Comprehensive multidimensional liquid chromatography: Theory and applications

Available online at www.sciencedirect.com

Journal of Chromatography A, 1184 (2008) 353–368

Review

Comprehensive multidimensional liquid chromatography: Theory and applications Paola Dugo a,∗ , Francesco Cacciola b , Tiina Kumm b , Giovanni Dugo b , Luigi Mondello b a

Dipartimento di Scienza degli alimenti e dell’ambiente, Facolt`a di Scienze, Universit`a di Messina, Salita Sperone 31, 98166 Messina, Italy b Dipartimento Farmaco-chimico, Facolt` a di Farmacia, Universit`a di Messina, Viale Annunziata, 98168 Messina, Italy Available online 6 July 2007

Abstract Comprehensive two-dimensional (2D) liquid chromatographic (LC × LC) techniques can be considered innovative methods only recently developed and adopted in many configurations. The revolutionary aspect of comprehensive two-dimensional techniques, with respect to classical multidimensional (MD) chromatography, is that the entire sample is subjected to the 2D advantage. The major benefit is that the separation capacities of each dimension are multiplied, offering a high peak capacity to resolve samples of great complexity. The first part of the present review briefly describes the theoretical and practical aspects related to the development of a multidimensional comprehensive liquid chromatographic method. Applicational experiences in comprehensive liquid chromatography are then described, divided into four groups, according to the HPLC modes used in the two dimensions and to the nature of the samples analyzed. © 2007 Elsevier B.V. All rights reserved. Keywords: Two-dimensional separation; Comprehensive LC

Contents 1. 2.

3.

4.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . General aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Column selectivity, orthogonality, peak capacity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Resolution and sampling rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Method development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Comprehensive LC techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Comprehensive 2D LC separation of synthetic and natural polymers and oligomers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Comprehensive 2D LC separation of biological and organic compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. NP × RP LC of fats, essential oils, alcohols, hydrocarbons and pharmaceutical compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. RP × RP LC separation of antioxidants, natural and environmental compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction One-dimensional chromatography is widely applied to the analysis of real-world samples in several fields. However, such separation methods often do not provide sufficient resolving power for the separation of target components in many real∗

Corresponding author. Tel.: +39 090 676 6541; fax: +39 090 676 6532. E-mail address: [email protected] (P. Dugo).

0021-9673/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2007.06.074

353 354 354 355 355 357 357 363 364 366 367 367

world samples. It is well known that even in samples of low complexity there is generally a random peak distribution which requires high plate number values for total peak resolution [1,2]. If the number of components exceeds 37% of the peak capacity, peak resolution is statistically compromised [2]. A possible solution to this problem can be the use of multidimensional systems (MD), where the dimensions are based on different separation mechanisms [3,4]. Multidimensional chromatographic techniques are characterized by a greatly increased resolving power as compared to

354

P. Dugo et al. / J. Chromatogr. A 1184 (2008) 353–368

one-dimensional methods. In particular, LC techniques offer a wide variety of separation mechanisms, such as normal phase (NP), reversed phase (RP), size exclusion (SEC), ion exchange (IEX) or affinity chromatography (AC), characterized by different selectivities. Consequently, two-dimensional (2D) liquid chromatography can be theoretically employed in many multidimensional combinations, generating increased peak capacity, selectivity and resolution, especially in the comprehensive LC mode. However, the combination of certain LC modes can present a series of difficulties, if not impossibilities, such as, for example, mobile phase immiscibility or mobile phase of first dimension and stationary phase of second dimension incompatibility. Two-dimensional HPLC can be performed either off-line or on-line. The former, although easier, can be time consuming, difficult to automate and reproduce, susceptible to sample loss and contamination and formation of artefacts. The on-line approach is faster and more reproducible, but needs specific interfaces and is more difficult to operate. The difference between a conventional heart-cutting multidimensional chromatographic technique and a comprehensive one is that the first enables the re-injection of a certain number of multi-component effluent fractions from a primary to a secondary column [5,6], while in the second the entire sample is subjected to separation in both dimensions [7,8]. Other requirements of a comprehensive 2D separation are that any two components separated in the first dimension must remain separated in the second dimension and that elution profiles from both dimensions are preserved [7,8]. Comprehensive 2D HPLC is, currently, performed using one or two HPLC systems equipped with columns connected via a transfer device (called interface or modulator) located between them. The interface cuts the fractions of the primary column effluent and releases them onto the secondary column, where usually a fast separation takes place. The fraction injected onto the secondary column should be completely analyzed before the successive transfer occurs. The second dimension analysis time should be at least equal or less than the duration of a modulation period. The importance of performing at least three or four modulations per each first dimension peak, in order to avoid a serious loss of information in the two-dimensional separation due to under-sampling of first dimension peaks, has been demonstrated [9]. The development and optimization of a comprehensive 2D HPLC method requires the time adjustment of many parameters in order to accomplish successful separations, as will be explained in the following sections. Apart from the enhanced resolving power, a further substantial benefit is related to the great identification power due to the formation of 2D chemical class patterns [8,10]. These unique capabilities have attracted many researchers, who have applied comprehensive HPLC methods for the characterization of their samples from a variety of origin. The first comprehensive two-dimensional liquid system was introduced by Erni and Frei [11] who analyzed a complex plant extract with an SEC column as the first dimension and a reversed phase column as the second dimension, connecting the two

columns with an eight-port valve. In the last 20 years, comprehensive HPLC methods have been developed and applied mainly to the separation of peptides and proteins [12–29], as well as polymers [30–37], but also to small molecules such as antioxidants [38–43], triacylglycerols [44–46] and bioactive and pharmaceutical samples [47–49]. Comprehensive HPLC has been reviewed recently, with attention devoted mainly to separation in the peptide and protein field [50–53], as well as biomedical and pharmaceutical areas [53–54]. Other specific reviews have treated food analysis in general [55], lipids in particular [56] and various theoretical and practical aspects [57–59]. The first section of the present review will briefly focus on the general aspects that have to be considered when developing a comprehensive HPLC method, while the second section will give an overview on the applications developed in different areas of research up to now. For each application, type of interface, column sets and detector used are specified. 2. General aspects 2.1. Column selectivity, orthogonality, peak capacity Column selectivity is a primary concern when designing a 2D separation, as it has a direct effect on 2D system orthogonality and, as a consequence, on the peak capacity. Selectivity of the columns used in the two dimensions must be different to attain maximum gain in peak capacity. Furthermore, mobile phase composition, either isocratic or gradient, flow rate and in some cases temperature are other parameters that require careful tuning during optimization. The best results are achieved in socalled “orthogonal” systems with non-correlated retention times in both dimensions. Column selectivity for two-dimensional liquid chromatography has been the subject on an extensive review [59]. Ideally, the total peak capacity, n2D , is equal to the product of the peak capacities in the first (n1 ) and in the second (n2 ) dimensions in fully orthogonal 2D systems with non-correlated selectivity in the first and in the second dimensions [8,10,60]: n2D = n1 × n2

(1)

For complex mixtures, characterized by random peak distribution, a very high value of n2D is required for resolution of all the compounds. The product of the peak capacity in each dimension, excluding the portion of separation space corresponding to void volume and re-equilibration, gives a n2D value that overestimates the real peak capacity achieved for a given separation, as it includes the region where orthogonality is not achieved. Methods for calculating a truer n2D were proposed and reviewed by Dixon and Perrett [54]. Whatever approach the method used, it is important to note that an accurate calculation of n2D is complex and that, in practice, theoretical n2D values are difficult if not impossible to attain. Two-dimensional systems with fully noncorrelated selectivities are rarely found in practice and hence, to obtain a significant increase in resolving power, the operational conditions in both dimensions should be carefully matched and optimized. The most important aspects affecting the results of an on-line 2D separation include:

P. Dugo et al. / J. Chromatogr. A 1184 (2008) 353–368

355

1. Stationary phase, mobile phase and temperature effects on separation selectivity. 2. Design of solvent or temperature gradients in the first and in the second dimension to increase the peak capacity. 3. Mobile phase compatibility in the first and in the second dimension and its effects on the fraction transfer between the two dimensions. 4. Matching the column dimensions and the flow rates in the first and in the second dimension, the volume of the transferred fractions and the frequency of the sample transfer cycles. 5. Sample transfer modulation in order to suppress band broadening and achieve peak focusing. 2.2. Resolution and sampling rate The improvement in resolving power of multidimensional chromatography is expressed also in terms of resolution. Giddings [10] showed that in a two-dimensional system where RsD1 and RsD2 are the resolution between a pair of peaks in each dimension, the overall resolution Rs will be given as: 1/2 Rs ∼ = (R2sD1 + R2sD2 )

A fundamental rule to follow in a comprehensive 2D separation is to maintain the first dimension resolution, which may be achieved performing a sufficient number of samplings per peak (three to four according to Murphy et al.) [9]. This is of particular importance if the first dimension contribution towards the entire multidimensional separation is high. In practice, second dimension separations should be fast enough to ensure an adequate number of samplings. The sampling period should be less than 1.5 times the first dimension peak standard deviation to determine first dimension peak retention times with the necessary precision [61]. After defining the second dimension analysis time, the first dimension can be optimized at such a low flow rate to get three to four samplings per peak. It is ideally achieved using a gradient program, that maintains peak widths approximately constant during the run. 2.3. Method development In the most common set-up for comprehensive 2D systems, small volume fractions of the effluent from the first dimension are transferred via a multi-port switching valve into the second dimension. Fig. 1 shows an example of such a comprehensive LC system. The valve is usually equipped with two identical-volume sampling loops, one of which collects the effluent from the first dimension, while the previous effluent fraction contained in the second loop is transferred onto the second dimension and is subjected to separation [7,30]. Both dimensions are operated continuously, as the two loops are periodically and alternately switched between the collection and the re-injection modes, the modulation period is controlled by the switching valve frequency. However, resolution in the second dimension is usually rather limited when the interface with two sampling loops is employed, as the time available for the second dimension separation is strongly limited by the small volume of the sampling

Fig. 1. Conventional LC × LC set-up using one multi-port switching valve between the two dimensions (reprinted from [45] with permission from Elsevier, 2006).

loops; this is dictated by the necessity to deposit a narrow analyte band at the head at the secondary column. Furthermore, it is advantageous if the mobile phase used in the first dimension has a lower elution strength than that of the second dimension, which allows the re-concentration of the primary column fraction in a narrow zone at the head of the second dimension column (on-column focusing) [62]. In this approach, the use of a microbore HPLC column in the first dimension, operated at a low flow rate, and a conventional size column in the second dimension, operated at a high flow rate, is convenient: - the small column i.d. ensures a minimization of dilution and provides flow rates that are compatible with secondary dimension injection volumes; - a pre-concentration step at the head of the secondary column is not necessary and solvent incompatibility between different separation modes is avoided [6,63]. Other advantages in using microbore HPLC columns are the low mobile phase consumption and higher separation efficiency. Although the lower sample capacity of these columns can be considered a disadvantage, it must be noted that a large volume injection can be performed, with a tolerable decrease in efficiency. An even stronger fraction-transfer effect is achieved using the interface with two trapping columns instead of sampling loops [23–25,39,41]. Here, each fraction is adsorbed alternately on one of the trapping columns; in the following cycle the retained compounds are back-flushed from the trapping columns onto the second dimension analytical column in a very small volume of mobile phase with minimum band broadening. In a different LC × LC configuration the interface can be equipped with two or more parallel fast secondary columns [13,14,17–19,22,27,39,40,43,64,65] especially with 1.5 ␮m pellicular columns [13,14,18,19,22,27,43] rather than storage loops. The use of loops to entrap 1D fractions is always possible, mostly when the first dimension flow rate is much lower than that of the secondary dimension, even if the disadvantage of a possi-

356

Table 1 Comprehensive 2D LC separation of synthetic and natural polymers and oligomers Reference

[30]

[9] [32] [70]

[33]

10-port 2-position valve equipped with two sampling loops 8-port 2-position valve with two sampling loops 10-port 2-position valve equipped with two sampling loops Two 6-port 2-position valves equipped with two alternative C18 columns. Use of supplementary flow mixed after D-1 to enhance trapping of hydrophilic compounds on D-2 10-port 2-position valve equipped with two sampling loops 10-port 2-position valves with sampling loops

Materials, dimensions (L × i.d., mm) and flows of columns

Detection

Application

UV

Functional polymers and co(polymers)

First dimension, D-1

Second dimension, D-2

Silica, 250 × 1.0; 3 or 4 ␮L/min; IP

SEC; 50 × 7.5; 0.4 mL/min; IP

C18; 150 × 1.0; 4 ␮L/min; GP

75 × 4.6; 0.4 mL/min; IP

C18; 150 × 3.0; 0.1 mL/min; Splitter (1:3–1:20) GP Two silica; 150 × 1.0; 8 ␮L/min; IP

SEC; 50 × 8; 1 mL/min; IP

ELSD

Polyethylene glycols and surfactants

SEC; one or two; 50 × 4.6; 0.9 mL/min; IP

UV ELSD

Functional acrylate polymers

SEC; 250 × 4.6; 1 mL/min; IP

C18; 7.5 × 4.6; 1 mL/min; GP

UV ESI-IT

Various compounds with different hydrophobicity and size

Silica; 150 × 3.2; 10 ␮L/min; IP

SEC; 150 × 6.0; 0.8 mL/min IP

UV MALDI-IT

Poly(bisphenolA) carbonate sample

Silica; 150 × 3.9; 40 ␮L/min (after splitting); GP

SEC; 50 × 20; 6 mL/min

UV FTIR

Copolymers

UV

Polystyrenes

150 × 6.0; 0.8 mL/min 50 × 4.6; 0.3 mL/min [34]

[69]

10-port 2-position valves with sampling loops 10-port 2-position valves with sampling loops

SEC; 250 × 4.6; 10 ␮L/min; IP

SEC; 50 × 4.6; 0.6 mL/min; IP

250 × 1.0; 4 ␮L/min; IP

150 × 4.6; 1.5 mL/min; IP

Silica; 250 × 3.0; 10 ␮L/min; GP

SEC; 150 × 6.0; 0.8 mL/min; IP

UV MALDI-TOF-MS

Poly(bisphenol A) carbonate sample

C18; 250 × 4.6; 1.0 mL/min; IP

SEC; 50 × 20; 2.5–8 mL/min; IP

ELSD

PEG-g-PVAc copolymers

Silica; 2 (150 × 1.0)

SEC (one or two 50 × 4.6); 0.9 mL/min; IP

UV

Poly(methyl-methacrylate) polymers

C18; 150 × 4.6; 20 ␮L/min; IP

SEC; 50 × 20; 4.0 mL/min; IP

UV MALDI-TOF

Blanched polystyrenes

Amino; 150 × 4.6; 0.05 mL/min; GP

C18; 33 × 4.6; 1.5 mL/min; IP

ELSD

Alcohol ethoxylates

Amino-propyl; Silica; 150 × 1.0 or 50 × 1.0; 0.5 mL/min; IP ZR-Carbon; 30 × 4.6; 2 mL/min; GP

ELSD

Co(oligomers)

UV

ZR-Carbon; 30 × 4.6; 2.0 mL/min; IP

UV

ZR-Carbon; 50 × 4.6; 2.0 mL/min; IP

UV

Separation of isomers of oligostyrenes Separation of isomers of oligostyrenes Separation of isomers of oligostyrenes

150 × 3.2; 10 ␮L/min; IP [35] [36] [31] [74]

8-port 2-position valves with sampling loops 10-port 2-position valves with sampling loops 10-port 2-position valves with sampling loops 8-port 2-position valve with one two sampling loops

Silica; 150 × 3.0; 0.2 mL/min; GP [37] [71] [72] [73]

10-port 2-position valves with sampling loops 6-port 2-position valve with one storage loop Four 6-port 2-position valves fitted with micro-electric 2-position valve actuators Four 6-port 2-position valves fitted with micro-electric 2-position valve actuators

“IP”: isocratic program. “GP”: gradient program.

C18; 150 × 1.0; 10 ␮L/min; GP C18; 250 × 4.6; 0.1 mL/min; GP C18; 250 × 4.6; 0.1 mL/min; 0.7 mL/min; 1.0 mL/min; IP C18; 150 × 4.6; 0.1 mL/min; IP

P. Dugo et al. / J. Chromatogr. A 1184 (2008) 353–368

[68]

Type of interface

P. Dugo et al. / J. Chromatogr. A 1184 (2008) 353–368

ble additional peak broadening has to be considered. Trapping on the column has severe limitations. In this case the primary mobile phase should have a very low eluent strength so that analytes can be trapped at the head of the secondary column during the loading step. In this configuration, a fraction from the primary column is trapped alternately at the head of one of the two secondary column during the loading step in an on-column-focusing mode, meanwhile in the other column the solutes, transferred during the previous cut, are analyzed. Furthermore, the use of identical columns is critical in order to achieve retention time precision in consecutive second dimension runs. Whatever configuration is used, second dimension separations as stated before, should be fast enough to be completed before the successive transfer. Rapid second dimension analysis can be achieved using monolithic columns, that can be used at high flow rates without loss of resolution, under gradient conditions with very brief conditioning times, thus allowing the application of gradient programs during successive second dimension runs [39,41,44–47,63,66]. An alternative to monolithic columns in speeding up the 2D analysis, can be the use of high temperatures. Increasing temperatures to more than 100 ◦ C, ultra-fast gradient elution separations with excellent retention times repeatability has been obtained using narrow-bore wide pore carbon-coated zirconia columns [15,67]. The decrease of eluent viscosity allows fast column re-equilibration due to the higher flow rate at the same maximum pressure. Moreover, high temperatures mitigate the loss in efficiency which usually occurs at higher than optimum linear velocities. Conventional short columns have also been employed, but in this case column re-equilibration is longer and a repetitive gradient is difficult to perform in very short time. In this case, the second dimension run can be longer, and the number of sampling per each first dimension peak became lower than 3–4 [39–41]. With this approach, only one gradient program, spanning over the whole 2D separation time, is usually run during the total analysis time [41,64–65]. In comprehensive chromatography usually only one detector is installed after the second dimension column with the first dimension separation monitored only during the optimization step. Most of the traditional HPLC detectors can be used in LC × LC analyses, as can be seen from the information reported in Tables 1–4. If the second dimension is fast, data acquisition should be rapid enough to ensure the acquisition of narrow peaks, and then the proper reconstruction of the 2D chromatogram. The use of a PDA or MS detectors can add a third dimension to the LC × LC system, providing additional information to be used for component identification. An LC × LC application produces a great amount of data, with retention information for each second dimension separation. The quantity of data becomes even larger if an MS or a PDA are used as detector. Data handling is rather complicated, and requires dedicated softwares for the elaboration of two-dimensional or three-dimensional plots. Difficulties increase if peak quantification needs to be performed. Peak integration and quantitation can be performed by summing the areas of individual second dimension peaks belonging to one analyte peak, which are integrated using specific integration algorithms.

357

At present, the area of dedicated software for LC × LC is poorly developed and complete data processing packages are not yet available. A dedicated software should automatically process the acquired data and then transform it into a two-dimensional chromatogram, giving all the necessary information to perform qualitative and quantitative analysis. It must be noted, though, that progresses are being made in this field, that also concern the combination of multidimensional separation with chemometric data analysis, as recently reviewed by Dixon and Perrett [54].

3. Comprehensive LC techniques A large variety of LC modes can be exploited to resolve specific separation problems. The selectivity in the individual modes is principally based on the differences in size, polarity and shape, in acidities/basicities, or on the specific charge of ionic compounds. The molecular size represents the separation mechanism in SEC, even if it significantly contributes to the separation selectivity in RP and in some cases also in NP systems due to hydrophobic or polar interactions of the structural elements in large molecules. The polar separation selectivity may differ in various phase systems especially in both RP and NP modes, based on the differences in the selective dipole–dipole or proton–donor/acceptor interactions that affect retention. The differences in the charge, acid–base properties and non-ionic interactions are the principal sources of selectivity in IEX or in ion-pairing systems. When planning 2D separations, a criterion enabling the evaluation of the separation system selectivity with respect to the distribution of the characteristic sample properties is necessary in order to get a maximum gain in peak capacity. Tables 1–4 summarize comprehensive 2D LC applications in the field of synthetic and natural polymers and oligomers (Table 1), biological and organic components (Table 2), natural and pharmaceutical components using NPLC × RPLC (Table 3) and antioxidants, natural and environmental components using RPLC × RPLC (Table 4).

3.1. Comprehensive 2D LC separation of synthetic and natural polymers and oligomers Most comprehensive LC separations of synthetic and natural polymers and oligomers have been carried out by using LC × SEC, exploiting the different functionalities (end-groups with different polarities “chemical composition” distribution) in the first dimension and molar mass distribution in the second dimension [9,30,32,33,35,36,68,69]. In general, on-line detection in LC or SEC is accomplished through ultraviolet (UV) [30–34,36,68–73], Fourier transform infra-red (FTIR) [33], evaporative light scattering (ELS) [9,32,35,37,74] or MS detection [31,68–70]. All of these detectors are characterized by some disadvantages: for instance, UV detection is limited only to UV active polymers, RI detectors exhibit low sensitivity, ELS detectors yield a non-linear response and LC–MS is limited to polar and relatively small polymers. However, despite these

358

Table 2 IEX × RP, SEC × RP, IEX × SEC LC separation of biological and organic compounds Reference

[11] [16] [17] [18] [19]

[13] [15] [12] [79]

[14]

[21]

[22] [23] [24] [25] [26] [78] [27]

8-port 2-position valve equipped with two sampling loops 8-port 2-position valve equipped with two sampling loops 4-port 2-position valve with two alternative secondary columns 10-port 2-position valve equipped with two alternative secondary columns 10-port 2-position valve equipped with four parallel short secondary columns. Integrated clean-up of extract on RAM No interface is used; 2D LC is obtained by number of gradient elutions 10-port 2-position valve equipped with two alternative secondary columns 10-port 2-position valve equipped with two sampling loops 8-port 2-position valve equipped with two sampling loops Two electronically controlled valves in which the effluent from the D-1 is collected in a sample loop and then concentrated onto the head of the RP microcolumn Two 4-port 2-position valves equipped with two alternative C18 columns Two electronically controlled valves in which the effluent from the D-1 is collected in a sample loop and then concentrated onto the head of the RP microcolumn 10-port 2-position valve equipped with two alternative C18 columns 10-port 2-position valve equipped with two C18 trapping columns (5 × 0.3) 10-port 2-position valve equipped with two C18 trapping columns (5 × 0.3) 10-port 2-position valve equipped with two C18 trapping columns (5 × 1.0) 10-port 2-position valve equipped with one C18 trapping column (1 × 0.3) 10-port 2-position valve equipped with two sampling loops Three 10-port 2-position valves connected to four alternative RP columns

Materials, dimensions (L × i.d., mm) and flows of columns

Detection

Application

First dimension, D-1

Second dimension, D-2

SEC; 2000 × 4.0; 1.2 mL/h; GP

C18; 250 × 4.0; 2.0 mL/min; GP

UV

Plant extracts

SCX; 125 × 0.75; 10 ␮L/min; GP

RP-POROS; 100 × 0.5; 50 ␮L/min (after splitting); GP PSDVB; 33 × 2.1; 1.5 mL/min; GP

UV; ESI-MS

Protein and peptide mapping

UV; ESI-MS; MALDI-TOF-MS UV; MS

Protein mapping

UV; MALDI-MS

Protein and peptide mapping

ESI-MS; MS

Protein and peptide mapping

C18; 14 × 4.6; 2.5 mL/min; GP

UV

Protein mapping

SCX, PO4 3− ZrO2 ; 50 × 2.1; 0.1 mL/min; GP IEX; 250 × 1.0; 10 ␮L/min; GP

C18; 50 × 2.1; 3.0 mL/min; GP

UV

Peptide mapping

SEC; 250 × 9.4; 60 ␮L/min; IP

UV

Protein mapping

SAX; 900 × 0.1; 33 nL/min; GP

C18; 30 × 0.1; 6 ␮L/min; GP

Fluorescence

Biological amines

Six SECs; 300 × 7.8; 1 mL/min (first 40 min of run followed by 100 ␮L for the next 140 min); GP IEX; 1000 × 0.1; 33 nL/min; GP

C18; 33 × 4.6; 1 mL/min; GP

UV; ESI-MS

Peptide mapping

C18; 35 × 0.15; 6 ␮L/min; GP

Fluorescence

Peptide mapping

SCX; 35 × 4.6; 400 ␮L/min; GP

UV; ESI-TOF-MS

Protein mapping

SCX; 150 × 0.3; 400 ␮L/min; GP

C18; 33 × 4.6; 50 × 2.1; 0.5 mL/min; GP C18; 150 × 0.075; 300 nL/min; GP

UV

Protein and peptide mapping

SAX; 250 × 0.32; 5 ␮L/min; GP

PSDVB; 100 × 0.3; 20 ␮L/min; GP

UV; ESI-TOF-MS

Protein mapping

SAX; 150 × 1.0; 50 ␮L/min; GP

PSDVB; 150 × 0.3; 15 ␮L/min; GP

Protein mapping

SCX; 150 × 0.3; 15 ␮L/min; GP

C18; 150 × 0.075; 200 nL/min; GP

UV; MALDI-TOFMS; ESI-TOF-MS ESI-TOF-MS

SAX; 250 × 4.0; 1 mL/min; GP

C18; 33 × 7.0; 3.5 mL/min; IP

UV

Organic acids

SCX; 35 × 4.6; 0.5 mL/min; GP

C18; 14 × 4.6; 2 mL/min; GP

UV; MALDI-TOF

Peptide mapping

SEC; 7 × (300 × 7.8); 250 ␮L/min; GP IEX; 35 × 4.6; 1 mL/min; GP IEX; 35 × 4.6; 0.5 mL/min; GP

SCX + C18; (100 + 40) mm × 0.1 mm approximately; 0.3 ␮L/min; GP IEX; 35 × 4.6; 1 mL/min; GP

C18 NPR; 14 × 4.6; 2.5 mL/min; GP Four NPR C18; 14 × 4.6; 2.5 mL/min; GP

Protein and peptide mapping

Protein mapping

P. Dugo et al. / J. Chromatogr. A 1184 (2008) 353–368

[20]

Type of interface

[28]

A 6-port valve with continuous and stop flow operation

SEC; 300 × 7.8; 0.37 or 0.50 mL/min

C18 Chromolith; 25 × 4.6

UV

Protein mapping

100 × 4.6; 2 mL/min; GP [80] [80] [29]

10-port 2-position valves with two sampling loops 10-port 2-position valves with two sampling loops A multi-channel interface equipped with three-way microsplitter valves used as stop and flow matrix

SCX; 150 × 1.0; 40 ␮L/min; GP

C18; 50 × 3.0; 1.1 mL/min; GP

ESI-TOF

Plant extract

Amino; 50 × 3.0; 100 ␮L/min; IP

SCX; 20 × 2.0; 1.5 mL/min; IP

ESI-TOF

Plant extract

SCX; 70 × 0.32; 5 ␮L/min; GP

C8; 10 (250 × 0.25); 8 ␮L/min; GP

UV; MALDI-TOF-TOF

Protein and peptide mapping

“IP”: isocratic program. “GP”: gradient program.

Reference

[44] [45] [46] [63] [47] [47] [81] [87]

Type of interface

10-port 2-position equipped with two sampling loops 10-port 2-position equipped with two sampling loops 10-port 2-position equipped with two sampling loops 10-port 2-position equipped with two sampling loops 10-port 2-position equipped with two sampling loops 10-port 2-position equipped with two sampling loops 10-port 2-position equipped with two sampling loops 10-port 2-position equipped with two sampling loops

Detection

Application

First dimension, D-1

Second dimension, D-2

Nucleosil 100-5 SA; silvered in lab; 150 × 1.0; 13 ␮L/min; IP Nucleosil 100-5 SA; silvered in lab; 150 × 1.0; 11 ␮L/min; GP Nucleosil 100-5 SA; silvered in lab; 150 × 1.0; 11 ␮L/min; GP Bare silica; 300 × 1.0; 20 ␮L/min; IP

C18 Chromolith; 100 × 4.6; 4 mL/min; GP

APCI-MS

Lipidic samples

C18 Chromolith; 100 × 4.6; 4 mL/min; GP

ELSD (D-1); APCI-MS

Lipidic samples

C18 Chromolith; 100 × 4.6; 4 mL/min; GP

ELSD (D-1); APCI-MS

Lipidic samples

C18 Chromolith; 25 × 4.6; 4 mL/min; GP

UV; PDA

Lemon oil

Diol; 250 × 1.0; 40 ␮L/min; IP

C18 Chromolith; 100 × 4.6; 5 mL/min; GP

PDA

Pharmaceutical mixture

Diol; 250 × 1.0; 40 ␮L/min; GP

C18; 50 × 4.6; 5 mL/min; GP

PDA

Citrus oil extracts

Bare silica; 300 × 1.0; 15.4 ␮L/min; IP

C18 Chromolith; 25 × 4.6; 4 mL/min; GP

PDA

Allergens

Bare silica; 300 × 1.0; 10 ␮L/min; GP

C18 Chromolith; 100 × 4.6; 4.7 mL/min; GP

UV; PDA

Carotenoids

359

“IP”: isocratic program. “GP”: gradient program.

Materials, dimensions (L × i.d., mm) and flow-rates of columns

P. Dugo et al. / J. Chromatogr. A 1184 (2008) 353–368

Table 3 NP × RP LC of natural and pharmaceutical compounds

360

Table 4 RP × RP LC separation of antioxidants, natural and environmental compounds Reference

[38] [39]

Type of interface

6-port 2-position valve with stop flow on primary column during separation in D-2 10-port 2-position valve equipped with (1) Two sampling loops (2) Two C18 trapping columns (30 mm × 4.6 mm) (3) Two alternative ZR-Carbon columns (4) Two alternative ZR-Carbon columns

[40]

(2) Two C18 trapping columns (30 mm × 4.6 mm)

First dimension, D-1

Second dimension, D-2

PEG; 50 × 2.1; 0.4 mL/min; IP

C18; 125 × 2.0; 0.4 mL/min; GP

PEG; 150 × 4.6; 0.1 mL/min; GP PEG; 150 × 4.6; 0.3 mL/min; IP

C18; 10 × 2.1; 1.0 mL/min; IP C18 Chromolith; 50 × 4.6; 2.0 mL/min; GP ZR-Carbon; 50 × 2.1; 1.0 mL/min; IP ZR-Carbon; 50 × 2.1; 1.0 mL/min; IP

PEG; 150 × 4.6; 0.2 mL/min; GP PEG (50 × 2.1) + C18 (125 × 4.6); 0.2 mL/min; GP C18; 150 × 0.5; 10 ␮L/min; GP

[48] [49]

10-port 2-position valve equipped with two sampling loops 8-port 2-position valve equipped with two sampling loops 8-port 2-position valve equipped with two sampling loops

Application

PDA

Natural antioxidants

PDA

Natural antioxidants

UV

Natural antioxidants

UV

Phenolic and flavone antioxidants

PEG + C18; (50 × 2.1); (250 × 3.0); 0.3 mL/min; GP PEG + C18; (150 × 4.6); (50 × 4.6); 0.3 mL/min; GP Phenyl; 50 × 3.9; GP; 0.3 mL/min; GP C18; 150 × 2.1; 0.1 mL/min; GP

C18 Chromolith; 50 × 4.6; 2.0 mL/min; GP C18 Chromolith; 100 × 4.6; 2.0 mL/min; GP C18 Chromolith; 100 × 4.6; 2.0 mL/min; GP C18 Chromolith; 100 × 4.6; 2.0 mL/min; GP C18 Chromolith; 100 × 4.6; 2.0 mL/min; GP C18 Chromolith; 100 × 4.6; 2.0 mL/min; GP CN; 75 × 4.6; 1.9 ␮L/min; IP

ESI-TOF-MS

CN; 200 × 2.0; 40 ␮L/min; GP

C18; 50 × 2.0; 0.7 mL/min; IP

PDA; APCI-MS

Antioxidant and phenolic acids (herb extracts) Chinese medicines

CN; 150 × 4.6; 0.133 mL/min; GP

C18 Chromolith; 50 × 4.6; 3.0 mL/min; GP

PDA; APCI-MS

Chinese medicines

C18; 150 × 4.0; 1 mL/min; IP

Safrole silicaa ; 125 × 4.0; 1 mL/min; IP

UV

Explosives and by-products in water

PBBb ; 150 × 4.6; 1 mL/min; IP

C18 Chromolith; 50 × 4.0; 16 mL/min; GP

UV

Polycyclic aromatic hydrocarbons

C18; 150 × 4.6; 0.5 mL/min; GP

C18 Chromolith; 100 × 4.6; 4 mL/min; GP

UV

Aromatic amines and non-amines

Cyano; 33 × 7.0; 0.5 mL/min C18; 150 × 4.6; 0.5 mL/min; GP C18; 150 × 4.6; 0.5 mL/min; GP

Amino; 50 × 4.6; 2.5 mL/min Amino; 50 × 4.6; 1.5 mL/min Amino/Cyano; 50 × 4.6; 1.5 mL/min 33 × 7.0; 2.2 mL/min

PEG; 150 × 4.6; 0.1 mL/min; GP PEG; 150 × 4.6; 0.3 mL/min; GP PEG; 150 × 4.6; 0.3 mL/min; GP

[42]

ZR-Carbon; 50 × 2.1; 1.0 mL/min; IP at 120 ◦ C

Detection

SCX; 150 × 4.6; 0.133 mL/min; GP [88]

[89]

[64]

Three 6-port 2-position valves and one seven-way six position valve that permitted stepwise transfer of C18 effluent to the secondary column; stop flow on primary column was held during separation in D-2 10-port 2-position valve equipped with two alternative D-2 columns. Top column focussing in D-2 by mixing water after D-1 12-port 2-position valve with alternated sampling of the D-1 effluent onto two equal secondary columns through equivalent dual sample loops. A single pump delivers solvent to the two dimensions

P. Dugo et al. / J. Chromatogr. A 1184 (2008) 353–368

[41]

10-port 2-position valve equipped with two alternative ZR-Carbon columns 10-port 2-position valve equipped with: (1) Two sampling loops

Materials, dimensions (L × i.d., mm) and flows of columns

[65]

[90]

12-port 2-position valve with alternated sampling of the D-1 effluent onto two equal secondary columns through equivalent dual guard phenyl columns (20 mm × 3.9 mm). A single pump delivers solvent to the two dimensions Two 6-port valves equipped with 500 ␮L storage loop

[91]

Effluent from D-1 (1) Directly loaded into a 500 ␮L loop of D-2 HPLC injector (2) Two 6-port valves each equipped with a 500 ␮L storage loop (3) Two 6-port valves each equipped with one 500 ␮L loop and a D-2 column

[80]

[67] [92]

Two 6-port 2-position valves equipped with dummy columns which produce flow resistances and protect the pumps from a pressure drop when the valves are switched for fraction transfer and D-1 flow interruption 10-port 2-position valves with sampling loops

Two 6-port injection valves equipped with two sampling loops 10-port 2-position valve equipped with two sampling loops

“IP”: isocratic program. “GP”: gradient program. a (3 ,4 -Methylendioxyphenyl)propyl silica. b (Pentabromobenziloxy)propylsilyl-bonded phase. c Fluoroalkylsilyl-bonded phase. d Tetrachlorophthalimidopropyl phase. e Pentafluorophenylpropyl phase.

SB-Phenyl; 50 × 4.6; 1.3 mL/min;GP

UV; MS

Drug mixtures

C18 Chromolith; 100 × 4.6; 0.65 mL/min; IP; 0.5 mL/min; GP

C18 Chromolith; 50 × 4.6; 9.5 mL/min; IP; 9.5 mL/min; GP

UV

Aromatic compounds

UV

Hydrocarbons and benzene derivatives

UV

Phenols

ESI-TOF ESI-TOF

Plant extract

PDA

Indole-3-acetic acid derivates

UV; ESI-TOF-MS

Organic acids in atmospheric aerosols

FR silicab ; 150 × 4.6; 0.4 mL/min; GP FR silicac ; 150 × 4.6; 0.4 mL/min; GP FR silicac ; 150 × 4.6; 0.4 mL/min; GP FR silicac ; 150 × 4.6; 0.4-0.8 mL/min; GP TCPd Silica; 200 × 0.32; 5 ␮L/min; IP

C18 Chromolith; 30 × 4.6; 10 mL/min; GP C18 Chromolith; 30 × 4.6; 10 mL/min; GP Two C18 Chromolith; 30 × 4.6; 10 mL/min; GP C18 Chromolith; and PBBa ; 30 × 4.6; up to 10 mL/min; GP C18; 30 × 4.6; 1 mL/min; IP

Amino; 50 × 3.0; 100 ␮L/min; IP C18; 150 × 2.1; 100 ␮L/min; GP

C18; 50 × 3.0; 1.0 mL/min; IP Amino; 30 and 50 × 2.0; 1.8 mL/min; IP ZR-Carbon; 50 × 2.1; 3.0 mL/min; GP; at 110 ◦ C C18 Chromolith; 50 × 4.6; 50 ␮L/min; GP 2.5 ␮m C18; 50 × 3.0; 50 ␮L/min; GP

F5e ; 50 × 2.1; 0.1 mL/min; GP SCX; 150 × 1.0; 40 ␮L/min; GP

P. Dugo et al. / J. Chromatogr. A 1184 (2008) 353–368

[43]

C18; 150 × 4.6; 0.8 mL/min; GP

361

362

P. Dugo et al. / J. Chromatogr. A 1184 (2008) 353–368

limitations each detection technique has been widely adopted for the characterization of polymers. Under NP conditions, usually a silica column [30,32,33,36, 37,68,69,74] is used either with isocratic [30,32,37,68,69] or with gradient elution [33,69,74], while under RP conditions a conventional octadecyl silica is normally employed [9,30,31,35,37,70–74] either with isocratic [31,35,72–74] or with gradient elution [9,30,37,70,71]. As the monomer units in polymers are more or less polar, each repeated monomer unit contributes more or less to the polarity of polymers, so that retention in both NP and RP systems depends to a certain extent on the polymer molecular weight. Although a series of comprehensive LC systems in this field have been developed by employing a SEC column in the first dimension, due to the increased length and resulting extended analysis times [34,70], it is advantageous to use SEC in the second dimension in combination with either isocratic or gradient LC in the first dimension in order to accomplish an improved separation according to the chemical composition distribution of polymers. For this reason short columns (50 mm or less) [9,30,32–36] should be used for fast SEC separations, usually in isocratic elution, using THF which is the most frequently used solvent for SEC separations. With regards to SEC × LC separations, an interesting way to avoid severe band broadening and solute loss connected to the sample transfer from the SEC to the RP columns is represented by the application of a supplementary flow proposed by Winther and Reubsaet [70]. Secondary interactions on SEC, normally, require a high methanol concentration in the mobile phase that is less compatible with the loading of the RP columns. To circumvent this problem a supplementary flow was applied by adding a phase without organic modifier in a mixing Tee between the outlet of the SEC column and the inlet of the first column switch enhancing the trapping of hydrophilic compounds on 2D. Fig. 2 shows the schematic of the instrument used in this application. NP × SEC separations methods have been applied to the separation and characterization of synthetic copolymers [30] and functional acrylate polymers [32], styrene–methylacrylate (co)polymers [33], poly(bisphenol A) carbonate samples [68–69] while less frequent RP × SEC separations have been applied to polyethylene glycols and surfactants [9] and poly(ethylene glycol)–poly(vinyl acetate) graft copolymers (PEG-g-PVAc) [35]. As tool for studying band broadening in SEC chromatography a comprehensive 2D SEC × SEC has been investigated for polystyrene separation [34]. If narrow fractions are collected from the first dimension, a clear distinction between chromatographic band broadening (column and extra-column) and SEC selectivity (band broadening due to sample polydispersity) can be made as function of molecular weight. For some polar samples, mainly low molecular weight block (co)polymers with great differences in polarities of two blocks, RP × NP, can be employed: Jandera et al. [37] developed an RP × NP system for the separation of ethylene oxide–propylene oxide (EO–PO) (co)oligomers by employing a C18 microbore column with an acetonitrile–water mobile phase in the first dimension and an aminopropyl silica column (APS) with an ethanol–dichloromethane–water mixture in the second dimen-

Fig. 2. Schematic presentation of the two-dimensional HPLC system with supplementary flow for SEC × RP separation (reprinted from [70] with permission from Wiley, 2005).

sion. The system developed revealed to be almost orthogonal according to the different retention behaviour of non-polar (PO) and polar (EO) groups in both the separation modes. Furthermore, in contrast to non-aqueous NP, hydrophilic interaction liquid chromatography (HILIC) with an APS stationary phase enabled the transfer of aqueous organic fractions from the RP dimension without deteriorating the normal phase separation by disactivation of the polar adsorbent. Fig. 3 shows the comprehensive 2D RPLC × NPLC of an EO–PO co(oligomer) mixture. A 2D NP × RP system was used by Murphy et al. for the separation of alcohol ethoxylates with a dual distribution of non-polar alkyls and polar EO units by using either a silica or an amino column in the first dimension and a conventional C18 column in the second dimension [74]. A useful combination of a C18 column and methanol as the mobile phase in the first dimension and a carbon-clad zirconia

Fig. 3. The comprehensive 2D LC × LC contour plot of an EO–PO (co)oligomer. X-axis—RP retention times; Y-axis—NP (HILIC) retention times (reprinted from [37] with permission from Elsevier, 2006).

P. Dugo et al. / J. Chromatogr. A 1184 (2008) 353–368

column and ACN in the second dimension was used by Gray et al. [71,72] and Toups et al. [73] for the RP × RP separations of isomers in mixtures of oligostyrenes with various numbers of styrene units and various alkyl end-groups. In particular, the ZR-Carbon column provided the separation of diastereoisomers, whereas the C18 column resolved the species with various numbers of styrene units and alkyl lengths. 3.2. Comprehensive 2D LC separation of biological and organic compounds The wide majority of biopolymer separation and in particular proteins and peptides has been accomplished by two-dimensional electrophoresis [75–77]. It is usually based on isoelectric focusing (IEF) followed by sodium dodecyl sulfate polyacrylamide gel (2D IEF-SDS-PAGE), which are two orthogonal separation modes. However, 2D gel electrophoresis has some limitations: extensive sample handling, time consuming, difficult to automate, decreased resolving power for proteins with a molecular mass of <15 kDa as a result of their high mobility in the gel and difficult in coupling the gel to a mass spectrometer. Some of these problems can be easily overcome by using two-dimensional liquid chromatography in connection with UV absorbance [11–19,22–25,27–29,78], laser-induced fluorescence (LIF) [21,79] or MS detection [14,16–20,22,24–27,29,80]. Among the LC × LC methods employed for biopolymer separation, the most common set-up uses ion exchange chromatography in the first dimension and RP chromatography in the second dimension [13,15,16,18–27,29,80]. The first comprehensive system employed for the separation of these compounds was pioneered by Bushey and Jorgenson [12]. The system employed for the separation of protein standards and serum proteins used a 1D microbore cation exchange column and a 2D size exclusion column. Five years later, in 1995, Holland and Jorgenson [79] reported a two-dimensional liquid chromatography system based on the combination of a surprisingly long (90 cm) anion exchange microcolumn coupled to a 3 cm long reversed phase microcolumn. Both microcolumns were interfaced by two electronically controlled valves in which the effluent from 1D was collected in a sample loop and then concentrated onto the head of the RP microcolumn. The resolving power of the system was demonstrated with a two-dimensional chromatogram of peptides obtained from a tryptic digest of porcine thyroglobulin. When dealing with a complex sample an efficient sample preparation process is needed in order to fractionate it before the first dimension separation. Wagner et al. [19] used silica-based restricted access materials (RAM) with ion exchange functionalites that made them ideally suitable for sample preparation of small sized biopolymers. In fact, sensitivity was enhanced for less abundant components by loading high sample amounts, enabling the exclusion of the higher molecular weight fraction. An interesting alternative to salt gradient in the first dimension for protein separation was investigated by Pepaj et al. who used pH gradients for separations of proteins according to

363

their pIs representing a promising alternative to 2D CE in proteomic research because it is fast and can be easily automated [24,25]. An innovative method for shotgun proteomics is named multidimensional protein identification technology (MudPIT) and combines multidimensional liquid chromatography with electrospray ionization tandem mass spectrometry. The multidimensional liquid chromatography method integrates a strong cation exchange (SCX) resin and reversed phase resin in a biphasic column. In this approach no interface is used and 2D LC is obtained by a number of gradient elutions. The method was used to separate and identify the peptides from a digested soluble S. cerevisiae protein mixture [20]. Other separation modes employed for biopolymers have been carried out by using size exclusion chromatography for separation according to the molar mass distribution in the first dimension and RP chromatography in the second dimension [14,17,28]. One example of this approach was investigated by Opiteck et al. [14,17] who used a novel interface equipped with two pellicular RPLC columns in parallel rather then storage loops allowing the use of conventional analytical diameter columns for SEC and for RPLC. The addition of a mass spectrometer further reduced the possibility of undetected coeluting peaks, producing molecular weight information to accurately identify the peptides derived from the digests of ovalbumin and serum albumin [14] and E. coli lysate [17]. Fig. 4 shows a SEC × RPLC UV chromatogram of an E. coli lysate obtained using 12 SEC columns in series (300 mm × 7.8 mm i.d. each) for a total length of 3.6 m and RP columns in parallel (100 mm × 2.1 mm i.d.).

Fig. 4. A 2D SEC × RP LC UV chromatogram of an E. coli lysate showing a selection of native proteins isolated using a 2D HPLC system, where the first dimension total column length was 3.6 m (12 SEC columns in series), the flow rate was 150 ␮L/min, and the length of the RPLC columns was 10 cm (reprinted from [17] with permission from Elsevier, 1998).

364

P. Dugo et al. / J. Chromatogr. A 1184 (2008) 353–368

IEX × RP and SEC × RP have been also investigated for the separation of organic acids [78] and plant extracts [11,80] in connection with UV and mass detection. 3.3. NP × RP LC of fats, essential oils, alcohols, hydrocarbons and pharmaceutical compounds Of all the LC × LC approaches, the combination of NP and RP modes is probably the most orthogonal in nature, but also one of the most difficult to realize. For this reason, only few NPLC × RPLC applications have been published. However, the combined use of normal and reversed phase modes in the two dimensions of a comprehensive system can be very useful in the separation of complex mixtures that contain uncharged molecules of comparable dimension, different in polarity and hydrophobicity. The main problem in the interfacing of normal and reversed phase system is mobile phase immiscibility, that can cause broad and distorted peaks. When performing NPLC × RPLC analyses, using incompatible solvents in the two dimensions, the use of a microbore HPLC column in the first dimension can greatly help in avoiding peak distortion caused by mobile phase immiscibility. The microbore column used in the first dimension provides flow rates which are compatible with the sample volume injected onto the second conventional bore column, operated at a higher flow rate. Under these conditions, the dilution of 1D solvent occurs more rapidly through the 2D column, and therefore, band spreading is minimized [81]. A further complication is due to the fact that the mobile phase used in the first (normal phase) dimension is not only immiscible, but also stronger than that used in the second dimension, operated under reversed phase conditions. Under such conditions, the trapping of the solutes at the head of the secondary column becomes critical. It has been demonstrated that this effect, which generates band broadening and distorted peaks, is dependent on the volume injected and is mainly linked to the initial conditions before the injection plug becomes highly diluted by the mobile phase [81]. In this case, it is again important to use a microcolumn in the first dimension and to reduce as much as possible the strength of the initial mobile phase in the second dimension, to improve peak focusing. All the comprehensive NPLC × RPLC applications reported in literature have been developed using a similar set-up. A microbore column has been used in the first dimension, while a C18 column, monolithic in most of the cases, in the second dimension. The valve used as interface was equipped with sample loops, because the working conditions did not enable the direct entrapment at the head of the secondary columns. The first NPLC × RPLC system using microbore silica column operated at a very low flow rate in the first dimension, a monolithic C18 column operated at a very high flow rate in the second dimension and a 10-port valve equipped with two sample loops was developed and applied for the analysis of oxygen heterocyclic components in a lemon oil [63]. The relative position of the peaks in the 2D plane varied in relation to their chemical structure and according to the separation mechanism of the first and second dimensions, thus enabling a positive peak identification. In addition, characteristic UV spectra greatly supported

Fig. 5. NPLC × RPLC analysis of a citrus oil extract under optimized (A) and non-optimized (C) conditions. (B) Off-line NPLC × RPLC of fractions between 44 and 67 min, injected under the same conditions as (C) but with sample solvent with a higher amount of n-hexane (reprinted from [47] with permission from Wiley, 2006).

the identification of the analyzed components. A similar set-up was used for the analysis of a pharmaceutical mixture and citrus oil extracts [47]. It is interesting to note that performing a gradient program (using n-hexane and ethyl acetate) also in the first dimension, severe peak distortion and broadening of the components eluting at a high ethyl acetate concentration were observed. Higher concentration of n-hexane, immiscible with the RP solvents (water and acetonitrile), gave better focusing of the solutes. For the transfer of higher amount of ethyl acetate, the second dimension was started with higher water concentration, reducing the initial 2D eluent strength, but also reducing the available chromatographic 2D separation space. Fig. 5 shows the NPLC × RPLC analysis of a citrus oil extract under optimized (A) and non-optimized (C) conditions. Fig. 5B shows the off-line NPLC × RPLC of fractions between 44 and 67 min, injected under the same conditions as (C) but concentrated to dryness and diluted in a solvent with a higher amount of nhexane. These examples show how the solvent immiscibility in NP × RP comprehensive techniques is very challenging. However, the investigated NP × RP comprehensive method, is only valid for sample that the method was designed for and additional work has to be done in order to be universally adopted. Triacylglycerols (TAGs), the main constituents of food lipids, represent a typical sample for comprehensive LC analysis, due to the complexity of the fraction for the high number of combination of different fatty acids (FA) in the three glycerol positions.

P. Dugo et al. / J. Chromatogr. A 1184 (2008) 353–368

The approach for the comprehensive LC analysis of TAGs used silver ion (Ag)-HPLC in one dimension, and non-aqueous reversed phase HPLC in the second dimension. This approach is entirely orthogonal because the two separation modes are based on different retention mechanisms. Ag-HPLC separates TAGs on the basis of their double bonds number (DB), geometrical configuration and position of the DB with retention increasing as the number of DB increases [82]. The separation of TAGs with the same number of DB is critical. NARP-HPLC separates TAGs on the basis of their partition number (PN) [or equivalent carbon number (ECN) as some authors prefer], defined as: PN = CN − 2DB, where CN is the carbon number of the three FA chains. The separation of TAGs with the same PN (or ECN)

365

is critical, while positional isomers coelute completely [83]. MS systems equipped with APCI interfaces operated in positive mode are commonly used as detectors, as they can provide information on FA position and chemical structure [84,85]. An Ag-HPLC × NARPLC-APCI-MS system has been applied to the analysis of vegetable oil [44,45] and donkey milk TAGs [46]. The use of the Ag-HPLC separation in the first dimension was mandatory, as it is not possible, at the moment, to use such a column for very fast analyses. The micro-Ag column was silvered in the laboratory, starting from a strong anion exchange commercial column according to the method proposed by Christie [86] adapted for microcolumns. The obtained 2D chromatograms showed the formation of TAG group-type

Fig. 6. Schematic of a 2D LC system using one pump for both dimensions, two parallel secondary columns and guard columns in the 12-port valve (reprinted from [65] with permission from Wiley, 2006).

366

P. Dugo et al. / J. Chromatogr. A 1184 (2008) 353–368

patterns, located in characteristic positions of the 2D space in relation to their PN and DB values. The additional third MS dimension was of support for peak identification, due to the absence of peak coelutions and to the restricted number of components that can be assigned to a 2D peak spot located in a well defined position. Recently, NPLC × RPLC has been applied to the analysis of carotenoids in citrus products [87]. This is a particularly challenging application, due to the complex composition of carotenoids in natural matrices, their great structural diversity and extreme instability. Also in these applications, carotenoids were located in the 2D space according to their structural characteristics: hydrocarbons (carotens) were well separated from oxygenated counterparts (xantophylls). Furthermore, xanthophylls were separated into mono, diols and triols, and their corresponding epoxides. Retention behaviour and UV–vis absorption spectra were used to identify carotenoids, most of which are not commercially available as pure standards. 3.4. RP × RP LC separation of antioxidants, natural and environmental compounds Most RP × RP separations have been developed and applied for the determination of antioxidants [38–42], but also for the analysis of natural and environmental compounds, such as Chinese medicines [48,49], explosives and by-products in water [88], polycyclic aromatic hydrocarbons [89], aromatic amines and non-amines [64], drugs [65], aromatic compounds [90], hydrocarbons and benzene derivatives [91], phenols [43], plant extracts [42,80], indole-3-acetic acid derivates [67] and organic acids [92]. When planning a coupled column liquid chromatography system, operated in reverse phase mode, one might expect a strongly correlated system and, hence, an inefficient result. However, it has been demonstrated, that some degree of orthogonality can be achieved by using either two different sets of mobile phase solvents and one type of RP column, or a single mobile phase and two LC columns packed with different RP stationary phases [64,65,90]. In fact, different selectivities using two C18 columns can be obtained by changing the organic modifier in the two dimensions, which causes differences in polar interactions between a solute and the organic modifier existing in the stationary phase [90]. Cross-correlation between the two RP dimensions can be minimized by tuning mobile phase strength applying a solvent gradient in the primary dimension and by progressively incrementing the solvent strength in the secondary dimension [64]. The effects of increasing hydrophobicity of late eluting components is nullified, enabling small differences in stationary phases to dominate 2D retention [64,65]. Venkatramani and Patel [65] developed a system that used only one pump operated in both dimensions, as shown in Fig. 6. They coupled RP columns of different selectivity using an electronically controlled twelve-port, dual position valve (V) with guard columns (G1, G2 and G3) which enable continuous, alternating sampling of the primary column (C) eluate onto dual secondary columns (S1 and S2). Further, the 2D LC system, employed four unions with the function either of splitting (V1, V2, V4) or of merg-

Fig. 7. RP × RP LC of indolic metabolite standard mixture (reprinted from [67] with permission from Elsevier, 2006).

ing (V3). In position 1, the eluate from the primary column flows through the guard column G2 before exiting through the detector or to the waste, meanwhile the content of the guard column G1 from the previous cycle is sampled onto the secondary column S1. When the valve position is switched, the primary column eluate flows through the guard column G1 to the detector or to the waste, meanwhile the content of the guard column G2 from the previous cycle is sampled onto the secondary column S2. The flow through G3 maintains an interrupted flow alternatively through the secondary columns. A high degree of orthogonality, which indicates strong differences in the retention mechanism, can be obtained by the combination of a PEG-silica and C18 columns in the first and second dimension, respectively. This combination has been used for the separation of antioxidants [38,39,41]. Low correlation and high orthogonality can be obtained also by coupling a C18 [40] or a pentafluorophenylpropyl [67] column with a ZR-Carbon column used in the second dimension. The advantage of the ZR-Carbon column is the possibility to accelerate the second dimension analysis (which can be as fast as 21 s [67]), by using high temperatures together with a gradient program. Due to the high temperature (110 ◦ C) used in the second dimension, very fast analysis was achieved. Fig. 7 reports the separation of indolic methabolite standard mixture using C18 column in the first dimension and ZR-Carbon in the second dimension. The elucidation of these biologically relevant compounds is quite challenging as they are involved in many physiological phenomena including: flowering, fruit ripening, root initiation and senescence. A wide variety of stationary phases have been used in the first dimension separation, in combination with a C18 column used for the second dimension separation: SCX [49,92], cyano [48,49], amino [80] columns with (pentabromobenziloxy)propylsilyl-bonded phase (PBB) [89], fluoroalkylsilyl-bonded phase (FR silica) [91], tetrachloroph-

P. Dugo et al. / J. Chromatogr. A 1184 (2008) 353–368

thalimidopropyl (TCP) phase [43]. RP × RP separations have been obtained also using a C18 column in the first dimension and Cyano [42], safrole silica [88], amino [64] or SB-phenyl [65] in the second dimension. While most of RP × RP separations have been carried out using C18 at least in one of the two dimensions, several researchers have developed systems without this column type, for example, the coupling of cyano in the first and amino in the second dimension for the analysis of mixture of aromatic amines and non-amines [64]; FR silica in the first and PBB in the second dimension for the separation of hydrocarbons and benzene derivatives [91]. RP × RP separations are obtained through a slow analysis in the first and fast analysis in the second dimension. In some cases, a conventional size column (i.d. ≥ 2.1 mm) in the first dimension and a fast monolithic C18 column in the second dimension [39,41,49,64,89–91] have been used. Columns with reduced internal diameters have also been used both in the first [38,40,42,43,48,67,80,92], and in the second dimension [38–40,48,67,80]. The two dimensions are generally coupled by a multi-port valve equipped either with sampling loops or trapping columns.

[6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23]

4. Conclusions Comprehensive HPLC can be carried out simply by addition of a multiport valve(s) to one or two conventional HPLC systems. Many different configurations have been developed and applied to the analysis of real complex samples of different origin. However, most of these methods are only valid for a limited number of samples for the moment, and it is not possible to develop a generic multidimensional LC × LC approach for the separation of all sample types. Method development in LC × LC can be more difficult and time consuming then in 1D LC or off-line 2D LC. However, the great potential in terms of peak separation and identification justifies the effort due to the increased number of information obtained and will certainly attract an increasing number of researchers. The large number of publications already found in the literature confirms that the technology is no longer in the exploratory mode, but has already evolved to more routine use. Progress in the development of commercially available instruments, ready-to-use, with dedicated software for the direct conversion of raw data into 2D or 3D chromatograms, and for qualitative and quantitative analysis will undoubtfully allow the scientific community to benefit from this technique, who will experience how large is still the amount of undiscovered information relative to the composition of their samples. References [1] G. Guichon, J. Chromatogr. A 1126 (2006) 6. [2] J.M. Davis, J.C. Giddings, Anal. Chem. 55 (1983) 418. [3] H.J. Cortes (Ed.), Multidimensional Chromatography: Techniques and Applications, Marcel Dekker, New York, 1990. [4] L. Mondello, C. Lewis, K.D. Bartle (Eds.), Multidimensional Chromatography, John Wiley and Sons, Chichester, 2001. [5] C. Corradini, in: L. Mondello, C. Lewis, K.D. Bartle (Eds.), Multidimensional Chromatography, John Wiley and Sons, Chichester, 2001, p. 109.

[24] [25] [26] [27]

[28] [29] [30] [31] [32] [33] [34] [35] [36] [37] [38] [39] [40] [41] [42] [43] [44] [45] [46] [47] [48]

367

H.J. Cortes, J. Chromatogr. 626 (1992) 3. P. Schoenmakers, P. Marriott, J. Beens, LC-GC Eur. 16 (2003) 335. J.C. Giddings, Anal. Chem. 56 (1984) 1258A. R.E. Murphy, M.R. Schure, J.P. Foley, Anal. Chem. 70 (1998) 1585. J.C. Giddings, J. High Resolut. Chromatogr. Chromatogr. Commun. 10 (1987) 319. F. Erni, R.W. Frei, J. Chromatogr. 149 (1978) 561. M. Bushey, J.W. Jorgenson, Anal. Chem. 62 (1990) 161. K. Wagner, K. Racaityte, K.K. Unger, T. Miliotis, L.E. Edholm, R. Bischoff, G. Marko-Varga, J. Chromatogr. A 893 (2000) 293. G.J. Opiteck, J.W. Jorgenson, R.J. Anderegg, Anal. Chem. 69 (1997) 2283. D.R. Stoll, P.W. Carr, J. Am. Chem. Soc. 127 (2005) 5034. G.J. Opiteck, K.C. Lewis, J.W. Jorgenson, R.J. Anderegg, Anal. Chem. 69 (1997) 1518. G.J. Opiteck, S.M. Ramirez, J.W. Jorgenson, M.A. Moseley III, Anal. Biochem. 258 (1998) 349. K.K. Unger, K. Racaityte, K. Wagner, T. Miliotis, L.E. Edholm, R. Bischoff, G. Marko-Varga, J. High Resolut. Chromatogr. 23 (3) (2000) 259. K. Wagner, T. Miliotis, G. Marko-Varga, R. Bischoff, K.K. Unger, Anal. Chem. 74 (2002) 809. D.A. Wolters, M.P. Washburn, J.R. Yates, Anal. Chem. 73 (2001) 5683. L.A. Holland, J.W. Jorgenson, J. Microcol. Sep. 12 (2000) 371. H. Liu, S.J. Berger, A.B. Chakraborty, R.S. Plumb, S.A. Cohen, J. Chromatogr. B 782 (2002) 267. G. Mitulovic, R. Swart, R. van Ling, T. Jakob, J.P. Chervet, LC Packings (2004) 61. M. Pepaj, S.R. Wilson, K. Novotna, E. Lundanes, T. Greibrokk, J. Chromatogr. A 1120 (2006) 132. M. Pepaj, A. Holm, B. Fleckenstein, E. Lundanes, T. Greibrokk, J. Sep. Sci. 29 (2006) 519. Y. Wang, J. Zhang, C.-L. Liu, X. Gu, X. Zhang, Anal. Chim. Acta 530 (2005) 227. E. Machtejevas, H. John, K. Wagner, L. St¨andker, G. Marko-Varga, W.G. Forssmann, R. Bischoff, K.K. Unger, J. Chromatogr. B 803 (2004) 121. F. Bedani, W.Th. Kok, H.-G. Janssen, J. Chromatogr. A 1133 (2006) 126. C. Liu, X. Zhang, J. Chromatogr. A 1139 (2007) 191. A. van der Horst, P.J. Schoenmakers, J. Chromatogr. A 1000 (2003) 693. K. Im, Y. Kim, T. Chang, K. Lee, N. Choi, J. Chromatogr. A 1103 (2006) 235. X. Jang, A. Van der Horst, V. Lima, P.J. Schoenmakers, J. Chromatogr. A 1076 (2005) 51. S.J. Kok, Th. Hankemeier, P.J. Schoenmakers, J. Chromatogr. A 1098 (2005) 104. S.T. Popovici, A. van der Horst, P.J. Schoenmakers, J. Sep. Sci. 28 (2005) 1457. D. Knecht, F. Rittig, R.F.M. Lange, J. Chromatogr. A 1130 (2006) 43. G. Viv´o-Troyols, P.J. Schoenmakers, J. Chromatogr. A 1120 (2006) 273. ˇ P. Jandera, J. Fischer, H. Lahovsk´a, K. Novotn´a, P. Cesla, L. Kolaˇrov´a, J. Chromatogr. A 1119 (2006) 3. E. Blahov´a, P. Jandera, F. Cacciola, L. Mondello, J. Sep. Sci. 29 (2006) 555. F. Cacciola, P. Jandera, E. Blahov´a, L. Mondello, J. Sep. Sci. 29 (2006) 2500. F. Cacciola, P. Jandera, L. Mondello, J. Sep. Sci. 30 (2007) 462. ˇ F. Cacciola, P. Jandera, Z. Hajd´u, P. Cesla, L. Mondello, J. Chromatogr. A 1149 (2007) 73. M. Kivilompolo, T. Hy¨otyl¨ainen, J. Chromatogr. A 1145 (2007) 155. A.P. Kohne, T. Welsch, J. Chromatogr. A 845 (1999) 463. L. Mondello, P.Q. Tranchida, V. Stanek, P. Jandera, G. Dugo, P. Dugo, J. Chromatogr. A 1086 (2005) 91. P. Dugo, T. Kumm, M.L. Crupi, A. Cotroneo, L. Mondello, J. Chromatogr. A 1112 (2006) 269. P. Dugo, T. Kumm, B. Chiofalo, A. Cotroneo, L. Mondello, J. Sep. Sci. 29 (2006) 1146. I. Franc¸ois, A. de Villiers, P. Sandra, J. Sep. Sci. 29 (2006) 492. X. Chen, L. Kong, X. Su, H. Fu, J. Ni, R. Zhao, H. Zou, J. Chromatogr. A 1040 (2004) 169.

368

P. Dugo et al. / J. Chromatogr. A 1184 (2008) 353–368

[49] L. Hu, X. Chen, L. Kong, X. Su, M. Ye, H. Zou, J. Chromatogr. A 1092 (2005) 191. [50] T. Stroink, M.C. Ortiz, A. Bult, H. Lingeman, G.J. de Long, W.J.M. Underberg, J. Chromatogr. B 817 (2005) 49. [51] C.R. Evans, J.W. Jorgenson, Anal. Bioanal. Chem. 378 (2004) 1952. [52] H. Wang, S. Hanash, J. Chromatogr. B 787 (2003) 11. [53] M. Gao, C. Deng, S. Lin, F. Hu, J. Tang, N. Yao, X. Zhang, J. Sep. Sci. 30 (2007) 785. [54] S.P. Dixon, I.A.D. Perrett, Biomed. Chromatogr. 20 (2006) 508. [55] P.Q. Tranchida, P. Dugo, G. Dugo, L. Mondello, J. Chromatogr. A 1054 (2004) 3. [56] P.Q. Tranchida, P. Donato, P. Dugo, G. Dugo, L. Mondello, Trends Anal. Chem. 26 (3) (2007) 191. [57] Z. Liu, M.L. Lee, J. Micro Sep. 12 (2000) 241. [58] E. Hogendoorn, P. van Zoonen, F. Hernandez, LC-GC Eur. (2003) 2. [59] P. Jandera, J. Sep. Sci. 29 (2006) 1763. [60] B.L. Karger, L.R. Snyder, Cs. Horvath (Eds.), An Introduction to Separation Science, Wiley Interscience, New York, 1973, p. 560. [61] J.V. Selley, J. Chromatogr. A 962 (2002) 21. [62] N.E. Hoffman, S.-L. Pan, A.M. Rustum, J. Chromatogr. 465 (1989) 189. [63] P. Dugo, O. Favoino, R. Luppino, G. Dugo, L. Mondello, Anal. Chem. 76 (2004) 2525. [64] C.J. Venkatramani, Y. Zelechnook, Anal. Chem. 75 (2003) 3484. [65] C.J. Venkatramani, A. Patel, J. Sep. Sci. 29 (2006) 510. [66] N. Tanaka, H. Kobayashi, N. Ishizuka, H. Minatuchi, K. Nakanishi, K. Hosoya, T. Ikegami, J. Chromatogr. A 965 (2002) 35. [67] D.R. Stoll, J.D. Cohen, P.W. Carr, J. Chromatogr. A 1122 (2006) 123. [68] L. Coulier, E.R. Kaal, Th. Hankemeier, Polym. Degrad. Stab. 91 (2006) 271. [69] L. Coulier, E.R. Kaal, Th. Hankemeier, J. Chromatogr. A 1070 (2005) 79. [70] B. Winther, J.L.E. Reubsaet, J. Sep. Sci. 28 (2005) 477. [71] M.J. Gray, A.P. Sweeney, G.R. Dennis, P.J. Slonecker, R.A. Shalliker, Analyst 128 (2003) 598.

[72] M.J. Gray, G.R. Dennis, P.J. Slonecker, R.A. Shalliker, J. Chromatogr. A 1041 (2004) 101. [73] E.P. Toups, M.J. Gray, G.R. Dennis, N. Reddy, M.A. Wilson, R.A. Shalliker, J. Sep. Sci. 29 (2006) 481. [74] R.E. Murphy, M.R. Schure, J.P. Foley, Anal. Chem. 70 (1998) 4353. [75] A. Sickmann, W. Dormeyer, S. Workelkamp, D. Woitalla, W. Kuhn, H.E. Meyer, Electrophoresis 21 (2000) 2721. [76] I. Nilsson, M. Utt, H.O. Nilsson, A. Ljungh, T. Wadstrom, Electrophoresis 21 (2000) 2670. [77] K. Marcus, D. Immier, J. Sternbeger, H.E. Mejer, Electrophoresis 21 (2000) 2622. [78] C.G. Fraga, C.A. Carley, J. Chromatogr. A 1096 (2005) 40. [79] L.A. Holland, J.W. Jorgenson, Anal. Chem. 67 (1995) 3275. [80] J. P´ol, B. Hohnov´a, T. Hy¨otyl¨ainen, J. Chromatogr. A 1150 (2007) 85. [81] P. Dugo, M. del Mar Ram´ırez Fern´andez, A. Cotroneo, G. Dugo, L. Mondello, J. Chromatogr. Sci. 44 (2006) 561. [82] W.W. Christie, J. Chromatogr. 454 (1998) 273. [83] N.K. Andrikopoulos, Crit. Rev. Food Sci. 42 (2002) 473. [84] M. Holˇcapek, M. Lisa, P. Jandera, N. Kab´atov´a, J. Sep. Sci. 28 (2005) 1315. [85] M. Holˇcapek, P. Jandera, P. Zderadicka, L. Hruba, J. Chromatogr. A 1010 (2003) 195. [86] W.W. Christie, J. High Resolut. Chromatogr. Chromatogr. Commun. 10 (1987) 148. ˇ r´ıkov´a, T. Kumm, A. Trozzi, P. Jandera, L. Mondello, Anal. [87] P. Dugo, V. Skeˇ Chem. 78 (2006) 7743. [88] A.P. K¨ohne, U. Dornberger, T. Welsch, Chromatographia 48 (1998) 9. [89] T. Murahashi, Analyst 128 (2003) 611. [90] T. Ikegami, T. Hara, H. Kimura, H. Kobayashi, K. Hosoya, K. Cabrera, N. Tanaka, J. Chromatogr. A 1106 (2006) 112. [91] N. Tanaka, H. Kimura, D. Tokuda, K. Hosoya, T. Ikegami, N. Ishizuka, H. Minakuchi, K. Nakanishi, Y. Shintani, M. Furuno, K. Cabrera, Anal. Chem. 76 (2004) 1273. [92] J. P´ol, B. Hohnov´a, M. Jussila, T. Hy¨otyl¨ainen, J. Chromatogr. A 1130 (2006) 64.