representation methods to be mentioned later in this
223
topic.
224
< Table 1>
225
Giddings proposed dimensionless metrics in order to correlate the performance of
226
different forms of chromatography [29]. The dimensionless metrics were denominated as
227
reduced terms. The reduced plate height (h) and the reduced linear velocity (υ) take into
228
account the particle diameters of the packing material (Equations 5 and 6, Table 1). Knox
229
and Parcher [30] proposed that by plotting log h as a function of log υ, a powerful tool to
230
assay the packing quality would be available. This relationship, known latter as the Knox
231
equation (equation 4, Table 1), correlated the h and the υ in a way similar to the van
232
Deemter equation. The major difference between the equations is related to the dependence
233
of the multipath term on the υ (see equations 1 and 4 to compare). At low υ values, the
234
multipath diffusion tends to have a lower impact on h until it reaches a constant value at
235
high υ values. The longitudinal diffusion and the mass transfer resistance present the same
236
behavior as proposed by van Deemter.
9
237
Recently, the kinetic plots are receiving more attention in order to characterize
238
columns and separation performances. A kinetic plot is a graphical representation method
239
that expresses a metric of separation quality (efficiency, peak capacity, resolution, and
240
others) as a function of a metric of time (retention time, analysis time, dead time, and
241
others). The initial concepts of kinetic plots were proposed by Giddings in his work
242
comparing the theoretical efficiency limits of GC and LC [31], which was followed by a
243
report by Poppe in the 90's, proposing the use of the now called Poppe plots to correlate
244
speed and efficiency of the modern liquid chromatography techniques [32]. The kinetic
245
plots started to be used as a valuable tool to evaluate separation and assay column
246
performance in the works reported by Desmet [33,34]. The kinetic plots can be applicable
247
in both isocratic and gradient analysis; due to this reason, it presents a series of uses and
248
applications [35–37]. Among the main kinetic plots used today, kinetic performance limits
249
plots (KPL) receives particular attention. This type of plot represents an imaginary set of
250
columns of various lengths operating under conditions in which their performances are
251
maximum [38]. Although the KPL plot represents an imaginary set of columns, it is
252
constructed using experimental data transformed by a correction factor (see Table 1,
253
equations 6 to 11 to the more usual kinetic plots metrics). The correction factor (Equation
254
7, Table 1) takes into account the pressure drop of the column, which makes it useful to
255
compare columns of different lengths, diameters, particle diameters, and even filled with
256
different supports.
257
Another critical factor for capillary columns is the internal diameter of the
258
capillary. Although it has no direct effect on the interaction with the analyte, smaller
259
diameters result in higher linear velocities because the smaller cross-section forces the flow
260
to be more intense. The column i.d. effect has been a focus of the investigation since the
261
beginning of the instrumentation development for chromatography. It was observed that
10
262
when the size is reduced to the capillary and nanoscale, the i.d. influences the column
263
performance (Fig. 2A).
264
265
Bruns and coworkers [39,40] published a series of papers on the evaluation of
266
column porosity by using confocal laser scanning microscopy (CLSM) and packing profile
267
of the particles close to the wall, and its consequences over the separation. The observation
268
showed that the i.d. of the capillary affects the packing porosity closer to the wall.
269
Columns with larger i.d. presented higher porosity on the wall region and consequently
270
lower performance. Adversely, smaller columns i.d. presented lower porosity and better
271
results (Fig. 2B). The CLSM also demonstrated that columns of smaller i.d. presents more
272
homogenous packing than larger i.d. columns [41]. Additionally, Gritti has demonstrated
273
that for certain combinations of i.d. and dp, some conditions of poor packing are formed,
274
reducing the efficiency (Fig. 2C) [42].
275
The thermal effects are also minimized in the miniaturized packed columns. The
276
viscous heating, generated by the friction of the mobile phase with the particles, is easily
277
dissipated in capillary columns. The heating distribution inside the column is not
278
homogenous; the center of the column becomes hotter than the walls, generating band
279
dispersion. For columns packed with particles of dp = 1.0 µm operating at 2000 bar, a
280
regular column of 4.6 mm i.d. produces 2.8 W (at 820 µL/min) of heating. For the same
281
conditions, a capillary column of 0.1 mm i.d. generates 1.3 mW (at 380 nL/min) and a
282
nano column of 0.05 mm i.d. results in only 0.33 mW (at 96 nL/min) of power dissipation
283
[43].
284
Miniaturized liquid chromatography has as its most notable advantage over
285
conventional HPLC, better compatibility with MS, especially the electrospray ionization
11
286
(ESI) interface. The small column diameters allow reduced mobile phase flow rates, which
287
are more compatible with the optimal values of flow for the ESI-MS. The sampling
288
efficiency increases significantly at low flow rates for ESI sources (Fig. 2D) [44].
289
Additionally, works have been carried out to allow the coupling of miniaturized LC with
290
electron ionization (EI) sources, which is practically impossible to be achieved with
291
conventional LC due to the high flow rates used and the back-pressures generated [45].
292 293
3. Packed columns
294
Packed columns represent the most significant slice of columns used in liquid
295
chromatography, being the most popular kind of commercial column available in all scales
296
(conventional and miniaturized LC). Other types of LC columns, such as the monolithic
297
and OT columns are still in the state-of-the-art; its main characteristics are described and
298
explored in the following sections of this review. Packed LC columns can be obtained in a
299
large variety of stationary phases commercially available, operating in several
300
chromatographic separation modes, including (i) reversed-phase, (ii) normal phase, (iii)
301
hydrophilic
302
chromatography (HIC), (v) ion-exchange chromatography, and others varieties. The
303
primary particles used in LC can be produced using silicates-like structures generating
304
phases such as C18, C8, cyano, amino, and bare silica particles, or be produced as organic
305
resins. The particles' morphology can be divided into three types: (i) the fully porous (FP)
306
particles, which is, as its name describe, a fully porous sphere; (ii) the superficially porous
307
(SP) particles, which is a sphere of solid (non-porous) core surrounded by a thin porous
308
layer; and (iii) the non-porous particles (NP), that is a non-porous sphere of the stationary
309
phase. Majors summarized those and other more types of column packing for LC [46].
interaction
chromatography
(HILIC),
(iv)
hydrophobic
interaction
12
310
A significant advancement in columns for LC was the optimization of the packing
311
material, in particular, the reduction of particle size. The reduction in particle diameter dp
312
resulted in a more significant performance incensement due to the considerable
313
improvement in the mass transfer between the phases and the reduction in trans-particle
314
and trans-channel diffusions. On the other hand, these small dp demands a higher mobile
315
phase pressure to percolate along its packed bed [47]. To overcome the limitation of the
316
higher back-pressure produced by the stationary phase, more powerful equipment were
317
developed, resulting in the UHPLC concept. Some commercially available models can
318
withstand pressures higher than 1500 bar.
319
The SP particles, also known as core-shell, porous-shell, or fused-core particles,
320
were developed as an alternative to the UHPLC system. The presence of a solid core
321
reduces the trans-porous diffusion because the porous layer is thinner than in the FP
322
particles. This reduction in the porous structure allows to bigger SP particles to reach
323
comparable efficiency as smaller FP particles. NP particles are also availed but are less
324
used than FP and SP particles.
325
The packed columns are composed of three main parts: (i) the column tubing, (ii)
326
the packed stationary phase, and (iii) the frits (responsible for retaining the stationary phase
327
inside the column). The type of frit used affects the separation performance and detection
328
possibilities. Franc and coworkers have evaluated the performance of several types of frit
329
for capillary columns produced with different materials in columns of 0.32 and 0.25 mm
330
i.d. [48]. Four different types of frits were produced (Fig. 3). The type A frit was composed
331
of a glass wool filter frit placed inside the column capillary and secured by a smaller id
332
outlet capillary emended with epoxy resin. The type B frit used the same glass wool frit but
333
secured by a polyether ether ketone (PEEK) union. The type C frit was produced by
334
placing a glass wool frit, stainless steel, or titanium frit inside the union and then secure the
13
335
frit by pressing the capillary. Type D was produced with different monolithic frits, which
336
allows on-column detection. It was concluded that both the material and the frit type affect
337
the separation efficiency significantly and must be taken into account when preparing the
338
column based on the experiment target. For an analysis not demanding on-column
339
detection, the type A frit is recommended. The coupling of packed capillary columns to
340
MS detection requires a capillary tubing connecting the column outlet to the MS inlet. A
341
highlighted work concerning capillary columns is presented by Santos-Neto and
342
coworkers, who used a fully operational system consisting of a capillary column switching
343
restricted-access media-liquid chromatography-electrospray ionization-tandem mass
344
spectrometry system to analyze drugs in biofluids [49]. The use of nano-LC columns
345
coupled with MS has increasing relevance in the field of metabolomics; Chetwynd and
346
David summarized several examples in a recent review [50].
347
348
The interface between miniaturized columns and MS is so relevant that several
349
groups (and companies) are making substantial efforts to produce LC chips that replace the
350
conventional columns [51]. Independently of the company that produces, the chip column
351
has as an essential characteristic, its placement very close to the ionization source (and in
352
some cases, it may contain an ESI tip) [52,53]. This approach is used to reduce the post-
353
column band diffusion significantly.
354
The packing procedure well performed is vital to ensure highly efficient columns.
355
The most used type of column packing procedure is the slurry packing, which consists of a
356
dispersion of the stationary phase in a solvent (the slurry solvent) been flushed through the
357
column. The slurry solvent nature and slurry concentration play an essential role in column
358
packing. The nature of the solvent used affects the distribution of the particles in the slurry.
359
It was found that the most aggregated solution produces the most efficient columns for the
14
360
packing of 0.03 mm i.d. columns with particles of 1.1 µm dp [54]. Similar results were
361
found by Wahab and coworkers [16].
362
Different packing approaches are employed to pack columns using slurry solutions.
363
The simplest, but very effective system, is prepared in a set up very similar to the one
364
employed for packing regular HPLC columns. In this setup, the slurry reservoir is
365
connected directly to an air driven-liquid pump, and the column is connected to the slurry
366
reservoir by using an empty capillary tube (Fig. 4A). Though it is expected to produce
367
pressure pulses during the packing, the columns produced by this system result in efficient
368
columns [49,55]. An alternative set up was proposed for packing capillary columns,
369
avoiding pressure pulses (Fig. 4B). In this setup, the slurry is placed in the chamber and
370
stirred during the application of the high pressure; a microscope can be used to evaluate the
371
packing process [16]. Berg and coworkers used a similar approach in which the chamber is
372
filled with the slurry solution, but nitrogen gas is used to flow the material through the
373
capillary [56].
374
375 376
Dry packing and supercritical fluid packing are also alternatives to pack columns.
377
The dry packing procedure is straightforward; the particles are deposited in the reservoir,
378
and then a gas (usually nitrogen) is flushed through the system resulting in the packed bed
379
[57]. The supercritical fluid packing combines the best of the slurry and dry packing
380
methods, using supercritical CO2 as a solvent. The setup of supercritical fluid packing is
381
different from dry or slurry packing because the outlet of the column is connected to a
382
vacuum pump [58]. Other possibilities of packing procedures are described and discussed
383
by a practical viewpoint by Lanças and coworkers [17].
15
384
Alternatives to packing are also presented in the literature. The pillar array columns
385
(µPAC) are produced by milling pillars on silicon, followed by the functionalization with
386
the stationary phase. The significant advantage of this layout is that the milled pillars result
387
in a high ordinated filling, which allows a more homogenous flow inside the column and a
388
reduction on the multipath diffusion [59,60]. 3D printing has also been investigated as a
389
way to produce ordinated fillings for columns. Although it seems to be promissory, mainly
390
because they use of 3D manufacturing allows the construction of different geometry for the
391
inner part of the column, the instrumental (3D printers) limitations are still the bottleneck
392
for this use [61].
393 394
4. Monolithic Columns
395
Monolithic columns consist of a small i.d. tubing, all filled with a robust porous
396
polymer. The polymeric phase consists of pores of different sizes that provide retention
397
and separation selectivity. The porous skeleton of the monolithic columns gives the mobile
398
phase high permeability and low back pressure, which enables the use of high flow rates
399
allowing faster mass transport when compared to particle-filled columns. Therefore, the
400
central positive aspect claimed in favor of monolithic columns is their ability to maintain a
401
proper separation efficiency at high flow rates [62].
402
The monolithic columns stationary phases are made of polymers that may have
403
inorganic, organic, or hybrid characteristics. Silica monoliths from inorganic stationary
404
phases have a porosity higher than 80%, resembling silica particle packed columns. Thus,
405
the main advantages related to the use of silica monoliths are the high separation efficiency
406
associated with low backpressure. Also, the main disadvantage is the sensitivity to pH
407
variations characteristic of silica-based materials.
16
408
A general procedure for producing monolithic columns with silica polymer phase
409
employs the sol-gel approach, consisting in the hydrolysis and polycondensation processes
410
catalyzed by tetraalkoxysilanes acids in the presence of an inert compound that acts as a
411
porogenic agent. Such a reaction results in a continuous silica mesh with a defined pore
412
structure that can be modified according to the reactant concentration, temperature, and
413
polymerization time. It is also possible to perform interventions on the surface of the
414
polymeric phase through washing with solutions containing modifiers, in order to improve
415
selectivity and extend the application of these columns [63].
416
Due to the high porosity characteristics of silica monoliths, these phases are ideal
417
for the separation of small molecules. Thus, monolithic columns with inorganic stationary
418
phases are generally used in the separation of environmental, forensic, natural products,
419
and pharmaceutical samples, among others [64].
420
Another aspect that stands out in the use of monolithic columns containing
421
inorganic stationary phases is their high efficiency at flow rates higher than the
422
theoretically expected value, without loss of separation efficiency. This ability to perform
423
quick separations qualifies it to be used for multidimensional LC separations, proving to be
424
an excellent tool for separating complex samples. As an example, Espina-Benitez et al.
425
developed an in-line coupling of a monolithic boronate extracting column with a
426
monolithic silica-based analytical column to analyze uridine, cytidine, adenosine, and
427
guanosine. The monolithic silica analytical column had its activated surface functionalized
428
by thiol-one photo click [65].
429
In turn, organic monoliths are extensively used due to their qualities, such as easier
430
preparation and stability at high temperatures and pH variations. As a disadvantage, the
431
low mechanical strength stands out.
17
432
The general procedure for producing an organic polymeric phase for monolithic
433
columns is to subject a solution containing functional organic monomers, porogenic
434
solvents, and a radical initiator to either heat or a light source to release free radicals which
435
will trigger the monolith formation. The morphology of the formed monolith is a
436
consequence of the reagent concentration, temperature or light intensity, and
437
polymerization time. As in silica monoliths, organic monoliths can be surface modified to
438
increase stationary phase selectivity. It is noteworthy that, besides the mentioned thermal
439
initiation method, there are also other techniques such as polycondensation, emulsion
440
polymerization, and ring-opening metathesis [66].
441
Organic monoliths have a nonporous granular characteristic interspersed with large
442
pores, as can be observed by scanning electron microscopy (SEM) (Fig. 5) [67]. In this
443
type of monolith, the pores are too miniaturized, making it impossible to separate small
444
molecules. Thus, this stationary phase is ideal for the separation of large molecules, such
445
as proteins and other biomolecules, as the passage pores retain these analytes. However,
446
there are scientific advances aimed at the development of organic monoliths capable of
447
separating small molecules. Initial efforts are focused on pore morphology changes
448
through studies that adapt the polymerization time and reagent concentration. Also, post-
449
polymerization modifications and the addition of nanostructures to the polymer may be
450
performed [68].
451
452
Echevarría et al. developed monolithic capillary columns with organic stationary
453
phase coated with tris cellulose (3,5-dimethyl phenyl carbamate) for the separation of
454
several compounds such as β-blockers and pesticides. The synthesis of the stationary phase
455
was done by thermal polymerization of 2-hydroxyethyl methacrylate and ethylene glycol
456
dimethacrylate in the presence of a porogenic mixture [69].
18
457
Monolithic columns consisting of hybrid stationary phase combine the advantages
458
shown by organic polymer-based and silica-based monolithic columns. As such, they have
459
a high surface area and mesoporous structure, such as the inorganic ones, and stability at a
460
higher temperature and pHs, like organic ones, still retaining the general advantages of
461
monolithic columns that are low flow resistance. Sol-gel or ring-opening methodologies
462
can prepare most such monoliths, which have a wide range of applications depending on
463
the analytes of interest.
464
Recently, Zhao et al. used a monolithic column consisting of hybrid characteristics
465
aiming at the separation of inorganic arsenic. The thiol functionalized organic-inorganic
466
phase was synthesized using a unique ternary weak basic solvent system by a sol-gel
467
process [70]. Other application examples encompassing inorganic, organic, and hybrid
468
monoliths have been selected and are included at the application table in the following
469
sections.
470
Monolithic columns, after its laboratory preparation, can be evaluated by physical,
471
chemical, and chromatographic means. The phase characterization is made by physical
472
evaluation through SEM photos and methods able to evaluate the porosity and dimensions
473
of the polymers. In turn, the chemical and chromatographic analyses are obtained through
474
chromatographic runs to evaluate the selectivity and separation power of the produced
475
column.
476
The use of capillary monolithic columns in LC is growing and is being established
477
as a great alternative to packed columns, as they often have superior results. The main
478
advantages of both organic and silica polymeric stationary phases for monolithic columns
479
are its ease synthesis, rapid analysis, no need for use frits, and a variety of stationary
480
phases that can be available. The existence of mass spectrometry coupled micro/nano-LC
19
481
systems also represents a gain for miniaturized monolithic columns to analyze samples
482
with small volumes and excellent detection efficiency [62]. Therefore, it is expected that
483
the miniaturized monolithic column in LC will continuously be further explored, and
484
phases will continue to be developed in order to increase the selectivity of analyzing
485
specific compounds.
486 487
5. Porous Layer Open Tubular Columns (OT-PLOT)
488
The technological advances in the miniaturization of LC and the commercial
489
availability of adequate instrumentation made it possible to explore the use of OT
490
analytical columns, allowing its successful coupling to mass spectrometry using nano-
491
electrospray ionization (nano-ESI) or electron ionization (EI) sources.
492
The use of open tubular columns in LC is theoretically advantageous compared to
493
the use of filled columns. Desmet et al. [11] comparing the kinetic efficiency of OT-PLOT
494
columns with packed columns found that at any retention time, OT analytical columns
495
have at least ten times more theoretical plates, as can be seen in Fig. 6. Other advantages of
496
using OT-PLOT LC over conventional LC columns include the ability to use higher flow
497
rates without significant column pressure increase; column efficiency increase; lower
498
mobile phase and sample consumptions; lower waste generation –all in accordance to the
499
green chemistry principles– and ease of coupling to MS [3].
500
501
Among the open tubular columns, PLOT columns present more compatibility with
502
LC at present. The porous nature of the stationary phase enables a larger surface area of
503
contact, which promotes a more significant interaction of the stationary phase with the
504
analytes and, consequently, increases the retention factor making the chromatographic
505
separation more efficient.
20
506
It is noteworthy that after production, PLOT columns can be evaluated by physical,
507
chemical, and chromatographic parameters. The physical parameters refer to the stationary
508
phase morphology; SEM is one of the main tools in this type of evaluation, as it provides
509
information on the thickness, uniformity, and adhesion of the stationary phase to the tube
510
wall. Fig. 7 shows three SEM images corresponding to three OT-PLOT columns prepared
511
by Hara et al. having 5 µm i.d. with a thin layer of mesoporous silica [27]. These columns,
512
produced utilizing a sol-gel process, fundamentally differ one from the other in the amount
513
of TMOS utilized. From the SEM figures, it is possible to determinate for each column the
514
layer thickness and the flow-throw diameter, as exemplified in Fig. 7A. It is also noticed in
515
this figure that the layer thickness increased with increasing the amount of TMOS
516
employed [27]. The chemical analysis aims to verify the selectivity of the developed
517
column and is performed by comparing the chromatograms resulting from the separation of
518
analytical standards injected into a regular column with those obtained by an OT-PLOT
519
column. The chromatographic analyses are performed by applying dimensionless or
520
reduced parameters such as impedance (E), reduced plate height (h), flow resistance (φ),
521
and reduced linear velocity (ʋ) (Table 1).
522
523
In the last decade, there has been significant progress in the production of OT-
524
PLOT LC columns. However, to date, there is no general protocol that can be used to
525
synthesize the coating phase utilized in such columns, in part due to the vast diversity of
526
stationary phases that can be used. Though, there are three fundamental steps in the
527
fabrication of any OT-PLOT column: (i) a pre-treatment of the inner wall of the tube,
528
followed by (ii) its silanization for use as a support for the porous layer, and (iii) the final
529
polymerization or adsorption of the stationary phase.
21
530
The pre-treatment and silanization steps refer to the preparation of the inner wall of
531
the PLOT column support assembly, which generally consists of a fused silica material.
532
These steps are significant because the characteristics of the internal wall of the capillary
533
are the governing factors in the formation of adequate immobilized stationary phases since
534
a secure connection of the stationary phase to the capillary wall is necessary - otherwise,
535
the coating bleeding will occur.
536
The polymerization or adsorption of the stationary phase into the inner wall is
537
considered as a central topic involving the development of PLOT columns at present. In
538
polymerization, a fused silica capillary is filled with a polymeric mixture, its ends are
539
sealed, and the supply of heat or light causes the polymerization reaction. Such processes
540
are respectively called thermal initiation and photoinitiation, and both give rise to
541
stationary phases linked to the capillary wall by covalent bonding.
542
OT-PLOT LC columns developed using thermal initiation are the most used, after
543
Luo et al. reintroduced the use of OT-PLOT in LC in 2007. They demonstrated the
544
development and application of a stationary phase based on poly (styrene-divinylbenzene)
545
(PS-DVB) in a 10 µm internal diameter column for peptide separation, awakening the
546
interest of other researchers [71]. More recently, Knob et al. used thermal initiation on
547
small diameter columns to make OT-PLOT analytical columns and suggested that the
548
polymeric stationary phase morphology is significantly impacted by internal diameters
549
lower than 10 µm [72].
550
The stationary phase can also be adhered to the capillary wall by adsorptive
551
processes. In these cases, they are termed as physically adsorbed stationary phases, being
552
connected, like layers, to the capillary wall through electrostatic and hydrophobic
553
interactions. Just a single layer can be adsorbed, forming a monolayer, or several
22
554
overlapping layers can be built, called the "layer by layer" (LbL) approach. The application
555
of several overlapping layers against the monolayer has the advantages of increased
556
durability, stability, and loadability.
557
Kubáň et al. have developed a column containing as a stationary phase a multilayer
558
structure of methylamine (MA) and 1,4-butanedioldiglycidyl ether (BDDE) copolymer
559
(MA-BDDE) demonstrating its usability by separating inorganic anions [73]. Another
560
example was the work reported by Wang et al. in which the researchers describe the
561
development of a stationary phase based on hydroxypropyl cellulose (HPC) monolayer in
562
an OT-PLOT column employed to separate proteins [74].
563
Depending on the nature of the polymerization reagents, the stationary phase
564
formed in PLOT columns may be organic or inorganic. Similar to monolithic columns,
565
PLOT columns made up of organic polymers are the most used in miniaturized LC. This
566
fact might be explained by considering that this type of stationary phase was the first to be
567
developed, and therefore was the most reproduced, to date. The main advantages related to
568
the use of these phases are the synthesis simplicity, control of material porosity, stability
569
against extreme pH conditions, high thermal stability, and the possibility of making
570
modifications to its surface with several active groups. As a disadvantage, the low
571
mechanical strength stands out. In turn, the stationary phases of most inorganic polymers
572
are formed by silica-based polymers. This type of stationary phase has a higher surface
573
area and mechanical resistance than must organic polymer phases. However, they are more
574
sensitive to sudden pH variations [3].
575
There are also “less conventional” stationary phases that envision the incorporation
576
of new materials capable of providing higher selectivity. The primary new materials used
577
for the preparation of OT-PLOT LC columns are based on different metal oxides, ion
23
578
exchange materials, molecularly imprinted polymers, and hybrid materials, which contain
579
both organic and inorganic characters [3].
580
Peng et al. developed a new zwitterionic stationary phase (ZIC) by thermal
581
initiation for HILIC separations [75]. Also, Aydoğan developed a stationary phase based
582
on β-cyclodextrin (β-CD) reporting enantioseparation with high theoretical plate numbers -
583
up to 26 000 plates/m [76].
584
In short, the use of OT-PLOT analytical columns in LC has not yet reached its
585
climax. However, it has shown great potential and is increasingly being the focus of the
586
interest of different research groups. There is a wide variety of new stationary phases
587
preparation techniques under development. Also, the natural compatibility for coupling
588
these miniaturized columns with MS evidences the full potential of OT-PLOT-LC-MS.
589 590
6. Wall Coated Open Tubular Columns (WCOT)
591
Another type of open tubular column is termed as "wall coated open tubular"
592
(WCOT). These columns have as a stationary phase a non-porous thin film adhered to the
593
interior of the tube, which is often made of fused silica. This coating is obtained after
594
extensive treatments of the tubing interior surface, enabling the immobilization of the film
595
on the column wall through cross-linking processes [77–79].
596
Nowadays, WCOT columns with capillary dimensions are consolidated in gas
597
chromatography and have many advantages over packed columns such as high durability,
598
superior performance, more inertness, ease of use, and from the theoretical perspective, the
599
highest chromatographic potential [80].
600
However, for miniaturized LC, there are few reports in the literature employing OT
601
columns [81–83]. One of the main reasons would be the fact that these columns present
24
602
low sample capacity because of their low surface area of contact, reducing the interaction
603
of the analyte with the stationary phase. As a consequence, it is more instrument
604
demanding than all other forms of miniaturized LC [84].
605
Although this type of column has promoted highly efficient separations in GC, it
606
has still been little exploited in miniaturized LC. WCOT columns were first successfully
607
used in LC by Hibi et al. [85] in 1978, when the authors were promoted highly efficient
608
separations of polynuclear aromatic hydrocarbons (PAHs) using 15 cm long and 0.5 mm
609
i.d. polytetrafluoroethylene (PTFE), introducing the term "micro-LC".
610
The preparation of WCOT-LC columns is meticulous and requires great care to
611
obtain a uniform stationary phase that tightly adheres to the column tube wall. The initial
612
step in preparing a WCOT column consists of leaching the fused silica capillary. This
613
process consists in preparing the capillary wall to receive the phase of interest. Leaching
614
can be done in an acidic or basic medium, and its efficiency depends on the solution
615
concentration, temperature, and treatment time. In the second stage, the tube is dehydrated
616
by heating it in an oven while passing through its interior a gentle and controlled flow of
617
nitrogen [86,87].
618
Following the silica tube treatment procedures described above, the treated tubes
619
are usually subjected to a further persilylation step using a compound capable of producing
620
the appropriate "wettability" for the subsequent coating, which will result in the formation
621
of a thin film inside of the tube. For the persilylation process, a silica capillary tubing is
622
filled with a proper silanizing solution, its ends are closed, and the capillary is placed into
623
an oven. The most used silanizing agents are hexamethyldisilazane (HMDS),
624
tetraphenyldimethyldisilazane (TPDMDS), and diphenyltetramethyldisilazano (DPTMDS).
625
After the persilylation process, the capillary is covered with the stationary phase of
626
interest. The most employed WCOT stationary phases used so far to produce WCOT-LC
25
627
columns follow the same trends used for GC, with polydimethylsiloxane (PDMS) and its
628
various modifications being the most used liquid polymer evaluated to date. By inserting
629
distinct functional groups into the pendant siloxane groups, it is possible to obtain a variety
630
of new stationary phases (modified polysiloxanes). The most frequently inserted functional
631
groups are phenyl and cyano. In addition to PDMS, other stationary phases have been
632
investigated, including GeO2-PDMS, 3-mercaptopropyltrimethoxylanosilica, polypyrrole,
633
TiO2-PDMS, and β-CD [88,89]. Besides, the sol-gel process, as well as alternative
634
procedures, may render different stationary phases for the coating process.
635
The interior wall of the tube can be covered with the stationary phase in two
636
different ways: static coating or dynamic coating. In the dynamic process, the stationary
637
phase solution containing the stationary phase is pushed with the aid of gas through the
638
capillary tube, forming a thin film on the tube wall. Although it is an easy and
639
straightforward technique, it results in a smaller phase ratio. In the static coating, the tube
640
is kept at a high temperature so that the phase solution solvent evaporates, and steam is
641
displaced out of the column with the aid of vacuum exerted at one end of the column [86].
642
The static coating procedure has gained prominence and is the most used coating
643
type, allowing the use of higher molar mass phases. Moreover, with this methodology, it is
644
possible to more accurately predict the thickness of the stationary phase film by varying
645
the concentration of the polymer in solution.
646
After the coating process, a cross-linking step is performed to immobilize the
647
stationary phase on the tubing wall, preventing its detachment during the chromatographic
648
run. The cross-linking process is usually done by using compounds such as azo-tert-butane
649
(ATB) or azo-tert-octane (ATO), among others.
26
650
Figure 8 shows a schematic drawing representing the capillary filling system with
651
the solutions of interest (Fig. 8A), coating tube set up (Fig. 8B), and the static coating
652
resulting process inside the tube of a capillary column (Fig. 8C).
653 654
655
Table 2 shows the characteristics of some commercially available stationary phases
656
derived from polydimethylsiloxane, which are the most used materials to prepare WCOT
657
columns to date.
658
S0165-9936(19)30120-7
DOI:
https://doi.org/10.1016/j.trac.2019.115735
Reference:
TRAC 115735
To appear in:
Trends in Analytical Chemistry
Received Date: 7 March 2019 Revised Date:
13 November 2019
Accepted Date: 14 November 2019
Please cite this article as: K. Mejía-Carmona, J. Soares da Silva Burato, J.V. Basolli Borsatto, A. Lúcia de Toffoli, F.M. Lanças, Miniaturization of liquid chromatography coupled to mass spectrometry. 1. Current trends on miniaturized LC columns Trends in Analytical Chemistry, https://doi.org/10.1016/ j.trac.2019.115735. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.
1
Miniaturization of liquid chromatography coupled to
2
mass spectrometry.
3
1. Current trends on miniaturized LC columns
4 5
Karen Mejía-Carmona, Juliana Soares da Silva Burato, João Victor Basolli Borsatto, Ana
6
Lúcia de Toffoli and Fernando Mauro Lanças*
7 8
University of São Paulo, São Carlos, Institute of Chemistry of São Carlos, SP, Brazil
9 10
*Corresponding author. Tel: +(55) 16 3373 9983; Fax: +(55) 16 3373 9984
11
E-mail address: [email protected] (F. M. Lanças)
12 13 14
Highlights
15
•
Recent advances in miniaturized liquid chromatography
16
•
New materials employed for capillary columns separations
17
•
Recent advances in the development of capillary columns for miniaturized liquid
18
chromatography
19 20
Contents
21
1. Introduction
22
2. Theoretical aspects
23
3. Packed columns
24
4. Monolithic columns
25
5. Porous Layer Open Tubular Columns (OT-PLOT)
26
6. Wall Coated Open Tubular (WCOT) Columns
27
7. Recent applications of miniaturized LC
28
8. Concluding remarks
1
29
Abstract
30
Over the last decade, the use of miniaturized columns in liquid chromatography (LC) has
31
seen intense growth in a wide range of research areas. This report brings an overview of
32
the current developments and applications of miniaturized columns in LC, focused on the
33
type of columns used. Following a short introductory section on some theoretical aspects,
34
the fundamental bases on their design and fabrication, new stationary phases and
35
incorporated materials, performance evaluation/characterization, main advantages, and
36
recent applications of filled capillary and open tubular (OT) capillary columns are
37
introduced and critically discussed. Prospects on the different types of miniaturized
38
columns and their hyphenation with mass spectrometry (MS) reported in the last years are
39
also introduced and discussed.
40 41
Keywords: miniaturized liquid chromatography, particle packed capillary column,
42
monolithic capillary columns, open-tubular columns, wall coated open-tubular columns,
43
porous layer open tubular columns, mass spectrometry.
44 45
Abbreviations
46 47 48 49 50 51 52 53 54 55 56 57 58 59
β-CD, β-cyclodextrin; capillary-LC, capillary liquid chromatography; CLSM, confocal laser scanning microscopy; CTCs, circulating tumor cells; dp, particle diameter; ESI, electrospray ionization; EI, electron ionization; FP, fully porous particles; GC, gas chromatography; h, reduced plate height; H, plate height; HILIC, hydrophilic interaction chromatography; HIC, hydrophobic interaction chromatography; HPLC, high-performance liquid chromatography;
2
60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79
i.d., inner diameter; KPL, kinetic performance limits plots; LC, liquid chromatography; MS, mass spectrometry; MS/MS, tandem mass spectrometry; nano-ESI, nano-electrospray ionization; nano-LC, nano liquid chromatography; NP, non-porous particles; OT, open tubular; PDMS, polydimethylsiloxane; PLOT, porous layer open tubular; SEM, scanning electron microscopy; SP, superficially porous particles; TMOS, tetramethoxysilane; UHPLC, ultra-high pressure liquid chromatography; υ, reduced linear velocity; WCOT, wall coated open tubular;
1. Introduction
80
Liquid chromatography (LC) has become a widespread analytical technique used in
81
many fields of natural and health sciences [1–3]. Following the current trends in analytical
82
chemistry, miniaturization is among the main LC innovation areas along the last ten years
83
[4].
84
Horvárth et al. [5] were one of the pioneers in LC miniaturization. In 1967 they
85
compared a stainless-steel uncoated capillary tubing of inner diameter (i.d.) of 0.3 mm with
86
a 50 µm pellicular particle packed capillary of 1.0 mm i.d. for the separation of
87
nucleotides. This study drove the authors to the conclusion that packed small-bore
88
capillaries were more efficient than uncoated capillary ones. One decade later, Tsuda and
89
Novotny [6,7] established the required equipment for working with capillary LC, by
90
miniaturizing the overall capillary LC system. They diminished the column i.d. to 50–200
91
µm, and the particle size down to ~30 -10 µm, as well as have implemented modifications
92
on conventional injection and detector systems aiming to reduce the band-broadening
3
93
effects. At the same time, Ishii et al. [8] worked in the development of a micro-high
94
performance liquid chromatography (MHPLC) system, successfully applied to both packed
95
[9] and open tubular (OT) capillary columns. These seminal works were an essential
96
contribution that allowed miniaturized LC to significantly advance in the 80s, along with
97
other
98
chromatography, waking up the interest of researchers from different fields [10]. Recently,
99
Novotny et al. [10] reported a comprehensive review of the historical aspects, advances,
100
techniques
as
capillary
electrophoresis
and
capillary
supercritical
fluid
and efforts of capillary LC developments from the beginning until the present.
101
Nowadays, the separation in miniaturized LC is performed in capillary columns
102
similar to those used in high-resolution gas chromatography. As a result of all the
103
developments made in miniaturized LC, it is possible now to find commercially available
104
capillary and nano liquid chromatography systems (capillary-LC and nano-LC), including
105
–and principally– using ESI-MS as a detector.
106
One of the most important advantages offered by miniaturized LC is the viability to
107
work in applications were just a small sample volume may be collected and analyzed,
108
covering areas such as forensic, clinical, and omics. Besides, the low flow rate employed
109
means lower solvent consumption, allowing the use of specialized solvents, and leading to
110
enhanced detection as a result of the lower chromatographic dilution [11,12].
111
Miniaturized LC is a general term employed for describing liquid chromatography
112
using i.d. columns below to 1.0 mm. In practice, to make an accurate description of this
113
approach, it is necessary to consider both the internal column diameter and the mobile
114
phase flow rate used [13]. In general terms, for practical purposes, capillary-LC employs
115
capillary columns with i.d. between 100–500 µm and flow rates of 1–10 µL/min, while
4
116
nano-LC employs capillary columns with i.d. < 100 µm with flow rates from 10 to 1000
117
nL/min [2].
118
The design of columns has been one of the main drivers on LC miniaturization, as
119
exposed by Desmet et al. [11]. There are two main types of capillary columns: filled
120
capillary columns and open tubular capillary columns (Fig. 1). The former can be filled
121
with a particle bed or a monolith, and the second has a thin layer of stationary phase
122
attached to the inner surface of the column, leaving an open tubular space.
123
124
Particle packed capillary columns commonly use the same particles employed in
125
conventional HPLC columns; silica-based particles are the most employed. Silica particles
126
can be prepared in different ways, producing unique characteristics, resulting in a wide
127
range of packed capillary columns commercially available [12]. In LC, it is known that
128
reducing the particle size and the internal diameter of columns improves the separation
129
performance; the use of sub-2 µm particles enables greater efficiency at faster flows,
130
reducing analysis time [14]. Nevertheless, this advantage is limited by the high back-
131
pressure generated, requiring specialized instrumentation similar to that used in ultra-high
132
pressure liquid chromatography (UHPLC) [15]. Additional information about particles and
133
support materials, as well as packing capillary columns methods, can be found in several
134
reviews [2,14–17].
135
Monolithic capillary columns - another way to prepare filled columns - since their
136
introduction in the early 90s, have gained particular interest because of their easy in-situ
137
preparation and high permeability that decrease the back-pressure when compared to
138
particle packed capillary columns. A monolithic column consists of a continuous
139
polymeric block structure made from either silica-based or organic-based polymers that fill
5
140
the whole capillary tube [1]. Capillaries with i.d. ranging from 20–500 µm are commonly
141
employed. The efficiency of monolithic columns depends on the stationary phase
142
morphology, macropore size, uniform skeleton, and kind of polymers employed.
143
Improvements in the efficiency –similarly by that obtained by the reduction particle sizes
144
in packed columns– can be achieved by controlling both the external porosity and pore size
145
[14]. Currently, the synthesis of a wide range of monolithic materials containing metallic
146
nanoparticles, metal-organic frameworks, carbon-based nanomaterials, as well as
147
monoliths functionalized with boronic acid, zwitterionic monomers and other
148
functionalities [18], extended the range of applications of capillary LC.
149
On the other hand, open tubular (OT) columns, firstly introduced for gas
150
chromatography (GC) in 1958 by Golay [19], became an unique type of column which
151
have a thin layer of stationary phase on the inner wall of the capillary. By the late 1970s,
152
Tsuda et al. [20] explored the OT concept in LC studies, including the design, theory, and
153
practical aspects of the technique. However, the research interest decayed quickly, mainly
154
due to its lower performance compared to particle packed columns, and the lack of suitable
155
miniaturized instrumentation and detectors at the time. Recently, with the commercial
156
availability of highly sensitive detectors, reduced volume injectors, and micro and
157
nanoflow LC instruments, the interest in applications and fundamental research in OT
158
columns emerged again.
159
In contrast to particle packed columns, the most critical advantages of OT columns
160
is the exclusion of eddy dispersion contribution –counting roughly to one-half of band-
161
broadening in packed columns– and their high permeability, which allows the use of longer
162
columns [11]. However, to achieve a high chromatographic efficiency are required
163
columns with downsized inner diameters (~5 µm), a fact that severely compromises the
6
164
backpressure and column length [21]. OT columns are classified into two main types: wall
165
coated open tubular (WCOT), and porous layer open tubular (PLOT).
166
Although the former is the most employed in GC, consisting of a thin cover film of
167
non-porous stationary phase, WCOT columns were poorly explored in LC because of their
168
lower sample capacity and efficiency, mainly due to their non-porous film. Later efforts
169
aiming to obtain thicker films and increase their sample capacity were made, employing
170
multilayer coatings and unique chemical bonding processes. Despite this, WCOT columns
171
were overshadowed by a new type of open tubular LC column termed PLOT columns,
172
once they showed to be superior to the LC purposes at that time [1]. In practice, LC-type
173
WCOT capillary columns have been to the moment employed more successfully in
174
capillary electrochromatography (CEC) than in micro and nano-LC [22].
175
In a PLOT column, also initially developed for GC, the stationary phase is a porous
176
material that offers a higher surface area and better retention capacity compared to WCOT
177
columns. Among the stationary phases employed, organic polymeric or silica polymeric-
178
based materials are the most utilized. Polystyrene (PS), polydivinylbenzene (DVB), and
179
polymethacrylate (PMMA) are the current phases applied to proteomic analyses. Thin
180
layer polymeric stationary phases are usually deposited by the free-radical polymerization
181
method, either by thermal or photo-initiation approaches, yielding several layer structures
182
and morphologies by modifying such as the energy supplied through the polymerization
183
time [3,23,24].
184
The last generation of silica-based PLOT columns was recently obtained by the sol-
185
gel method, as reported by Forster et al. [25]. They employed a solution of
186
tetramethoxysilane (TMOS), urea and polyethylene oxide (PEO) as progeny agent in
187
preparing fused silica capillaries of 10–100 µm i.d. The authors obtained PLOT columns
7
188
with a good mass loadability that provided high-efficiency separations (>170,000 plates),
189
successfully applied to normal phase separations [25,26]. Recently, Hara et al. [27]
190
demonstrated that capillary columns with 5 µm of i.d. coated with mesoporous silica, 550
191
nm layer width, and 2.6 m length, achieved ~950,000 plates.
192
In this first review of a series on coupling miniaturized LC columns to mass
193
spectrometry (MS), we provide an overview of the recent advances on capillary-LC and
194
nano-LC, focused on the capillary columns, stationary-phases synthesis and packing
195
methods, and their recent applications with particular emphasis on the coupling to mass
196
spectrometry (MS). In a second review, we will discuss the miniaturized instrumentation,
197
hyphenation with mass spectrometry, and the current status of chip-based systems.
198 199
2. Theoretical aspects
200
The behavior of the analyte's molecules inside a column and how it affects the
201
analysis performance have been one of the most important topics to be understood in all
202
chromatography techniques. The main fundaments were first developed for GC and further
203
transferred to LC. Among the researchers that have developed the theory of
204
chromatography in the last century, J. J. van Deemter and coworkers receive the main
205
attention. In their work, the authors described how the molecules of analytes are distributed
206
along the column during the separation based on three terms [28]. The first term (A-term),
207
also called eddy diffusion, demonstrates how the molecules of analytes spread by flowing
208
through the column. It is independent of the flow but depends on the column morphology
209
(particle diameter (dp) for packed columns and i.d. for OT columns, as an example). The
210
second term (B-term) is related to the longitudinal diffusion and depends on the diffusion
211
coefficient of the mobile phase, but also on the flow rate. The last term, (C-term) called the
212
mass transfer resistance, depends on the transference of the analytes between the mobile 8
213
and stationary phase and presents a linear dependence on the flow rate. Those terms were
214
used to correlate the plate height and the flow rate of analysis generating Equation 1 (Table
215
1). The plate height (H) (Equation 2, Table 1), an important metric to be considered in the
216
evaluation of the separation performance, is defined as the rate of increase of the peak
217
variance per unit of length of the column. The lower the H, the better the separation
218
performance. The van Deemter equations not only brought a deeper view of the
219
chromatography fundamentals but also resulted in a powerful tool to evaluate the
220
performance of the columns. The so-called van Deemter plots have been applied for
221
generations of scientists to evaluate the performance of the chromatographic separation in
222
combination with other graphical representation methods to be mentioned later in this
223
topic.
224
< Table 1>
225
Giddings proposed dimensionless metrics in order to correlate the performance of
226
different forms of chromatography [29]. The dimensionless metrics were denominated as
227
reduced terms. The reduced plate height (h) and the reduced linear velocity (υ) take into
228
account the particle diameters of the packing material (Equations 5 and 6, Table 1). Knox
229
and Parcher [30] proposed that by plotting log h as a function of log υ, a powerful tool to
230
assay the packing quality would be available. This relationship, known latter as the Knox
231
equation (equation 4, Table 1), correlated the h and the υ in a way similar to the van
232
Deemter equation. The major difference between the equations is related to the dependence
233
of the multipath term on the υ (see equations 1 and 4 to compare). At low υ values, the
234
multipath diffusion tends to have a lower impact on h until it reaches a constant value at
235
high υ values. The longitudinal diffusion and the mass transfer resistance present the same
236
behavior as proposed by van Deemter.
9
237
Recently, the kinetic plots are receiving more attention in order to characterize
238
columns and separation performances. A kinetic plot is a graphical representation method
239
that expresses a metric of separation quality (efficiency, peak capacity, resolution, and
240
others) as a function of a metric of time (retention time, analysis time, dead time, and
241
others). The initial concepts of kinetic plots were proposed by Giddings in his work
242
comparing the theoretical efficiency limits of GC and LC [31], which was followed by a
243
report by Poppe in the 90's, proposing the use of the now called Poppe plots to correlate
244
speed and efficiency of the modern liquid chromatography techniques [32]. The kinetic
245
plots started to be used as a valuable tool to evaluate separation and assay column
246
performance in the works reported by Desmet [33,34]. The kinetic plots can be applicable
247
in both isocratic and gradient analysis; due to this reason, it presents a series of uses and
248
applications [35–37]. Among the main kinetic plots used today, kinetic performance limits
249
plots (KPL) receives particular attention. This type of plot represents an imaginary set of
250
columns of various lengths operating under conditions in which their performances are
251
maximum [38]. Although the KPL plot represents an imaginary set of columns, it is
252
constructed using experimental data transformed by a correction factor (see Table 1,
253
equations 6 to 11 to the more usual kinetic plots metrics). The correction factor (Equation
254
7, Table 1) takes into account the pressure drop of the column, which makes it useful to
255
compare columns of different lengths, diameters, particle diameters, and even filled with
256
different supports.
257
Another critical factor for capillary columns is the internal diameter of the
258
capillary. Although it has no direct effect on the interaction with the analyte, smaller
259
diameters result in higher linear velocities because the smaller cross-section forces the flow
260
to be more intense. The column i.d. effect has been a focus of the investigation since the
261
beginning of the instrumentation development for chromatography. It was observed that
10
262
when the size is reduced to the capillary and nanoscale, the i.d. influences the column
263
performance (Fig. 2A).
264
265
Bruns and coworkers [39,40] published a series of papers on the evaluation of
266
column porosity by using confocal laser scanning microscopy (CLSM) and packing profile
267
of the particles close to the wall, and its consequences over the separation. The observation
268
showed that the i.d. of the capillary affects the packing porosity closer to the wall.
269
Columns with larger i.d. presented higher porosity on the wall region and consequently
270
lower performance. Adversely, smaller columns i.d. presented lower porosity and better
271
results (Fig. 2B). The CLSM also demonstrated that columns of smaller i.d. presents more
272
homogenous packing than larger i.d. columns [41]. Additionally, Gritti has demonstrated
273
that for certain combinations of i.d. and dp, some conditions of poor packing are formed,
274
reducing the efficiency (Fig. 2C) [42].
275
The thermal effects are also minimized in the miniaturized packed columns. The
276
viscous heating, generated by the friction of the mobile phase with the particles, is easily
277
dissipated in capillary columns. The heating distribution inside the column is not
278
homogenous; the center of the column becomes hotter than the walls, generating band
279
dispersion. For columns packed with particles of dp = 1.0 µm operating at 2000 bar, a
280
regular column of 4.6 mm i.d. produces 2.8 W (at 820 µL/min) of heating. For the same
281
conditions, a capillary column of 0.1 mm i.d. generates 1.3 mW (at 380 nL/min) and a
282
nano column of 0.05 mm i.d. results in only 0.33 mW (at 96 nL/min) of power dissipation
283
[43].
284
Miniaturized liquid chromatography has as its most notable advantage over
285
conventional HPLC, better compatibility with MS, especially the electrospray ionization
11
286
(ESI) interface. The small column diameters allow reduced mobile phase flow rates, which
287
are more compatible with the optimal values of flow for the ESI-MS. The sampling
288
efficiency increases significantly at low flow rates for ESI sources (Fig. 2D) [44].
289
Additionally, works have been carried out to allow the coupling of miniaturized LC with
290
electron ionization (EI) sources, which is practically impossible to be achieved with
291
conventional LC due to the high flow rates used and the back-pressures generated [45].
292 293
3. Packed columns
294
Packed columns represent the most significant slice of columns used in liquid
295
chromatography, being the most popular kind of commercial column available in all scales
296
(conventional and miniaturized LC). Other types of LC columns, such as the monolithic
297
and OT columns are still in the state-of-the-art; its main characteristics are described and
298
explored in the following sections of this review. Packed LC columns can be obtained in a
299
large variety of stationary phases commercially available, operating in several
300
chromatographic separation modes, including (i) reversed-phase, (ii) normal phase, (iii)
301
hydrophilic
302
chromatography (HIC), (v) ion-exchange chromatography, and others varieties. The
303
primary particles used in LC can be produced using silicates-like structures generating
304
phases such as C18, C8, cyano, amino, and bare silica particles, or be produced as organic
305
resins. The particles' morphology can be divided into three types: (i) the fully porous (FP)
306
particles, which is, as its name describe, a fully porous sphere; (ii) the superficially porous
307
(SP) particles, which is a sphere of solid (non-porous) core surrounded by a thin porous
308
layer; and (iii) the non-porous particles (NP), that is a non-porous sphere of the stationary
309
phase. Majors summarized those and other more types of column packing for LC [46].
interaction
chromatography
(HILIC),
(iv)
hydrophobic
interaction
12
310
A significant advancement in columns for LC was the optimization of the packing
311
material, in particular, the reduction of particle size. The reduction in particle diameter dp
312
resulted in a more significant performance incensement due to the considerable
313
improvement in the mass transfer between the phases and the reduction in trans-particle
314
and trans-channel diffusions. On the other hand, these small dp demands a higher mobile
315
phase pressure to percolate along its packed bed [47]. To overcome the limitation of the
316
higher back-pressure produced by the stationary phase, more powerful equipment were
317
developed, resulting in the UHPLC concept. Some commercially available models can
318
withstand pressures higher than 1500 bar.
319
The SP particles, also known as core-shell, porous-shell, or fused-core particles,
320
were developed as an alternative to the UHPLC system. The presence of a solid core
321
reduces the trans-porous diffusion because the porous layer is thinner than in the FP
322
particles. This reduction in the porous structure allows to bigger SP particles to reach
323
comparable efficiency as smaller FP particles. NP particles are also availed but are less
324
used than FP and SP particles.
325
The packed columns are composed of three main parts: (i) the column tubing, (ii)
326
the packed stationary phase, and (iii) the frits (responsible for retaining the stationary phase
327
inside the column). The type of frit used affects the separation performance and detection
328
possibilities. Franc and coworkers have evaluated the performance of several types of frit
329
for capillary columns produced with different materials in columns of 0.32 and 0.25 mm
330
i.d. [48]. Four different types of frits were produced (Fig. 3). The type A frit was composed
331
of a glass wool filter frit placed inside the column capillary and secured by a smaller id
332
outlet capillary emended with epoxy resin. The type B frit used the same glass wool frit but
333
secured by a polyether ether ketone (PEEK) union. The type C frit was produced by
334
placing a glass wool frit, stainless steel, or titanium frit inside the union and then secure the
13
335
frit by pressing the capillary. Type D was produced with different monolithic frits, which
336
allows on-column detection. It was concluded that both the material and the frit type affect
337
the separation efficiency significantly and must be taken into account when preparing the
338
column based on the experiment target. For an analysis not demanding on-column
339
detection, the type A frit is recommended. The coupling of packed capillary columns to
340
MS detection requires a capillary tubing connecting the column outlet to the MS inlet. A
341
highlighted work concerning capillary columns is presented by Santos-Neto and
342
coworkers, who used a fully operational system consisting of a capillary column switching
343
restricted-access media-liquid chromatography-electrospray ionization-tandem mass
344
spectrometry system to analyze drugs in biofluids [49]. The use of nano-LC columns
345
coupled with MS has increasing relevance in the field of metabolomics; Chetwynd and
346
David summarized several examples in a recent review [50].
347
348
The interface between miniaturized columns and MS is so relevant that several
349
groups (and companies) are making substantial efforts to produce LC chips that replace the
350
conventional columns [51]. Independently of the company that produces, the chip column
351
has as an essential characteristic, its placement very close to the ionization source (and in
352
some cases, it may contain an ESI tip) [52,53]. This approach is used to reduce the post-
353
column band diffusion significantly.
354
The packing procedure well performed is vital to ensure highly efficient columns.
355
The most used type of column packing procedure is the slurry packing, which consists of a
356
dispersion of the stationary phase in a solvent (the slurry solvent) been flushed through the
357
column. The slurry solvent nature and slurry concentration play an essential role in column
358
packing. The nature of the solvent used affects the distribution of the particles in the slurry.
359
It was found that the most aggregated solution produces the most efficient columns for the
14
360
packing of 0.03 mm i.d. columns with particles of 1.1 µm dp [54]. Similar results were
361
found by Wahab and coworkers [16].
362
Different packing approaches are employed to pack columns using slurry solutions.
363
The simplest, but very effective system, is prepared in a set up very similar to the one
364
employed for packing regular HPLC columns. In this setup, the slurry reservoir is
365
connected directly to an air driven-liquid pump, and the column is connected to the slurry
366
reservoir by using an empty capillary tube (Fig. 4A). Though it is expected to produce
367
pressure pulses during the packing, the columns produced by this system result in efficient
368
columns [49,55]. An alternative set up was proposed for packing capillary columns,
369
avoiding pressure pulses (Fig. 4B). In this setup, the slurry is placed in the chamber and
370
stirred during the application of the high pressure; a microscope can be used to evaluate the
371
packing process [16]. Berg and coworkers used a similar approach in which the chamber is
372
filled with the slurry solution, but nitrogen gas is used to flow the material through the
373
capillary [56].
374
375 376
Dry packing and supercritical fluid packing are also alternatives to pack columns.
377
The dry packing procedure is straightforward; the particles are deposited in the reservoir,
378
and then a gas (usually nitrogen) is flushed through the system resulting in the packed bed
379
[57]. The supercritical fluid packing combines the best of the slurry and dry packing
380
methods, using supercritical CO2 as a solvent. The setup of supercritical fluid packing is
381
different from dry or slurry packing because the outlet of the column is connected to a
382
vacuum pump [58]. Other possibilities of packing procedures are described and discussed
383
by a practical viewpoint by Lanças and coworkers [17].
15
384
Alternatives to packing are also presented in the literature. The pillar array columns
385
(µPAC) are produced by milling pillars on silicon, followed by the functionalization with
386
the stationary phase. The significant advantage of this layout is that the milled pillars result
387
in a high ordinated filling, which allows a more homogenous flow inside the column and a
388
reduction on the multipath diffusion [59,60]. 3D printing has also been investigated as a
389
way to produce ordinated fillings for columns. Although it seems to be promissory, mainly
390
because they use of 3D manufacturing allows the construction of different geometry for the
391
inner part of the column, the instrumental (3D printers) limitations are still the bottleneck
392
for this use [61].
393 394
4. Monolithic Columns
395
Monolithic columns consist of a small i.d. tubing, all filled with a robust porous
396
polymer. The polymeric phase consists of pores of different sizes that provide retention
397
and separation selectivity. The porous skeleton of the monolithic columns gives the mobile
398
phase high permeability and low back pressure, which enables the use of high flow rates
399
allowing faster mass transport when compared to particle-filled columns. Therefore, the
400
central positive aspect claimed in favor of monolithic columns is their ability to maintain a
401
proper separation efficiency at high flow rates [62].
402
The monolithic columns stationary phases are made of polymers that may have
403
inorganic, organic, or hybrid characteristics. Silica monoliths from inorganic stationary
404
phases have a porosity higher than 80%, resembling silica particle packed columns. Thus,
405
the main advantages related to the use of silica monoliths are the high separation efficiency
406
associated with low backpressure. Also, the main disadvantage is the sensitivity to pH
407
variations characteristic of silica-based materials.
16
408
A general procedure for producing monolithic columns with silica polymer phase
409
employs the sol-gel approach, consisting in the hydrolysis and polycondensation processes
410
catalyzed by tetraalkoxysilanes acids in the presence of an inert compound that acts as a
411
porogenic agent. Such a reaction results in a continuous silica mesh with a defined pore
412
structure that can be modified according to the reactant concentration, temperature, and
413
polymerization time. It is also possible to perform interventions on the surface of the
414
polymeric phase through washing with solutions containing modifiers, in order to improve
415
selectivity and extend the application of these columns [63].
416
Due to the high porosity characteristics of silica monoliths, these phases are ideal
417
for the separation of small molecules. Thus, monolithic columns with inorganic stationary
418
phases are generally used in the separation of environmental, forensic, natural products,
419
and pharmaceutical samples, among others [64].
420
Another aspect that stands out in the use of monolithic columns containing
421
inorganic stationary phases is their high efficiency at flow rates higher than the
422
theoretically expected value, without loss of separation efficiency. This ability to perform
423
quick separations qualifies it to be used for multidimensional LC separations, proving to be
424
an excellent tool for separating complex samples. As an example, Espina-Benitez et al.
425
developed an in-line coupling of a monolithic boronate extracting column with a
426
monolithic silica-based analytical column to analyze uridine, cytidine, adenosine, and
427
guanosine. The monolithic silica analytical column had its activated surface functionalized
428
by thiol-one photo click [65].
429
In turn, organic monoliths are extensively used due to their qualities, such as easier
430
preparation and stability at high temperatures and pH variations. As a disadvantage, the
431
low mechanical strength stands out.
17
432
The general procedure for producing an organic polymeric phase for monolithic
433
columns is to subject a solution containing functional organic monomers, porogenic
434
solvents, and a radical initiator to either heat or a light source to release free radicals which
435
will trigger the monolith formation. The morphology of the formed monolith is a
436
consequence of the reagent concentration, temperature or light intensity, and
437
polymerization time. As in silica monoliths, organic monoliths can be surface modified to
438
increase stationary phase selectivity. It is noteworthy that, besides the mentioned thermal
439
initiation method, there are also other techniques such as polycondensation, emulsion
440
polymerization, and ring-opening metathesis [66].
441
Organic monoliths have a nonporous granular characteristic interspersed with large
442
pores, as can be observed by scanning electron microscopy (SEM) (Fig. 5) [67]. In this
443
type of monolith, the pores are too miniaturized, making it impossible to separate small
444
molecules. Thus, this stationary phase is ideal for the separation of large molecules, such
445
as proteins and other biomolecules, as the passage pores retain these analytes. However,
446
there are scientific advances aimed at the development of organic monoliths capable of
447
separating small molecules. Initial efforts are focused on pore morphology changes
448
through studies that adapt the polymerization time and reagent concentration. Also, post-
449
polymerization modifications and the addition of nanostructures to the polymer may be
450
performed [68].
451
452
Echevarría et al. developed monolithic capillary columns with organic stationary
453
phase coated with tris cellulose (3,5-dimethyl phenyl carbamate) for the separation of
454
several compounds such as β-blockers and pesticides. The synthesis of the stationary phase
455
was done by thermal polymerization of 2-hydroxyethyl methacrylate and ethylene glycol
456
dimethacrylate in the presence of a porogenic mixture [69].
18
457
Monolithic columns consisting of hybrid stationary phase combine the advantages
458
shown by organic polymer-based and silica-based monolithic columns. As such, they have
459
a high surface area and mesoporous structure, such as the inorganic ones, and stability at a
460
higher temperature and pHs, like organic ones, still retaining the general advantages of
461
monolithic columns that are low flow resistance. Sol-gel or ring-opening methodologies
462
can prepare most such monoliths, which have a wide range of applications depending on
463
the analytes of interest.
464
Recently, Zhao et al. used a monolithic column consisting of hybrid characteristics
465
aiming at the separation of inorganic arsenic. The thiol functionalized organic-inorganic
466
phase was synthesized using a unique ternary weak basic solvent system by a sol-gel
467
process [70]. Other application examples encompassing inorganic, organic, and hybrid
468
monoliths have been selected and are included at the application table in the following
469
sections.
470
Monolithic columns, after its laboratory preparation, can be evaluated by physical,
471
chemical, and chromatographic means. The phase characterization is made by physical
472
evaluation through SEM photos and methods able to evaluate the porosity and dimensions
473
of the polymers. In turn, the chemical and chromatographic analyses are obtained through
474
chromatographic runs to evaluate the selectivity and separation power of the produced
475
column.
476
The use of capillary monolithic columns in LC is growing and is being established
477
as a great alternative to packed columns, as they often have superior results. The main
478
advantages of both organic and silica polymeric stationary phases for monolithic columns
479
are its ease synthesis, rapid analysis, no need for use frits, and a variety of stationary
480
phases that can be available. The existence of mass spectrometry coupled micro/nano-LC
19
481
systems also represents a gain for miniaturized monolithic columns to analyze samples
482
with small volumes and excellent detection efficiency [62]. Therefore, it is expected that
483
the miniaturized monolithic column in LC will continuously be further explored, and
484
phases will continue to be developed in order to increase the selectivity of analyzing
485
specific compounds.
486 487
5. Porous Layer Open Tubular Columns (OT-PLOT)
488
The technological advances in the miniaturization of LC and the commercial
489
availability of adequate instrumentation made it possible to explore the use of OT
490
analytical columns, allowing its successful coupling to mass spectrometry using nano-
491
electrospray ionization (nano-ESI) or electron ionization (EI) sources.
492
The use of open tubular columns in LC is theoretically advantageous compared to
493
the use of filled columns. Desmet et al. [11] comparing the kinetic efficiency of OT-PLOT
494
columns with packed columns found that at any retention time, OT analytical columns
495
have at least ten times more theoretical plates, as can be seen in Fig. 6. Other advantages of
496
using OT-PLOT LC over conventional LC columns include the ability to use higher flow
497
rates without significant column pressure increase; column efficiency increase; lower
498
mobile phase and sample consumptions; lower waste generation –all in accordance to the
499
green chemistry principles– and ease of coupling to MS [3].
500
501
Among the open tubular columns, PLOT columns present more compatibility with
502
LC at present. The porous nature of the stationary phase enables a larger surface area of
503
contact, which promotes a more significant interaction of the stationary phase with the
504
analytes and, consequently, increases the retention factor making the chromatographic
505
separation more efficient.
20
506
It is noteworthy that after production, PLOT columns can be evaluated by physical,
507
chemical, and chromatographic parameters. The physical parameters refer to the stationary
508
phase morphology; SEM is one of the main tools in this type of evaluation, as it provides
509
information on the thickness, uniformity, and adhesion of the stationary phase to the tube
510
wall. Fig. 7 shows three SEM images corresponding to three OT-PLOT columns prepared
511
by Hara et al. having 5 µm i.d. with a thin layer of mesoporous silica [27]. These columns,
512
produced utilizing a sol-gel process, fundamentally differ one from the other in the amount
513
of TMOS utilized. From the SEM figures, it is possible to determinate for each column the
514
layer thickness and the flow-throw diameter, as exemplified in Fig. 7A. It is also noticed in
515
this figure that the layer thickness increased with increasing the amount of TMOS
516
employed [27]. The chemical analysis aims to verify the selectivity of the developed
517
column and is performed by comparing the chromatograms resulting from the separation of
518
analytical standards injected into a regular column with those obtained by an OT-PLOT
519
column. The chromatographic analyses are performed by applying dimensionless or
520
reduced parameters such as impedance (E), reduced plate height (h), flow resistance (φ),
521
and reduced linear velocity (ʋ) (Table 1).
522
523
In the last decade, there has been significant progress in the production of OT-
524
PLOT LC columns. However, to date, there is no general protocol that can be used to
525
synthesize the coating phase utilized in such columns, in part due to the vast diversity of
526
stationary phases that can be used. Though, there are three fundamental steps in the
527
fabrication of any OT-PLOT column: (i) a pre-treatment of the inner wall of the tube,
528
followed by (ii) its silanization for use as a support for the porous layer, and (iii) the final
529
polymerization or adsorption of the stationary phase.
21
530
The pre-treatment and silanization steps refer to the preparation of the inner wall of
531
the PLOT column support assembly, which generally consists of a fused silica material.
532
These steps are significant because the characteristics of the internal wall of the capillary
533
are the governing factors in the formation of adequate immobilized stationary phases since
534
a secure connection of the stationary phase to the capillary wall is necessary - otherwise,
535
the coating bleeding will occur.
536
The polymerization or adsorption of the stationary phase into the inner wall is
537
considered as a central topic involving the development of PLOT columns at present. In
538
polymerization, a fused silica capillary is filled with a polymeric mixture, its ends are
539
sealed, and the supply of heat or light causes the polymerization reaction. Such processes
540
are respectively called thermal initiation and photoinitiation, and both give rise to
541
stationary phases linked to the capillary wall by covalent bonding.
542
OT-PLOT LC columns developed using thermal initiation are the most used, after
543
Luo et al. reintroduced the use of OT-PLOT in LC in 2007. They demonstrated the
544
development and application of a stationary phase based on poly (styrene-divinylbenzene)
545
(PS-DVB) in a 10 µm internal diameter column for peptide separation, awakening the
546
interest of other researchers [71]. More recently, Knob et al. used thermal initiation on
547
small diameter columns to make OT-PLOT analytical columns and suggested that the
548
polymeric stationary phase morphology is significantly impacted by internal diameters
549
lower than 10 µm [72].
550
The stationary phase can also be adhered to the capillary wall by adsorptive
551
processes. In these cases, they are termed as physically adsorbed stationary phases, being
552
connected, like layers, to the capillary wall through electrostatic and hydrophobic
553
interactions. Just a single layer can be adsorbed, forming a monolayer, or several
22
554
overlapping layers can be built, called the "layer by layer" (LbL) approach. The application
555
of several overlapping layers against the monolayer has the advantages of increased
556
durability, stability, and loadability.
557
Kubáň et al. have developed a column containing as a stationary phase a multilayer
558
structure of methylamine (MA) and 1,4-butanedioldiglycidyl ether (BDDE) copolymer
559
(MA-BDDE) demonstrating its usability by separating inorganic anions [73]. Another
560
example was the work reported by Wang et al. in which the researchers describe the
561
development of a stationary phase based on hydroxypropyl cellulose (HPC) monolayer in
562
an OT-PLOT column employed to separate proteins [74].
563
Depending on the nature of the polymerization reagents, the stationary phase
564
formed in PLOT columns may be organic or inorganic. Similar to monolithic columns,
565
PLOT columns made up of organic polymers are the most used in miniaturized LC. This
566
fact might be explained by considering that this type of stationary phase was the first to be
567
developed, and therefore was the most reproduced, to date. The main advantages related to
568
the use of these phases are the synthesis simplicity, control of material porosity, stability
569
against extreme pH conditions, high thermal stability, and the possibility of making
570
modifications to its surface with several active groups. As a disadvantage, the low
571
mechanical strength stands out. In turn, the stationary phases of most inorganic polymers
572
are formed by silica-based polymers. This type of stationary phase has a higher surface
573
area and mechanical resistance than must organic polymer phases. However, they are more
574
sensitive to sudden pH variations [3].
575
There are also “less conventional” stationary phases that envision the incorporation
576
of new materials capable of providing higher selectivity. The primary new materials used
577
for the preparation of OT-PLOT LC columns are based on different metal oxides, ion
23
578
exchange materials, molecularly imprinted polymers, and hybrid materials, which contain
579
both organic and inorganic characters [3].
580
Peng et al. developed a new zwitterionic stationary phase (ZIC) by thermal
581
initiation for HILIC separations [75]. Also, Aydoğan developed a stationary phase based
582
on β-cyclodextrin (β-CD) reporting enantioseparation with high theoretical plate numbers -
583
up to 26 000 plates/m [76].
584
In short, the use of OT-PLOT analytical columns in LC has not yet reached its
585
climax. However, it has shown great potential and is increasingly being the focus of the
586
interest of different research groups. There is a wide variety of new stationary phases
587
preparation techniques under development. Also, the natural compatibility for coupling
588
these miniaturized columns with MS evidences the full potential of OT-PLOT-LC-MS.
589 590
6. Wall Coated Open Tubular Columns (WCOT)
591
Another type of open tubular column is termed as "wall coated open tubular"
592
(WCOT). These columns have as a stationary phase a non-porous thin film adhered to the
593
interior of the tube, which is often made of fused silica. This coating is obtained after
594
extensive treatments of the tubing interior surface, enabling the immobilization of the film
595
on the column wall through cross-linking processes [77–79].
596
Nowadays, WCOT columns with capillary dimensions are consolidated in gas
597
chromatography and have many advantages over packed columns such as high durability,
598
superior performance, more inertness, ease of use, and from the theoretical perspective, the
599
highest chromatographic potential [80].
600
However, for miniaturized LC, there are few reports in the literature employing OT
601
columns [81–83]. One of the main reasons would be the fact that these columns present
24
602
low sample capacity because of their low surface area of contact, reducing the interaction
603
of the analyte with the stationary phase. As a consequence, it is more instrument
604
demanding than all other forms of miniaturized LC [84].
605
Although this type of column has promoted highly efficient separations in GC, it
606
has still been little exploited in miniaturized LC. WCOT columns were first successfully
607
used in LC by Hibi et al. [85] in 1978, when the authors were promoted highly efficient
608
separations of polynuclear aromatic hydrocarbons (PAHs) using 15 cm long and 0.5 mm
609
i.d. polytetrafluoroethylene (PTFE), introducing the term "micro-LC".
610
The preparation of WCOT-LC columns is meticulous and requires great care to
611
obtain a uniform stationary phase that tightly adheres to the column tube wall. The initial
612
step in preparing a WCOT column consists of leaching the fused silica capillary. This
613
process consists in preparing the capillary wall to receive the phase of interest. Leaching
614
can be done in an acidic or basic medium, and its efficiency depends on the solution
615
concentration, temperature, and treatment time. In the second stage, the tube is dehydrated
616
by heating it in an oven while passing through its interior a gentle and controlled flow of
617
nitrogen [86,87].
618
Following the silica tube treatment procedures described above, the treated tubes
619
are usually subjected to a further persilylation step using a compound capable of producing
620
the appropriate "wettability" for the subsequent coating, which will result in the formation
621
of a thin film inside of the tube. For the persilylation process, a silica capillary tubing is
622
filled with a proper silanizing solution, its ends are closed, and the capillary is placed into
623
an oven. The most used silanizing agents are hexamethyldisilazane (HMDS),
624
tetraphenyldimethyldisilazane (TPDMDS), and diphenyltetramethyldisilazano (DPTMDS).
625
After the persilylation process, the capillary is covered with the stationary phase of
626
interest. The most employed WCOT stationary phases used so far to produce WCOT-LC
25
627
columns follow the same trends used for GC, with polydimethylsiloxane (PDMS) and its
628
various modifications being the most used liquid polymer evaluated to date. By inserting
629
distinct functional groups into the pendant siloxane groups, it is possible to obtain a variety
630
of new stationary phases (modified polysiloxanes). The most frequently inserted functional
631
groups are phenyl and cyano. In addition to PDMS, other stationary phases have been
632
investigated, including GeO2-PDMS, 3-mercaptopropyltrimethoxylanosilica, polypyrrole,
633
TiO2-PDMS, and β-CD [88,89]. Besides, the sol-gel process, as well as alternative
634
procedures, may render different stationary phases for the coating process.
635
The interior wall of the tube can be covered with the stationary phase in two
636
different ways: static coating or dynamic coating. In the dynamic process, the stationary
637
phase solution containing the stationary phase is pushed with the aid of gas through the
638
capillary tube, forming a thin film on the tube wall. Although it is an easy and
639
straightforward technique, it results in a smaller phase ratio. In the static coating, the tube
640
is kept at a high temperature so that the phase solution solvent evaporates, and steam is
641
displaced out of the column with the aid of vacuum exerted at one end of the column [86].
642
The static coating procedure has gained prominence and is the most used coating
643
type, allowing the use of higher molar mass phases. Moreover, with this methodology, it is
644
possible to more accurately predict the thickness of the stationary phase film by varying
645
the concentration of the polymer in solution.
646
After the coating process, a cross-linking step is performed to immobilize the
647
stationary phase on the tubing wall, preventing its detachment during the chromatographic
648
run. The cross-linking process is usually done by using compounds such as azo-tert-butane
649
(ATB) or azo-tert-octane (ATO), among others.
26
650
Figure 8 shows a schematic drawing representing the capillary filling system with
651
the solutions of interest (Fig. 8A), coating tube set up (Fig. 8B), and the static coating
652
resulting process inside the tube of a capillary column (Fig. 8C).
653 654
655
Table 2 shows the characteristics of some commercially available stationary phases
656
derived from polydimethylsiloxane, which are the most used materials to prepare WCOT
657
columns to date.
658