Polymer separations by liquid interaction chromatography: Principles – prospects – limitations

G Model CHROMA-354956; No. of Pages 18

ARTICLE IN PRESS Journal of Chromatography A, xxx (2013) xxx–xxx

Contents lists available at ScienceDirect

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

Review

Polymer separations by liquid interaction chromatography: Principles–prospects–limitations Wolfgang Radke ∗ Fraunhofer-Institut für Betriebsfestigkeit und Systemzuverlässigkeit LBF, Bereich Kunststoffe, Schlossgartenstraße 6, D-64289 Darmstadt, Germany

a r t i c l e

i n f o

Article history: Received 24 June 2013 Received in revised form 26 October 2013 Accepted 4 December 2013 Available online xxx Keywords: Polymer chromatography Critical chromatography Gradient chromatography Two-dimensional chromatography Polymer heterogeneity

a b s t r a c t Most heterogeneities of polymers with respect to different structural features cannot be resolved by only size exclusion chromatography (SEC), the most frequently applied mode of polymer chromatography. Instead, methods of interaction chromatography became increasingly important. However, despite the increasing applications the principles and potential of polymer interaction chromatography are still often unknown to a large number of polymer scientists. The present review will explain the principles of the different modes of polymer chromatography. Based on selected examples it will be shown which separation techniques can be successfully applied for separations with respect to the different structural features of polymers. © 2013 Elsevier B.V. All rights reserved.

Contents 1. 2.

3.

4.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Liquid chromatography of polymers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Peculiarities of polymer chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Elution modes of polymers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Barrier techniques and SEC-gradients . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. Two-dimensional liquid chromatography of polymers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. One-dimensional separations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1.1. Homopolymer separation according to end-group functionalization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1.2. Homopolymer separation according to topology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1.3. Separations by chemical composition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1.4. Separations by stereoregularity and microstructure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Two-dimensional separations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.1. Separation by functional end-groups and molar mass . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.2. Separation of statistical copolymers chemical composition and molar mass . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.3. Separation of segmented copolymers by chemical composition and molar mass . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.4. Separation of branched polymers by degree of branching and size . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions and outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

∗ Present address: PSS Polymer Standards Service, In der Dalheimer Wiese 5, 55120 Mainz, Germany, Tel.: +49 6131 96239 39; fax: +49 (0)6131 96239 11. E-mail address: [email protected] 0021-9673/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.chroma.2013.12.010

Please cite this article in press as: W. Radke, J. Chromatogr. A (2013), http://dx.doi.org/10.1016/j.chroma.2013.12.010

00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00

G Model CHROMA-354956; No. of Pages 18

ARTICLE IN PRESS W. Radke / J. Chromatogr. A xxx (2013) xxx–xxx

2

1. Introduction Polymers are part of our daily life. They are found in a huge number of very different applications ranging from packaging applications to microelectronics and from paints and nylon stocking to biodegradable implants. Although the applications of polymers seem to be unlimited, it is astonishing that a relatively small number of monomers are sufficient to produce these materials. The type and amount of the different monomers, the way they are linked together, the chain length and finally how the different polymers might be blended and modified by additives are key parameters which make the difference between a suitable and an inapplicable material. A selection of structures which can be synthesized and the structural parameters influencing polymer properties are schematically depicted in Fig. 1. Since the molecular structure transforms via self-organization into macroscopic properties of the materials, proper techniques to characterize the molecular structure are required to establish structure–property relations and to ensure a constant quality of the products. Schemes like the one in Fig. 1 are idealized. In general polymer samples are heterogeneous. In the simplest case heterogeneity due to molar mass, or equivalent due to chain length, exists. Other types of heterogeneity involve heterogeneity with respect to tacticity, functionalization, topology, chemical composition, etc. All these heterogeneities need to be characterized to allow establishing structure–property relations or to assure quality control. While average molecular properties like molar mass or composition can be obtained by spectroscopic techniques, the characterization of heterogeneities requires separation of the different structures. That is where separation methods, especially chromatographic ones, become important. It is not the intention of the article to provide a comprehensive review of the achievements and numerous applications of polymer chromatography. According to the authors experience it is a lucky exception if one finds the right chromatographic conditions for a specific separation problem in the literature. This is due to the huge number of different polymers that need to be characterized, according to specific features. Instead the present article would like to give guidelines on how to select a separation strategy suitable to tackle a general separation problem. A general separation problem means that details on the monomer units or the selected solvents and stationary phase will not be given. However, the guidelines will be explained and discussed on selected examples. Having identified a suitable strategy, the

Fig. 1. A selection of the multitude of polymer structures which can be synthesized. Reprinted from Ref. [1], Copyright (2012), with permission from Elsevier.

next step would be identification of suitable stationary and mobile phases. Suitable starting points might be critical conditions or gradient separations for the polymer class under investigation. A large compilation of critical conditions was published by Macko and Hunkeler [2]. Other valuable sources of separation conditions by polymer classes might be found in other reviews on polymer chromatography [3–7].

2. Liquid chromatography of polymers 2.1. Peculiarities of polymer chromatography One peculiarity of synthetic polymers, differentiating them from typical low molar mass analytes, is the heterogeneity inherent in any polymeric material. The number of different structures is considerable even for homopolymers prepared by (ideal) living polymerization techniques, which are often incorrectly referred to as being monodisperse. For example, a sample having a number average degree of polymerization, Pn = 100 and a dispersity of D = 1.01, which is close to the theoretical limit for an ideal living polymerization, contains all chains within the range 80 < P < 120, in order for account for 99% of all chains. This means that at least 40 different but very similar species have to be regarded. If the molar mass or the dispersity increases, the number of molecules differing in chain length grows dramatically. The introduction of a second comonomer (B) adds another level of complexity and increases the number of possible structures by magnitudes. In principle in a copolymer chain of degree of polymerization, P, between zero and P monomer units of type B can be incorporated, meaning P + 1 chains of different composition exist at a given degree of polymerization, without considering the different possible arrangements of the monomers along the chain. Thus, a typical polymer sample contains a huge number of different, but often very similar molecular structures. As a consequence, in chromatography of synthetic polymers individual separated chromatographic peaks for each species are rather the exception than the rule. Instead, broad peaks are observed. These are, however, no indication of a poor separation. Still today, size exclusion chromatography (SEC), which is based on steric exclusion of the macromolecules from the pores of the stationary phase, is the most widely used separation technique for macromolecules. However, methods of interaction chromatography have become increasingly important over the last decade, as these methods provide information on heterogeneities which are not accessible by SEC, despite the hyphenation of SEC with molar mass or chemically sensitive detectors. The reason for the late evolution of interaction chromatography of polymers is that establishing isocratic conditions for interaction chromatography of high molar mass polymers is rather difficult. The early isocratic experiments of interaction chromatography for polymers revealed that macromolecules elute either before the solvent peak at elution volumes which correspond to a separation by SEC, or the macromolecules adsorb very strongly to the stationary phase such that no or only incomplete elution was observed. The transition from SEC to complete adsorption was found to be very sensitive to small variations in the composition of the mobile phase. Therefore, isocratic adsorption chromatography showed similarities to an “on/off” mechanism, giving the impression that interaction polymer chromatography is non-controllable. Glöckner showed that this effect has its origin in the high molar mass nature of polymers and can be explained by a multiple attachment mechanism [8]. An individual repeating unit can be either in an adsorbed or in the desorbed state. Since even adsorption of a single repeating unit to the stationary phase prevents movement along the column, the macromolecule can only migrate through the chromatographic column, if all repeating units are desorbed at the same

Please cite this article in press as: W. Radke, J. Chromatogr. A (2013), http://dx.doi.org/10.1016/j.chroma.2013.12.010

ARTICLE IN PRESS

G Model CHROMA-354956; No. of Pages 18

W. Radke / J. Chromatogr. A xxx (2013) xxx–xxx

time. Assuming to a first approximation statistical independence of the repeating units results in the following interesting features: • At isocratic conditions the retention of a polymer molecule increases approximately exponentially with degree of polymerization. • A high molar mass molecule will have a very strong retention even if the retention of an isolated repeating unit is extremely weak. • A tiny variation of the interaction strength of the repeating unit with the stationary phase suffices to alter the retention behavior from non-adsorbed SEC behavior to extremely strong adsorption. Another problem complicating interpretation of polymer interaction chromatography results from the fact that polymers are often soluble only in a very limited number of solvents. Often gradient experiments were performed under conditions, where effects of interaction with the stationary phase and solubility were acting simultaneously. The combination of effects due to adsorption/desorption and precipitation/dissolution complicated understanding polymer chromatography. 2.2. Elution modes of polymers In general the retention volume, V, of an analyte in chromatography can be described as V = Vi + K × Vp

(1)

where Vi and VP are the interstitial and pore volume of the stationary phase. K is the distribution coefficient, which is defined as the ratio of the analyte concentrations in the stationary and in the mobile phase (K = cst /cm ). According to the modern theory of polymer interaction chromatography, as set up since the 1980s [9–17], the distribution coefficient of a homopolymer can be written as

 Y (−cR) − 1

K =1+

2R D

Y (−x) =

exp(x2 )[1 − erf (−x)]

cR

2 −√ 

 (2)

In Eq. (2) the distribution coefficient is a function of three parameters: the radius of gyration of the polymer chain, R, which scales with molar mass, the pore diameter D and an adsorption interaction parameter, c, which is a measure of the deviation of the interaction energy of the repeating unit with the stationary phase from a critical value. At this critical value of the interaction energy the entropic contributions resulting from steric exclusion effects are exactly compensated by the enthalpic contributions of the interaction of the repeating units with the stationary phase. Since the parameter c always shows up in a product cR, any deviation of the interaction energy from its critical value is amplified by the molar mass of the polymer chain, via its influence on the radius of gyration, R. Depending on the magnitude of the interaction parameter, c, three different molar mass dependences are observed for a homologues series. If c < 0, i.e. the interaction energy of the repeating units is weaker than the critical value, the distribution coefficient will be less than unity and will decrease with increasing size of the macromolecule, i.e. with increasing molar mass. According to Eq. (1) for K < 1 the polymer molecule elutes before the void volume of the column (Vi + VP ) in order of decreasing molar mass. Such an elution behavior is typical for a separation by size exclusion chromatography (SEC). If the interaction energy exceeds the critical value, i.e. c > 0, K exceeds unity and the polymer is adsorbed by the column. In the adsorption regime (liquid interaction chromatography, LAC) retention increases rapidly with molar mass, as predicted by

3

Glöckners multiple attachment mechanism. The nearly exponential increase in retention with the number of repeating units is well known as Martin’s rule in the literature [18]. A special situation arises if c = 0. At these conditions, K for a linear non-functionalized homopolymer becomes unity irrespective of the molar mass of the macromolecule. Consequently all linear polymers having the same type of repeating unit will elute at the void volume of the column. Since the repeating units do not contribute to the elution volume at these conditions, the repeating units are often referred to as being chromatographically invisible. Chromatography performed at these conditions is named critical chromatography (CC), liquid chromatography at critical conditions (LCCC) or liquid chromatography at the critical adsorption point (LC-CAP) [19,20]. If separations by small structural differences are to be performed, e.g. according to functionalized end-groups, such minor structural differences are usually hidden by the molar mass distribution. However, since at critical conditions the number of repeating units does not contribute to retention, LCCC allows performing separations by the type and number of functionalized end-groups. It is important to note that critical conditions in principle can be established on any porous stationary phase. There are no columns specifically dedicated to critical chromatography. The critical eluent conditions for a given homopolymer correspond to a specific eluent composition at a given temperature on a selected stationary phase. According to theory the critical point is independent of pore width. Although according to theory critical conditions exist even if the pore size is less than the size of the analyte, such a situation should be avoided, because at critical conditions retention is very sensitive toward small variations of eluent composition. This sensitivity becomes more important the larger the size of the molecule relative to the pore size [16]. This means a given solvent or solvent mixture might act as a critical solvent at lower molar masses, but will result in SEC or LAC type of elution for higher ones. There are still open questions concerning a thorough understanding of critical chromatography. The theory of critical conditions assumes an established equilibrium between the polymer concentrations inside and outside the pore. However, especially for analyte sizes exceeding the pore size, the question arises of how long it will take to establish the equilibrium? For high molar mass samples establishing and maintaining critical conditions is difficult. Sample recovery sometimes is a concern. Often experiments at critical conditions are performed at conditions close to the precipitation threshold which complicate interpretation of data. How will effects of excluded volume, which are ignored in the theory, alter the prediction of the chromatographic behavior? Simulations including excluded volume effects revealed a dependence of critical conditions on pore size and a stronger adsorption at the critical interaction energy for small pores [21]. How would specific interactions between unlike segments within the same molecule alter the predictions? However, despite these shortcomings and open questions, today critical chromatography still is the only method allowing separating high molar mass polymer chains by the types of end-group functionalization or to estimate the molar mass distribution of an individual block in a binary block copolymer. Fig. 2 summarizes the dependences of elution volume on molar mass in SEC, LAC and LCCC. As described above, retention at adsorbing conditions increases rapidly with molar mass. Keeping in mind that all synthetic polymers exhibit a molar mass distribution, it becomes clear that the higher molar mass fractions of the molar mass distribution might not elute from the column in a reasonable time. This is the reason for the incomplete recovery frequently found at isocratic adsorbing conditions. This is true not only for samples having a broad molar mass distribution, but also for those of low dispersity. Therefore, gradient experiments are usually conducted, when

Please cite this article in press as: W. Radke, J. Chromatogr. A (2013), http://dx.doi.org/10.1016/j.chroma.2013.12.010

G Model CHROMA-354956; No. of Pages 18 4

ARTICLE IN PRESS W. Radke / J. Chromatogr. A xxx (2013) xxx–xxx

Fig. 2. Schematic representation of the dependence of elution volume on molar mass for isocratic conditions (SEC, LAC and LCCC) as well as for a linear gradient.

samples of different chemical composition need to be separated. In gradient chromatography (also named gradient elution liquid chromatography (GELC), eluent gradient liquid chromatography (EGLC), gradient polymer elution chromatography (GPEC), solvent gradient interaction chromatography (SGIC)) the sample is dissolved and injected in a solvent which allows for adsorption of the sample components onto the stationary phase. Ideally the eluent strength is increased systematically during the course of the chromatographic experiment by changing the eluent composition. The adsorbed polymer molecules start moving through the column if it is surpassed by a sufficiently strong eluent composition. Due to the strong molar mass dependence of retention, desorption occurs only in a very narrow window of eluent compositions close to the critical one. Lower molar mass polymer molecules, being relatively weakly adsorbed, might start migrating already at an eluent composition below critical. In essence, with increasing molar mass the polymer molecules start moving in eluent compositions closer to the critical one. Due to this mechanism low molar mass samples elute in order of increasing molar mass. However, with increasing molar mass, the molar mass influence vanishes and high molar mass samples elute nearly independently of molar mass at an eluent composition close to the critical one. This behavior has been predicted and is also observed experimentally [22,23]. The typical dependence of retention volume on molar mass in a linear gradient is also given in Fig. 2. Instead of changing the interaction of the repeating units with the stationary phase by variation of the eluent composition, temperature changes can be applied for the same purpose. A major advantage of temperature gradient interaction chromatography (TGIC) over solvent gradients is the enhanced capability to fine tune the interaction energy, which allows for very sensitive separations. Another significant advantage of temperature gradients over solvent gradients is the higher freedom in choosing detectors. A proper temperature control of the detector allows, e.g. application of light scattering detection for characterization of the eluting fractions. This is not possible when using solvent gradients. On the other hand, the range of interaction energies that can be covered by temperature variations is very narrow. Thus, the application of TGIC is limited to (adsorbing) samples which do not differ strongly in adsorption energy. Another interesting detail of TGIC is that it allows decoupling solvent flow from the increasing interaction strength, while in solvent gradients changing the eluent strength requires an eluent flow. As a consequence, homopolymers might elute in TGIC in increasing or decreasing order or even nearly independent of molar mass. Which elution order is observed depends on rate of temperature increase relative to the flow rate. Mechanistic details and applications of TGIC are given in [4,24–29].

It should be noted that all above descriptions relate to pure adsorption/desorption equilibria. However, polymers are often soluble in only a limited number of solvents. Thus, gradient applications are often run at conditions which are influenced by precipitation/dissolution processes as well. Especially in the early days of polymer gradient chromatography the simultaneous occurrence of different separation mechanisms complicated interpretation of chromatographic experiments. Gradient polymer elution chromatography (GPEC) in its original form was applied to separations based on solubility differences of the components rather than on adsorption. The existence of critical conditions for a given stationary phase/mobile phase system is closely related to the question whether the retention in a gradient is based on an adsorption/desorption or on a precipitation/dissolution equilibrium. A very good explanation of the relations between solubility and critical conditions was given by Brun [22]. If a sample is precipitated within the column at injection, it will redissolve at its solubility threshold ˚sol . Dissolution of the precipitated samples might occur at a solvent composition above (˚sol > ˚cr ) or below (˚sol < ˚cr ) the critical eluent composition for the specific polymer/stationary phase/mobile phase system. In the former situation the sample will not experience adsorbing interaction after dissolution. Thus, it will be migrate through the column at the same velocity as the surrounding solvent. Consequently the sample will elute at ˚ = ˚sol. This implies that no critical conditions can be established in the selected phase system, as any composition capable to dissolve the sample will prevent interaction with the stationary phase rendering SEC like elution. In the latter case, the sample is dissolved but remains adsorbed onto the stationary phase. With increasing eluent strength it becomes desorbed and elutes close to the critical eluent composition as explained above. Since the sample is soluble in the critical eluent, critical conditions can be established for the selected phase system. Another problem arising from the limited number of solvents for most polymers is the occurrence of so called breakthrough peaks, the origin was thoroughly studied by Jiang et al. [30]. These peaks arise if the sample is dissolved in a solvent which at the same time is a strong eluent for the phase system under investigation. Upon injection into the weak eluent at the beginning of the gradient, parts of the sample may stay non-adsorbed and will pass the column within the solvent plug introduced upon injecting the sample. As a consequence, a peak around t0 , the retention time of the solvent, is observed, besides a regular peak which elutes within the gradient. Breakthrough peaks complicate data interpretation and quantitation of the amounts of the different components. Approaches to reduce or even eliminate the breakthrough effect by variation of concentration, injection volume or mixing the gradient components after the injector have been discussed [30,31]. A recent approach to eliminate breakthrough peaks is the application of SEC gradients, which will be discussed together with barrier techniques in the following. 2.3. Barrier techniques and SEC-gradients Closely related with the breakthrough phenomenon are limiting conditions, introduced in a series of papers by Berek [20,32,33]. Among the various limiting condition approaches, the most promising one is chromatography at limiting conditions of desorption (LC-LCD). LC-LCD refers to isocratic experiments in a strong eluent which results in SEC conditions. At SEC conditions, the polymer elutes before the solvent peak and thus its velocity is higher than that of the surrounding eluent. As a consequence the polymer might catch up with a solvent front (the barrier) of another liquid introduced shortly before the sample. When the sample components reach the barrier, molecules for which the barrier is a strong

Please cite this article in press as: W. Radke, J. Chromatogr. A (2013), http://dx.doi.org/10.1016/j.chroma.2013.12.010

G Model CHROMA-354956; No. of Pages 18

ARTICLE IN PRESS W. Radke / J. Chromatogr. A xxx (2013) xxx–xxx

5

gradients have also been run by applying solvent/non-solvent gradients. In that case the polymer adapts itself at the precipitation threshold. In analogy to LC-LCD columns with small pores but high porosity are required. It should be noticed that the separation range of SEC-gradients is restricted by the pore volume of the column. 2.4. Two-dimensional liquid chromatography of polymers

Fig. 3. Schematic representation of the mechanism of SEC-gradients. Lower part: at time of injection, upper: sample components have adjusted themselves at their respective adsorption thresholds.

eluent can migrate through the barrier, while molecules for which the injected barrier is a weak eluent or even a non-solvent cannot. These components will be either adsorbed or precipitated (liquid chromatography at limiting conditions of insolubility, LC LCI [34]) within it. Such molecules therefore can neither migrate through the barrier nor can they fall behind, because the strong eluent behind the molecule forces the molecules to move with a velocity higher than the surrounding solvent. Consequently such molecules pile up at the back of the barrier and elute nearly independently of molar mass right behind it. By this mechanism LC-LCD allows separating a sample in two fractions, those for which the injected barrier is a strong eluent and those for which it is a weak one, resulting in adsorption. It becomes clear that limiting conditions of desorption are closely related to the critical eluent composition, which separates the adsorption and the SEC regime. Drawbacks of LC-LCD are the limited number of fractions that can be separated and that the solvent or solvent mixture of the barrier as well as barrier width and the delay between barrier and samples need to be carefully optimized. In addition, the elution volume range useful for the separation is restricted to the pore volume of the column. Since the polymer molecules need to be much faster than the surrounding eluent molecules in order to catch up with the barrier, small pore size columns need to be applied. Therefore, the columns applied for barrier techniques should exhibit a large volume but small pore size to be efficient. In order to increase the pore volume columns with the dimensions typical for SEC are frequently applied. The drawbacks of the limited number of fractions and finding the proper barriers can be overcome using SEC-gradients [35–37]. In SEC-gradients a solvent gradient is started before the sample is injected at strong eluent conditions at the end of the gradient (Fig. 3). Similar to LC-LCD, the sample components experience strong eluent conditions at the time of injection. Due to the exclusion from the pores of the stationary phase, the macromolecules overtake the different eluent compositions of the gradient until they reach an adsorption threshold at which molecules of a given structure accumulate, similar to the barrier in LC-LCD. The adsorption threshold depends on the chemical structure or composition of the macromolecules. Thus, a SEC-gradient acts as a huge number of consecutive barriers of different eluent compositions and the macromolecules automatically adapt at their respective adsorption thresholds. From the discussion of the three isocratic modes of polymer chromatograph (SEC, LCCC, LAC) and on gradient chromatography, it becomes clear that the adsorption threshold in SEC-gradients should be closely related to the critical eluent composition of the respective polymer. SEC

The above described chromatographic approaches cannot completely separate complex mixtures, because the elution volumes in the different chromatographic modes are influenced by molar mass and chemical composition, etc. Thus, if a separation by a single structural parameter is achieved, information on the other structural parameters is usually lost. For example, LCCC allows separating homopolymers by functional end-groups. However, at critical conditions no information on molar mass and molar mass distribution can be obtained, since the effect of molar mass is eliminated. Similarly in gradient chromatography of copolymers or polymer blends, at low molar masses the elution volume is influenced by molar mass and chemical composition, resulting in coelution of species differing in both structural parameters. On the other hand, for high molar masses the influence of molar mass vanishes allowing separating by chemical composition at the cost, however, of coelution of molecules exhibiting identical composition but differing in molar mass. In order to separate such complex polymers combinations of different separation mechanisms in a two-dimensional approach can be applied. In two-dimensional chromatography the sample is fractionated in a 1st dimension and the fractions are further analyzed by a different chromatographic mechanism in a second chromatographic experiment. If the two-dimensional separation is performed such that the same percentage of all sample components are analyzed in the second dimension, without significant loss of the 1st dimension resolution, the separation is termed comprehensive [38]. Comprehensive two-dimensional chromatography is indicated by a multiplex sign (×), in contrast to a hyphen (-) which merely indicates a direct coupling of a chromatographic separation with a detection device or another chromatographic separation. Thus, LCCC × SEC indicates a separation by critical chromatography in the 1st dimension where all sample components are subjected to a SEC separation in the 2nd dimension, while at the same time the peak resolution of the 1st dimension chromatogram is maintained. In contrast, gradient chromatography–SEC merely indicates that fractions of a gradient separation are on-line subjected to a subsequent SEC analysis. Such an analysis might involve heart cutting of selected fractions. The easiest way to perform a two-dimensional separation is by manual fractionation or by use of a fraction collector (offline two-dimensional chromatography). The same chromatographic instrumentation can be used for the 2nd dimension separation after changing column and/or the eluent to achieve a different mechanism. Today, automated online two-dimensional chromatography is frequently applied. The automatic approach fulfills the requirements of reduced work load, higher sample throughput, unattended operation, increased reproducibility and reduces sources of human errors. A typical setup for online two-dimensional separations is schematically depicted in Fig. 4. The setup consists of a HPLCsystem, the detector of which is replaced by a software controlled transfer valve equipped with two injection loops of identical volume. A second pump is added to provide the 2nd dimension eluent flow through the valve to the 2nd dimension column and further to the detector. The heart of the two-dimensional setup is the transfer valve, which is either an eight or a ten port two position valve, equipped

Please cite this article in press as: W. Radke, J. Chromatogr. A (2013), http://dx.doi.org/10.1016/j.chroma.2013.12.010

G Model CHROMA-354956; No. of Pages 18 6

ARTICLE IN PRESS W. Radke / J. Chromatogr. A xxx (2013) xxx–xxx

Fig. 4. Setup of online two-dimensional chromatography (left). Configuration of a 10 port 2 position valve (right) in two-dimensional chromatography. (For interpretation of the references to color in the text, the reader is referred to the web version of this article.)

with two transfer loops of identical loop size. In Fig. 4 (right) the effluent of the 1st dimension fills the blue loop, while the content of the red loop is analyzed in the 2nd dimension column. When the 2nd dimension analysis is finished, the valve is switched such that the content of the blue loop is analyzed, while the red loop is getting filled. By switching the valve back and forth a series of 2nd dimension chromatograms is obtained. Each chromatogram is correlated via its injection time to the elution volume in the 1st dimension. The series of 2nd dimension chromatograms is converted by suitable software in contour plots or other appropriate data representations. The chromatograms of both dimensions can be reconstructed, to compare the obtained resolution with that obtained if only a 1st dimension or 2nd dimension separation would be performed. This allows evaluating whether the efficiency of the 1st dimension is maintained. By plotting the integral intensities of each SEC run as a function of the 1st dimension retention time, the 1st dimension chromatogram is reconstructed. In analogy summing up the intensities of the 1st dimension chromatogram at a given 2nd dimension retention time results in a reconstruction of the 2nd dimension chromatogram. It should be noted that these reconstructed chromatograms are sometimes incorrectly referred to as “projections”. As mentioned, the transfer valve is either a ten or an eight position valve. Application of a ten position valve has the advantage that the effluent enters and leaves the loops in the same direction, while in the eight port configuration filling and emptying of one loop happens in the opposite direction. This might present a small disadvantage in case of only partially filled loops, because the flow fronts of the two loops reach the column at slightly different times after injection. Two-dimensional separations require several optimizations sometimes at the expense of the separation efficiency in the individual dimension. For comprehensive two-dimensional analysis the flow speeds and loop volumes have to be carefully adjusted. The loops have to be filled in the time required for the 2nd dimension analysis. A too fast 1st dimension flow speed will result in overfilling the loop and consequently in material loss via the waste outlet. Thus, a fast flow rate in the 2nd and a very slow flow rate in the 1st dimension are required. The former requirement can be achieved using optimized column dimensions. Two approaches are presently applied. Short columns of a large diameter, having the same overall volume as conventional columns, allow separations

at high flow rates, while still staying close to the minimum in the Van Deemter curve. The advantage of this approach is that no or only little optimization for band broadening is needed. However, because high flow rates of up to 10 mL/min can be applied by these columns, appropriate pumps are required and detectors have to be chosen which can withstand the high backpressure, if no flow splitting is intended. The second approach uses columns of conventional diameters. The reduction in analysis time is obtained by shortening the column. The benefit of this approach is that conventional flow rates are applied. Thus, no problems arise from high flow rates. On the other hand the ratio of the stationary phase to the analyte becomes unfavorable, which might result in lower separation efficiencies. In addition, the lower column volume requires lower injection volumes and optimization for band broadening. Due to the repeated dilution in the two-dimensional separations, very sensitive detection is needed. Often rather high sample loads are used, despite a potential loss in resolution due to column overloading, in order to partially compensate for the reduction in concentration. Care has to be taken to select the right separation order. Since the eluent of the 1st dimension is injected into the 2nd dimension, the 1st dimension eluent has to be compatible with the 2nd dimension column. If one separation is to be performed by SEC, it is usually selected as the 2nd dimension, due to the following reasons: 1. Adsorption polymer chromatography seems to be less influenced by column overloading than SEC. Therefore larger sample amounts can be loaded allowing better detection. 2. Since SEC is performed isocratic, no equilibration is needed, allowing for short 2nd dimension analysis times. 3. Finally, SEC is performed in strong eluents. As explained above, if a separation is to be performed which requires adsorption or precipitation upon injection, a high risk for breakthrough peaks exists, when the sample is injected in the strong eluent of the SEC. Although comprehensive or online two-dimensional chromatography becomes increasingly popular, the offline approach has also benefits, besides cost issues. By repeated fractionations

Please cite this article in press as: W. Radke, J. Chromatogr. A (2013), http://dx.doi.org/10.1016/j.chroma.2013.12.010

G Model CHROMA-354956; No. of Pages 18

ARTICLE IN PRESS W. Radke / J. Chromatogr. A xxx (2013) xxx–xxx

and solvent evaporation offline two-dimensional chromatography allows injecting higher samples loads into the second dimension to overcome detection problems. In addition, the possibility of solvent exchange, reduces compatibility and breakthrough problems and allows application of RI-detection, which is prevented when injecting strongly differing solvents from the 1st into the 2nd dimension, due to the long time required for the RI-signal to return to the baseline. Despite all thoughts that have to be given to the above mentioned technical details of two-dimensional chromatography, the most crucial point is the proper section of the separation mechanisms for both dimensions.

7

Fig. 5. Chromatogram of a PBT-sample at critical conditions. Reprinted from [43], Copyright (1985), with permission from Springer-Verlag.

3. Applications Having laid down the basics of the different chromatographic modes and techniques applicable to polymers the following chapters will present which of the techniques can be applied to solve different separation problems. 3.1. One-dimensional separations 3.1.1. Homopolymer separation according to end-group functionalization In case of homopolymers the overwhelming numbers of separations are performed by molecular size using SEC. Other size based separation techniques like hydrodynamic chromatography or field flow fractionation can also be applied for very high molar masses. However, if separations by functionalization are to be performed separation by size is of no use. Since all synthetic polymers exhibit a molar mass distribution, the effect of molar mass usually results in broad peaks at size exclusion conditions as well as at adsorbing conditions. The peak width due to molar mass dispersity usually exceeds the effect of the end-group retention. In rare cases, if the retention of the end group is extremely strong, a separation by functionalization might be achieved, while the repeating units experience SEC conditions. At such conditions the sample elutes in order of decreasing molar mass but after the void volume of the column. Since this separation mode exhibits features of size exclusion (retention order) and adsorption (elution after the void volume) it is named LEAC (liquid exclusion-adsorption chromatography) [14,39]. In addition TGIC has been applied for the separation of end-group functionalized polymers of narrow molar mass distribution [40]. However, in the vast majority of cases the effect of molar mass on retention volume needs to be eliminated in order to separate by functionalization, since the additional adsorption or exclusion effects of the end-groups are much smaller than the effect of molar mass. As discussed above, at critical conditions of homopolymers, molar mass does not contribute any longer to retention. Thus, LCCC is the method of choice to separate by endgroup functionalization. Thus, establishing critical conditions is a prerequisite for performing the separations. However, establishing critical conditions will not automatically result in a successful separation. At critical conditions the end-group needs to have a stronger interaction with the stationary phase than the repeating units to achieve the desired separation. Therefore, in order to select the correct phase system not only the polarity of the repeating unit has to be taken into account but also the polarity of the end-group relative to the repeating unit. As an example Fig. 5 shows the chromatograms of poly(butyl terephthalate) (PBT) obtained at critical conditions for PBT. Tetrahydrofuran (THF)-heptane (65/35, V/V) was used as isocratic eluent on polar silica. Since the hydroxyl-groups are more polar than the repeating units, they are expected to interact more strongly with the stationary phase, resulting in stronger retention

than for less polar alkyl end-groups. Therefore the fraction containing no hydroxyl-groups elutes first, followed by the fraction with one and the fraction carrying two hydroxyl-functions. While the presented example shows a separation by the type of end-groups, separations by the number of functionalized endgroups and by differences in the placement within polylactide (PLA) stars polymers have been published [41]. These studies revealed increasing retention with the number of arms, carrying adsorbing OH-functionalities. The retention volume differs for PLA-stars bearing the same number of hydroxyl-groups, depending on whether the hydroxyl-groups are attached to the core of the star or to the arms. This behavior was quantitatively interpreted based on the presently accepted theory of polymer chromatography [42]. 3.1.2. Homopolymer separation according to topology Besides molar mass and functionality, topology provides another source of heterogeneity in homopolymers. Branched polyesters have been shown to be more strongly retained than the linear ones at the critical conditions of the corresponding linear polyester (Fig. 6) [44]. Retention increases with degree of branching (DB). Since at critical conditions of the linear sample a branched sample of a given DB experiences adsorbing conditions, it reveals the typical elution behavior of a polymer at LAC, i.e. a strong increase in retention volume with molar mass and thus broad peaks, which for high molar masses result in incomplete recovery [44]. Therefore gradient chromatography seems to be the better choice to separate linear and highly branched samples.

Fig. 6. Chromatograms of linear and branched polyesters of similar average molar mass at critical conditions of the linear one. Reprinted with permission from [44]. Copyright (2010) American Chemical Society.

Please cite this article in press as: W. Radke, J. Chromatogr. A (2013), http://dx.doi.org/10.1016/j.chroma.2013.12.010

G Model CHROMA-354956; No. of Pages 18 8

ARTICLE IN PRESS W. Radke / J. Chromatogr. A xxx (2013) xxx–xxx

Fig. 7. Chromatograms of cyclic polystyrenes and corresponding linear precursors at the critical conditions of linear polystyrene. Reprinted with permission from [45]. Copyright (2000) American Chemical Society.

However, critical conditions were applied for the effective separation of linear and cyclic polystyrenes. As shown in Fig. 7 rings are more strongly retained than their linear precursors at critical conditions of polystyrene, resulting in clear separations of both structures [45,46]. SEC separations of the linear precursors and the cyclic structures derived thereof are by far less effective and result in significant coelution for linear and cyclic species. In addition, the resolution in LCCC increases with increasing molar mass, while the molar mass has no pronounced effect on the resolution in SEC. Critical conditions have also been applied for the separation of star shaped polystyrenes [47]. However, the statistical theory of polymer chromatography predicts the same distribution coefficient for star polymers and linear polymers at the critical point [42]. Thus, it might be possible that the observed effects are rather due to slightly different interaction energies of the branching units or the larger number of end-groups present in any branched polymer than to a truly topological effect. Unfortunately the sensitivity of critical conditions to tiny variations in the eluent composition and ignoring the effect of excluded volume in the underlying theory makes it rather difficult to come to a final conclusion. However, computer simulations as those performed by Wang et al. [48] will yield additional information without suffering from either non-optimized chromatographic conditions or from structural imperfections of the model compounds. TGIC has been applied for the separation of star polymers [48–51]. 3.1.3. Separations by chemical composition 3.1.3.1. Polymer blends. Separations of polymer blends by chemical composition can be performed effectively either by critical or gradient chromatography. When critical conditions for component A are applied the molar mass distribution of the blend component B can be analyzed simultaneously besides determination of the relative amounts of the blend components. At critical conditions of component A, component B will elute either in LAC or SEC-mode, depending on the applied phase system and the polarity of B. Conditions of the latter type are to be preferred, due to the problems associated with isocratic polymer elution at LAC conditions. If component

B experiences SEC conditions it is separated from component A, which elutes at the void volume of the column. Thus, it is possible to determine the molar mass distribution of component B in a blend, after suitable SEC calibration at the same chromatographic conditions. In order to obtain reliable molar masses, the pore size distribution of the column must fit the molar mass range of component B. A large pore size will ease establishing critical conditions. However, component B might elute close to the void volume and thus close to component A, resulting in a too low resolution. If however the pore size is very small, establishing and maintaining critical conditions for component A is difficult. In addition component B might elute at or close to the exclusion limit, rendering unreliable molar masses. Fig. 8 shows separations of blends of polyvinyl chloride (PVC) and poly(methyl methacrylate) (PMMA) obtained at critical conditions of PMMA. The stationary phase was a 100 A˚ bare silica column. The mobile phase was a mixture of methyl ethyl ketone (MEK) and cyclohexane 73/27 (v/v). The more polar PMMA interacts more strongly with the stationary phase than PVC. Thus, at critical conditions of PMMA, PVC elutes in SEC mode, before the PMMA. A clear separation of both blend components is obtained, allowing determination of the relative peak amounts. However, all PVC peaks occur at approximately the same elution volume, without noticeable variation with molar mass. This is a consequence of PVC elution close to the exclusion limit of the small pore size column. However, by addition of another silica column with higher pore size, the authors were able to determine the molar masses of the PVCs from the respective chromatograms. The comparison of the expected molar masses and those determined at critical conditions is given in Table 1. As can be seen, a close agreement is obtained showing clearly the potential of LCCC for the characterization of polymer blends. As discussed, using critical conditions allows determination of the amounts of both components simultaneously with the molar mass averages of the non-critical blend component. However, setting up the chromatographic conditions in the proper way and maintaining critical conditions sometimes is cumbersome. Thus, gradient chromatography might be an alternative, if only determination of the blend components is aimed for. As explained above, elution of high molar mass samples in gradient chromatography occurs close to the critical eluent composition of the polymer under investigation. Having injected the blend onto the column at conditions which allow for adsorption of the blend components, an increase in eluent strength by variation of eluent composition will desorb and elute the blend components close to their respective critical compositions [22,23,53–57]. As an example the separation of different polymethacrylates is shown in Fig. 9. If the initial conditions of the gradient experiment are selected such that one components is not adsorbed at all and elutes in SEC mode, while the other blend component is initially adsorbed and subsequently desorbed by increasing the eluent strength, not only the amounts of both components can be determined, but also the molar mass distribution of the non-adsorbed component. Examples are given in [58,40]. Gradient chromatography requires using a gradient pump. More important however is that gradient chromatography requires the sample to be dissolved and injected in a solvent which allows for adsorption of all sample components onto the stationary phase, to avoid undesired breakthrough peaks. If either only isocratic equipment is available or the sample can only be dissolved in a strong eluent, blend separations might be successfully performed using barrier techniques. LC-LCD requires an additional valve allowing for injection of a suitable barrier in front of the injected polymer. As explained above, the injected sample components catch up with the barrier injected in front. A properly selected barrier will hold back some sample components which elute right after

Please cite this article in press as: W. Radke, J. Chromatogr. A (2013), http://dx.doi.org/10.1016/j.chroma.2013.12.010

G Model CHROMA-354956; No. of Pages 18

ARTICLE IN PRESS W. Radke / J. Chromatogr. A xxx (2013) xxx–xxx

9

Fig. 8. Chromatograms of PVC-PMMA blends at critical conditions of PMMA. Data extracted from [52]. Table 1 Comparison of molar masses of PVC-samples in PVC-PMMA blends determined at critical conditions of PMMA. PVC Mw /g/mol Nominal SEC 75, 300 36, 600

LCCC 79,900 38,400

Data taken from [50].

the adsorbing barrier, while other components might pass the barrier resulting in faster elution. An example of a separation of a 3 component blend composed of a polystyrene matrix with minority components of approximately 1 and 0.5% of PMMA and poly(vinyl acetate) (PVAC), respectively, is given in Fig. 10. A polar silica column was applied. Neither of the three components was adsorbed onto the stationary phase from a 70/30 (v/v) THF/toluene mixture. In contrast, if toluene was applied as solvent and eluent, both, PVAC and PMMA were retained on the column, while polystyrene was

not. Consequently using THF as eluent and a barrier of toluene is expected to separate polystyrene from both minority components, which will elute right behind the barrier. To separate the minority components the second barrier needs to be adjusted such that it allows for penetration of one minority component while the other should be adsorbed. A mixture of THF/toluene (40/60) was applied as a second barrier, as it retains PVAC but allows for penetration of PMMA. Therefore, after injection of the toluene barrier, a second barrier of THF/toluene (40/60) was introduced. Finally the sample is injected in the strong eluent THF/toluene (70/30). The sample components are faster than the surrounding solvent molecules and reach the later injected barrier (THF/toluene (40/60)). This barrier can be penetrated by the less polar PMMA and PS but not by PVAC. Both penetrating components will then reach the toluene barrier,

Fig. 9. Gradient separation of a blend of different polymethacrylates. (1) poly(methyl methacrylate), (2) poly(ethyl methacrylate), (3) poly(n-butyl methacrylate), (4) poly(n-hexyl methacrylate), (5) poly(lauryl methacrylate).

Fig. 10. LC-LCD separation of a ternary blend of polystyrene with 1 and 0.5% of PMMA and PVAC, respectively. The three curves correspond to variations of the injection times between the barriers and sample.

Reprinted from [22], Copyright (2002), with permission from Elsevier.

Reprinted from [59], Copyright (2009), with permission from Elsevier.

Please cite this article in press as: W. Radke, J. Chromatogr. A (2013), http://dx.doi.org/10.1016/j.chroma.2013.12.010

G Model CHROMA-354956; No. of Pages 18 10

ARTICLE IN PRESS W. Radke / J. Chromatogr. A xxx (2013) xxx–xxx

which will retain PMMA, but not PS. As result the chromatogram in Fig. 10 is obtained, showing a clear separation of all three components. However, the above explanation reveals a major drawback of the LC-LCD technique, namely the separate adjustment of barrier strength, barrier width and injection time for each of the fractions to be separated. The drawback of separately adjusting several barriers can be overcome by the self-alignment of the different sample components at the respective adsorption or precipitation thresholds which occurs in SEC-gradient. Therefore SEC-gradients were applied successfully for separations of polymer blends. The blend separations by SEC-gradients described in literature comprise those based on adsorption [36] and on precipitation [35]. Similar to the barrier techniques discussed above, SEC-gradients allow for sample dissolution and injection in a strong solvent, without the risk of breakthrough peaks. 3.1.3.2. Separations of copolymers by chemical composition. When two or more different comonomers are incorporated into polymer chains, the different chains may vary in their composition, resulting in a chemical composition distribution (CCD). In addition the different comonomers might be incorporated as longer or shorter segments of the same type, i.e. in a more or less blocky arrangement. These different microstructures introduce another level of heterogeneity, which is termed microstructural or sequence order distribution. In the following we will differentiate separations of statistical copolymers obtained by, e.g. radical copolymerization which are expected to result in more or less random placement of the monomers along the chains, and segmented copolymers which purposefully are prepared as block- or graft copolymers. 3.1.3.3. Separations of statistical copolymers by chemical composition. The chemical composition distributions of statistical copolymers obtained by copolymerization depend on the reactivity parameters, the monomer ratio and the reaction conditions. If one comonomer reacts much faster than the other one, the comonomer ratio will vary with reaction time and the chains produced at different reaction times will exhibit different chemical compositions. The drift in the comonomer composition and therefore the resulting chemical composition distribution may be compensated by proper feeding the faster reacting monomer to the reactor. The

chemical composition distribution of a copolymerization process can strongly affect the material properties. If, e.g. in tablet coating a specific dissolution profile is required, the average composition defines the average conditions for dissolution, while the width of the chemical composition distribution will define the steepness of the transition. Thus, determining the chemical composition distribution of statistical copolymers is of high importance. Brun theoretically showed that a statistical copolymer of a given composition behaves chromatographically similar to a homopolymer [55–57]. Most importantly a statistical copolymer has a critical adsorption point at which it elutes independently of molar mass. The critical adsorption point depends on the composition of the chain [56,60]. The effect of microstructure, arising from the statistical processes at each moment does not contribute significantly to retention [55,57,61]. Thus, a high conversion statistical copolymer can be regarded as a blend of homogenous copolymers each having a slightly different chemical composition and thus a slightly different critical point. The first experimental verification of the existence of a critical point for statistical copolymers was given by Brun, who used a series of chlorinated polyethylenes of similar compositions but varying molar masses to establish the typical “fan-like” diagram showing SEC, LAC and critical behavior as a function of eluent composition [56]. In the same paper it was shown that the critical eluent composition of the copolymer agrees well with the eluent composition at the time of gradient elution. Using radically polymerized styrene–ethyl acrylate copolymers Bashir et al. showed that the dependence of copolymer retention on eluent composition of copolymers is described by the same equations describing the retention of homopolymers. In particular they were able for each copolymer composition to identify an eluent composition at which the copolymers elute at the void volume as a narrow peak, i.e. without noticeable broadening due to molar mass [60]. Since SEC separates by size it is obvious that SEC separations will not be effective to determine the chemical composition distribution of copolymers. Although statistical copolymers strictly form complicated blends, the above approach of LCCC to separate polymer blends is not suited. If critical conditions have been established for one specific copolymer composition, chains of other compositions will elute either in LAC or SEC mode, depending on their composition (see Fig. 11). Thus, for all those copolymer compositions not eluting critically, coelution of chains having different chemical composition and different molar mass will occur. This

Fig. 11. Schematic representation of the elution behavior of a chemically heterogeneous copolymer at critical conditions for one copolymer composition (right) and in gradient chromatography (left). The different lines represent fractions of different chemical composition.

Please cite this article in press as: W. Radke, J. Chromatogr. A (2013), http://dx.doi.org/10.1016/j.chroma.2013.12.010

G Model CHROMA-354956; No. of Pages 18

ARTICLE IN PRESS W. Radke / J. Chromatogr. A xxx (2013) xxx–xxx

Fig. 12. High temperature gradient separation of EP-copolymers with ethylene contents ranging from 14 to 56 wt%. Reprinted from [67], © (2012), with permission from Wiley-VCH.

situation is schematically depicted on the left hand side of Fig. 11. In addition, for those chains eluting in LAC mode, incomplete recovery is expected to occur, similar to homopolymers. Therefore the solution to the problem is gradient chromatography, since each high molar mass fraction of a given composition is expected to elute close to its respective critical eluent composition. Linear to weakly curved dependences of the eluent composition at the time of elution on copolymer composition were observed in several studies [62–67], provided the separation is based on adsorption–desorption equilibria. An example of a copolymer separation by chemical composition is given in Fig. 12. High temperature gradient chromatography was applied to the separation of ethylene–propene-copolymers (EP-copolymers) with ethylene contents ranging between 14 and 56 wt%. The example has several interesting features. First a high temperature gradient chromatograph running at 160 ◦ C was required, due to the limited solubility of polyolefin samples. Such high temperature gradient separations have become possible only in the last decade [68]. Another feature is the selected stationary phase. The applied Hypercarb column and the gradient running from 1-decanol to trichlorobenzene (TCB) have been proven to the first time to result in an adsorption based separation [69], contrasting to earlier attempts to separate polyolefins by chemical composition, which have been mostly based on crystallizability or on precipitation/dissolution [68,70]. As discussed, conventional gradient separations require sample dissolution in a solvent allowing for adsorption onto the stationary phase. Sometime such solvents are not easily identified. E.g. in the separation of copolymers of n-butyl acrylate and acrylic acid, samples containing more than approx. 20% of acrylic acid are insoluble in chloroform (CHCl3 ). However, the copolymers are soluble in dimethyl acteamide (DMAc), irrespective of the content of acrylic acid. The acidic functions ask to select a polar stationary phase, in order to separate in order of increasing acrylic acid content. However, using CHCl3 as solvent and starting eluent is not possible due to the limited solubility. Dissolution in the highly polar DMAc allows for dissolution, but prevents adsorption onto the stationary phase even of pure acrylic acid. Dissolving and injecting samples of lower acrylic acid content in DMAc using CHCl3 as initial eluent results in severe breakthrough peaks. A solution to this problem is the application of a SEC gradient from CHCl3 to DMAc. The samples are dissolved in pure DMAc and are injected into the end of the gradient. Since the samples experience SEC conditions at the time of injection, they migrate at a higher velocity than the surrounding solvent, thereby passing through solvent zones of increasing CHCl3 content, i.e. of increasing adsorption strength. Finally the samples

11

Fig. 13. Separation of poly(n-butyl acrylate-stat-acrylic acid) in a 6 min SECgradient from 4% DMAC to 100% DMAC in CHCl3 . Stationary Phase PSS NOVEEMA 30 cm × 0.8 cm. Left to right; 100, 70, 40, 10% n-butyl acrylate.

align at their respective adsorption thresholds, which correspond to specific CHCl3 /DMAc compositions. The separation of poly(n-butyl acrylate-stat-acrylic acid) having different acrylic acid contents is shown in Fig. 13. A clear separation is observed without any indication of breakthrough peaks. Although the samples exhibit molar mass dispersities typical for conventional radical polymerization, the peaks are narrow. Thus, the molar mass effects are effectively suppressed by the SEC gradient and the main separation effect is by chemical composition. This effective reduction of molar mass effects is due to the small pore size of the column material. It should be noted that the samples remain completely soluble throughout the separation. Although samples move into the gradient in direction of decreasing DMAc content, which would result in precipitation of samples having a high content of acrylic acid, they are not precipitated, as they become adsorbed before they reach the precipitation threshold. 3.1.3.4. Separations of segmented copolymers by chemical composition. In case of segmented copolymers, such as block- or graft copolymers, it is often required to separate the corresponding homopolymers form the graft- or block structures. Proof of purity of block copolymers is often given based on SEC experiments, where shifts in elution volume to lower elution volumes and a monomodal curve is displayed. However, a good SEC separation typically provides a baseline separation of two narrowly distributed polymers if the molar masses differ by a factor of approximately 2. Thus, if the second block is of low molar mass, even significant fractions of the terminated first block will barely be visible in a SEC-chromatogram. For samples heterogeneous in molar mass, the situation is even worse. Therefore, the separations have to be performed by other means. Critical chromatography is often applied to the separation of block copolymers. Typically critical conditions are established for component A such that component B experiences SEC conditions. Since at critical conditions the segments of type A will not contribute to retention, the elution volume of the AB block copolymer should be determined solely by the length of the B block. Consequently the AB blockcopolymer should elute as if there is no block A attached. Hence the blockcopolymer is expected to coelute with homopolymer B before the void volume, while homopolymer A elutes at the columns void volume. Chromatography at critical conditions of block A can therefore provide only information on the amount of homopolymer A, but not on the amount of potential homopolymer B. To determine the amount of homopolymer B, the

Please cite this article in press as: W. Radke, J. Chromatogr. A (2013), http://dx.doi.org/10.1016/j.chroma.2013.12.010

G Model CHROMA-354956; No. of Pages 18 12

ARTICLE IN PRESS W. Radke / J. Chromatogr. A xxx (2013) xxx–xxx

Fig. 14. Schematic representation of separating block copolymer samples by combining LCCC at critical conditions of homopolymer A (left) and gradient chromatography (right). Solid: AB block copolymer, dashed: homopolymer B, dotted: homopolymer A.

Fig. 15. Separation of products of grafting PMMA onto EPDM by multistep gradient chromatography.

chromatographic system has to be inverted, i.e. now critical conditions for homopolymer B need to be applied, leaving homopolymer A and the copolymer to coelute. Adjusting critical conditions on another stationary phase of inverted polarity is however rather time consuming. An alternative to establishing critical conditions on two different stationary phases is given by applying a gradient using the same phase system as established for the first critical point. The gradient is started at conditions which allow for adsorption of all three components, both homopolymers and the block copolymer. The separation of both homopolymers will be identical to the gradient separation of the corresponding homopolymer blend, leaving us with the question, where the block copolymer will elute. Experimentally it is often observed that segmented copolymers especially of high molar mass elute close to the more strongly adsorbed homopolymer. A complete theoretical treatment has been given by Brun [57]. Thus, in the case under consideration the block copolymer is expected to elute close to homopolymer A. Thus, while at critical conditions of A, homopolymer B and the blockcopolymer are expected to coelute, homopolymer B is expected to be separated from homopolymer A and the AB blockcopolymer in a gradient experiment applying the same phase system. The analysis of block copolymers by combining of critical chromatography and gradient chromatography is schematically depicted in Fig. 14. An exceptional example of a separation of a segmented polymer, where all three components were separated in a single gradient run is shown in Fig. 15. The product obtained by grafting PMMA onto EPDM was analyzed using a gradient composed of isooctane and THF. A polar stationary phase was applied for the separation [71]. The example also shows that the graft copolymer elutes quite closely to the PMMA homopolymer. Thus, although a separation is achieved, the example does not violate the above conclusion that segmented copolymers usually elute close to the more strongly adsorbed homopolymer in gradient chromatography. This close elution often prevents separating all three components in a single gradient run. The above discussion concerned application of eluent gradients. However, eluent strength can be varied by temperature variations as well. Therefore TGIC has also been applied successfully to the separation of segmented copolymers. Examples can be found in references [29,72]. In the case of blend separations by LCCC it was shown that LCCC allows obtaining the blend composition as well as information on

the molar mass distribution and molar masses of the non-critical component in the presence of the second blend component. Similarly, separations of block copolymers at critical conditions allow obtaining additional information. If it can be assumed that no homopolymer B is present, as it is typically the case in sequential living polymerization if block B is initiated by block A, application of critical conditions provides additional information on the molar mass distribution and molar masses of block B, despite the fact that block B is attached to block A. Since block A is expected not to contribute to retention, the elution volume of the block copolymer should be identical to that of a homopolymer B of identical block length, allowing to at least estimate the molar mass distribution of block B. Fig. 16 shows a separation of poly(methyl methacrylate)block-poly(t-butyl methacrylate) at critical conditions of PMMA. From the upper graph it becomes clear that the blockcopolymer samples contain PMMA, which elutes at a retention time of 8.5 min, while the actual block copolymers elute in order of decreasing molar mass, in SEC mode. The lower graph shows identical elution volumes for three copolymers of identical PtBMA but varying PMMA block length. Apparently the variation of PMMA block length does not affect the retention volume. Thus, it is chromatographically “invisible”. The retention volume of the block copolymer is therefore obviously determined by the length of the non-critical block. There is still some discussion on the accuracy of the molar masses to be expected from such an analysis. While Falkenhagen et al. obtained a good agreement between the expected molar masses for block B and the ones obtained by the above approach [73], Chang and coworkers revealed significant differences in poly(styrene)-b-poly(isoprene) diblock copolymers [74]. Since the general elution behavior for various structures and at various chromatographic conditions is well described by the statistical theory of polymer chromatography [9–11,13,14,16,17,22,23,46,75–80] deviations between theory and experimental results can be assumed to result from simplifications of the theory, e.g. neglectance of excluded volume effects. Indeed, the influence of excluded volume effects as a major reason for the observed discrepancy has indeed been confirmed by recent computer simulations [81]. It should be mentioned that according to theory a prerequisite of obtaining chromatographic invisibility of a segment in a segmented copolymer it is the existence of a free chain end of the segment under consideration [10]. Thus, in case of ABA

Data extracted from [71], Wiley-VCH.

Please cite this article in press as: W. Radke, J. Chromatogr. A (2013), http://dx.doi.org/10.1016/j.chroma.2013.12.010

G Model CHROMA-354956; No. of Pages 18

ARTICLE IN PRESS W. Radke / J. Chromatogr. A xxx (2013) xxx–xxx

13

Fig. 16. Separation of PMMA–PtBMA blockcopolymers at critical conditions of PMMA. Upper: precursor PMMA (5) (Mn = 31,000 g/mol) with PtBMA block length of 46,300 (6) and 85,600 (7) g/mol. Lower: PtBMA blocklength 73,000 g/mol, PMMA blocklengths 29,600, 10,810, 167,500 g/mol. Adapted with permission from [73]. Copyright (2000) American Chemical Society.

triblockcopolymers the theory predicts that at critical conditions of A the copolymer elutes identical to a B-type homopolymer of identical molar mass. However, this is not the case for BAB triblockcopolymers, where the critical A-block has no free chain end. In such a case elution will be influenced by the molar mass of the A blocks as well [10]. This behavior has also been confirmed by simulations [81] and experiment [72]. The approach of selecting critical conditions for homopolymer A such that homopolymer B and thus the AB blockcopolymer elutes in SEC mode before the void volume is suitable, if block B is of sufficient length, since the separation is at least to a good approximation based on the size exclusion effect of block B. If B block is of low molar mass, the size exclusion effect of the B block might not suffice to gain enough resolution between the block copolymer and homopolymer A. At first glimpse decreasing the pore size might be an appropriate action to overcome the problem, since the SEC distribution coefficient decreases with increasing size to pore size ratio. However, with growing size to pore size ratio it becomes increasingly difficult to establish and maintain critical conditions. Therefore, if an AB blockcopolymer of a short B-block length is to be separated from a relatively large homopolymer A, critical conditions of homopolymer A should be set up in a way such that homopolymer B experiences adsorbing conditions. In that case, the block copolymer elutes after the void volume and thus after homopolymer A. Usually a gradient is required to prevent incomplete recovery, especially for high molar mass blocks B. The application of a gradient to elute the block copolymer will, however, usually result in eluent conditions at which homopolymer A is not invisible any longer. Thus, the retention of the pure block copolymer will be influenced by the lengths of block A and B, resulting in coelution of blockcopolymer fractions differing in composition and molar mass. A possibility to overcome this problem is using ternary gradients along the critical line. If a ternary gradient is run along the critical line of polymer A, chromatographic invisibility of polymer A is maintained throughout the gradient experiment, while the retention of block B is altered by the gradient. This approach was successfully applied to realize baseline separations of ethylene oxide–propylene oxide block copolymers according to the number of propylene oxide units without molar mass influence of the

ethylene oxide units [82]. Establishing the critical line is usually cumbersome, but can be significantly simplified by using a gradient approach [83]. LC-LCD has been applied for the separation of poly(styrene)block-PMMA from the corresponding homopolymers [84–87]. An example is given in Fig. 17. The separation was performed on a silica column, applying THF/toluene (60/40) as eluent. Pure toluene was used as first barrier. Since toluene is a strongly adsorbing solvent for PMMA but allows free elution of polystyrene, any PMMA containing structure will be adsorbed and will therefore elute later than barrier 1, while PS can freely penetrate both barriers. A second barrier therefore is required to separate the block copolymer from pure PMMA. This was achieved, using a barrier of THF/toluene

Fig. 17. LC-LCD separation of PMMA-PS blockcopolymers (c = 1 g/L) spiked with PMMA and PS of different molar masses (PMMA: 16 kg/mol, 103 kg/mol 294 kg/mol. PS = 17.5 kg/mol, 100 kg/mol, 233 kg/mol). Reprinted from [85], © (2008), with permission from Wiley-VCH.

Please cite this article in press as: W. Radke, J. Chromatogr. A (2013), http://dx.doi.org/10.1016/j.chroma.2013.12.010

G Model CHROMA-354956; No. of Pages 18 14

ARTICLE IN PRESS W. Radke / J. Chromatogr. A xxx (2013) xxx–xxx

(35/65). This composition is rather close to the reported critical conditions for PMMA on silica stationary phases using the same solvents (36–38% THF) [2,54,83]. This coincidence of critical conditions and barriers selection is not by accident. Since the block copolymer needs have a higher velocity than the surrounding eluent, it has to penetrate the barrier in SEC mode. However, the pure PMMA needs to become adsorbed by the barrier, i.e. the barrier has to have lower or equivalent eluent strength than the critical composition to retain the PMMA. However, if the adsorbing barrier is too strong, the “pulling effect” of the PS will not suffice for desorption of the block copolymer. Therefore the proper barrier must have a composition identical or only slightly weaker than the critical composition of the PMMA. The above interpretation is supported by the fact that a similar LC-LCD separation of PS-block-PMMA on pure silica was reported using THF/dichloromethane (DCM) (10/90) as barrier [84], while critical conditions for PMMA have been reported on pure silica for THF contents in DCM of 12.3 and 12.4%, i.e. close to the selected barrier [2]. In any case, two barrier compositions, their width and proper injection times as well as the eluent composition need to be carefully adjusted in order to obtain the desired separation by LC-LCD. Due to similar arguments, SEC gradients in the way described above are not expected to overcome the problem. Similar to conventional gradients successful separations are only expected between the less adsorbed homopolymer and the block copolymer, which in turn will elute close to the stronger adsorbed homopolymer. 3.1.4. Separations by stereoregularity and microstructure Retention in polymer chromatography is not only influenced by molar mass and chemical composition, but also by the microstructure of the chains. If separations are to be performed due to small differences in the structure critical chromatography might be an option, as molar mass effects are hidden. Therefore it is not surprising that chromatography at critical conditions has been applied to separate polymers by microstructure. Isotactic and syndiotactic poly(methacrylates) have been separated by Berek [88,89]. Hiller and coworkers succeeded in separating polyisoprenes containing mainly 1,4 units from those containing mainly 3,4 linkages [90]. It should be noticed that the above mentioned references describe blend separations at critical conditions of one of the components. For example in the latter publication critical conditions were established for 1,4 polyisoprenes. At these conditions polyisoprenes containing 1,3-linkages elute in SEC mode. However, if a separation is to be performed according to the amount of 1,3 units or of isotactic units, critical conditions might not be the appropriate choice. 1,4- and 3,4-units in polyisoprenes as well as the syndiotactic and isotactic diades can be regarded as comonomers. In that case each copolymer of a given composition has its own critical point and all fractions of different stereoregularity elute either in SEC or LAC mode and will thus coelute with fractions of slightly different composition and molar mass (see Fig. 11). A separation of polylactides according to enantiomeric purity was published by Li et al. [91]. Since no chiral stationary phase was used pure PLLAs and PDLAs eluted at the same retention volume but later than samples of lower enantiomeric excess. The separation was however based on solubility rather than on adsorption. Since PLAs of high optical purity are not soluble in THF, while chloroform is a good solvent for PLA irrespective of stereochemical composition, a separation based on the solubility was successful. A bare silica column was used and the samples were injected in pure chloroform, which results in strong adsorption onto the stationary phase. A gradient to THF would result in an adsorption/desorption dominated separation, which was not successful. Instead, flushing the column with hexane after adsorbing the PLA from CHCl3 resulted in precipitation of the

Fig. 18. Gradient separation of block and statistical copolymers containing approximately 50 mol% of styrene and methyl methacrylate. Molar masses: PS: 107 kg/mol, PMMA: 103 kg/mol, Stat: 109 kg/mol, Block 1 124 kg/mol block 2: 407 kg/mol. Reprinted from [57], © (2010), with permission from Wiley-VCH.

adsorbed polymer within the column. By applying a gradient from hexane to THF and further to CHCl3 , first fractions of lower optical purity were dissolved and eluted and finally the fractions of high optical purity. Interestingly blends of enantiomeric rich PLLA and PDLLA resulted in two peaks, the relative intensities of which varied with the ratio of the components. These peaks were assumed to be due to the stereocomplex formation between PLA chains of high optical purity of opposite configuration. Separations of statistical and segmented copolymers can also be performed. Block copolymers are longer retained in gradient than statistical copolymers of the same molar mass and composition [8]. A theory describing this effect as well as experimental evidence has been given by Brun [55,57]. Fig. 18 shows the separation of block and statistical copolymers composed of MMA and styrene of similar chemical composition. The figure shows a variety of interesting effects. As explained above, the block copolymers elute later than the statistical copolymers. However, while high molar mass statistical copolymers of a given composition are expected to elute nearly independent of molar mass at their respective critical composition, the block copolymers elute in order of increasing molar mass, i.e. they exhibit no critical behavior. The statistical copolymer elutes roughly in the middle between the two homopolymers, while the block copolymers elute much closer to the stronger adsorbing homopolymer (polystyrene), as discussed in the section on the separation of block copolymers by chemical composition. With increasing molar mass the block copolymers approach the elution volume of the polystyrene. 3.2. Two-dimensional separations In principle all of the above mentioned separations can be combined, resulting in a large variety of combinations and high potential for separating complex polymer mixtures. However, not all combinations have been realized. 3.2.1. Separation by functional end-groups and molar mass The separation by functional end-groups with simultaneous molar mass characterization of the fractions can be performed by LCCC × SEC. An example from the group of Schoenmakers is presented in Fig. 19. As explained in the section on critical chromatography, LCCC is probably the only method to achieve separations of high molar mass polymers according to end-group functionality. Thus, critical conditions for PMMA were applied in

Please cite this article in press as: W. Radke, J. Chromatogr. A (2013), http://dx.doi.org/10.1016/j.chroma.2013.12.010

G Model CHROMA-354956; No. of Pages 18

ARTICLE IN PRESS W. Radke / J. Chromatogr. A xxx (2013) xxx–xxx

15

dimensions. Alcohol ethoxylates were separated in the 1st dimension into individual ethylene oxide oligomers by a normal phase gradient. In the 2nd dimension a separation at critical conditions with respect to alkyl length of the initiating alcohol is performed on a reversed phase stationary phase. This separation order was selected, because of the short separation time in the 2nd dimension. LCCC, as isocratic separation mode, does not require equilibration. On the other hand, LCCC is rarely used in the 2nd dimension. This is because LCCC can crucially respond if the eluent composition differs from the composition used to dissolve the sample. If the eluent injected from the 1st dimension is stronger than the critical eluent, the samples will not become adsorbed and a breakthrough peak without separation might result close to the void volume. The success of the separation is rather surprising and seems to indicate that the transferred eluent from the 1st dimension must act as weak eluent in the 2nd.

Fig. 19. Comprehensive two-dimensional separation of PMMAs with OH-groups. 1,2 no OH-functionalization, 3,4 one hydroxyl functionality, 5 two hydroxyl-groups y-axis 2nd dimension (SEC) retention time, x-axis 1st dimension (LCCC) elution time. Reprinted from [92], Copyright (2005), with permission from Elsevier.

the 1st dimension. The selected bare silica stationary phase interacts more strongly with the polar OH-group as compared to the methacrylate units of lower polarity. The 1st dimension eluent was 48% acetonitrile (AN) in dichloromethane (DCM), while THF was used in the 2nd dimension. A reversal of the separation order would probably result in pronounced breakthrough peaks, since injecting the sample in the more polar THF would prevent adsorption onto the stationary phase in LCCC. The non-functionalized PMMAs show a slight change in retention time along the x-axis (LCCC), indicating a slight molar mass dependence. Thus, the separation was not performed at, but only close to critical conditions. Despite that, a separation of the different components is clearly achieved. A separation by SEC only would probably result in partial overlap of peaks 2,3,4,5 making data interpretation very complicated. The separation by LCCC alone, would probably lead to three peaks, due to partial overlap of the peakpairs 1,2 and 3,4, respectively. Therefore it can be concluded that only the two-dimensional separation can resolve the complexity of the sample mixture. In contrast to most two-dimensional separations, which combine a mode of interaction chromatography with SEC, Fig. 20 shows a separation where interaction chromatography is utilized in both 100

x=12 x=13 x=14 x=15 125

NPL C (min)

150

175

H-(CH2)x-O-(CHCHO) 2 2 12-H 200 0.00

0.25

0.50

0.75

1.00

RPL C (min) Fig. 20. Comprehensive two-dimensional separation of Alcohol ethoxylates x-axis 2nd dimension LCCC retention time, y-axis 1st dimension (normal phase gradient) elution time. Reprinted with permission from [93]. Copyright (1998) American Chemical Society.

3.2.2. Separation of statistical copolymers chemical composition and molar mass The separation of statistical copolymers by chemical composition is usually performed using gradient chromatography. As explained above, for high molar mass samples the separation in a linear gradient is expected to yield a nearly molar mass independent elution of fractions of identical composition, while at lower molar masses the elution volume is influenced by both, molar mass and chemical composition. The example in Fig. 21 presents a separation of a blend of three carboxymethyl celluloses (CMC), two of which have similar molar masses but differ in degree of substitution (DS), while two samples differ in molar muss but have approximately the same DS. In general cellulose derivatives are very complex copolymers, because even the introduction of a single type of substituent results in eight differently substituted anhydroglucose units. The characterization of cellulose derivatives is usually performed either by NMR or after complete or partial chain degradation. However neither of these techniques can provide information on first order heterogeneity, i.e. on the distribution of the substituents among the different chains. The separation in Fig. 21 shows that a separation with respect to DS and molar mass can be achieved by two-dimensional chromatography. In the 1st dimension gradient separation the retention increases with DS. It should be noted that the retention order of the CMCs crucially depends on pH. A reversal of elution order was observed at a pH = 4.54. The separation in the 2nd dimension is performed by SEC. The sharp peak at high 1st dimension elution volume corresponds to the higher DS sample. The two samples of lower DS merge to a single peak, due to their broad molar mass distributions. It can be seen that the 1st dimension elution volume increases slightly with decreasing 2nd dimension elution volume, i.e. with increasing molar mass. However, it approaches a constant 2nd dimension elution volume for high molar masses. This behavior therefore agrees with the expected molar mass dependence in linear gradients. 3.2.3. Separation of segmented copolymers by chemical composition and molar mass As explained above, the separation of segmented copolymers according to chemical composition might be achieved either by gradient chromatography, or by applying critical conditions for one of the corresponding homopolymers, while at the same time the non-critical homopolymer elutes in SEC mode. Such a situation is applied in Fig. 22, which shows the separation of poly(n-butyl acrylate)-graft-poly(methyl methacrylate). The sample was prepared by free radical copolymerization of n-BuA with a methacryloyl-terminated PMMA macromonomer. In the 1st dimension critical conditions were selected for PnBuA on a C18 phase. Thus pure PnBuA is expected to elute independent of molar mass without retention at the void volume. Thus, peak 4

Please cite this article in press as: W. Radke, J. Chromatogr. A (2013), http://dx.doi.org/10.1016/j.chroma.2013.12.010

G Model CHROMA-354956; No. of Pages 18 16

ARTICLE IN PRESS W. Radke / J. Chromatogr. A xxx (2013) xxx–xxx

Fig. 21. Comprehensive two-dimensional separation of a blend of carboxymethyl celluloses varying in DS and molar mass (DS = 0.98, low MW; DS = 0.95, high MW; DS = 1.25, high MW). y-Axis 2nd dimension (SEC) retention time, x-axis 1st dimension (gradient chromatography) elution time [63].

represents homo PnBuA. Due to the higher polarity the PMMA elutes in SEC mode, i.e. in order of decreasing molar mass and before the void volume. To a first approximation the PnBuAbackbone does not contribute to retention and elution volume in the 1st dimension (ordinate) should be determined only by the molar mass of the PMMA. Since the residual macromonomer has the same PMMA molar mass as the graft-copolymer with only one branch point (star), both structures are expected to elute at the same 1st dimension elution volume. Indeed two structures can be identified at a 1st dimension elution volume of approximately 3.2 mL. Because of the higher molar mass of the star polymer as compared to the macromonomer peak 2 is assigned to the star, while peak 3 represents the residual macromonomer. The graft copolymer has the highest total molar mass and the highest PMMA molar mass. Therefore it should elute at the lowest 1st dimension and at the lowest 2nd dimension elution volume. Consequently peak 1 is attributed to the graft copolymer.

Fig. 22. Separation of poly(n-butyl acrylate)-graft-poly(methyl methacrylate) by two dimension chromatography. y-axis critical conditions for PnBuA, x-axis SEC. Adapted with permission from [94]. Copyright (2000) American Chemical Society.

3.2.4. Separation of branched polymers by degree of branching and size Branched polymers are heterogeneous with respect to molar mass and topology. The characterization of branched polymers is usually performed by SEC with light scattering and/or viscosity detection. Since branched polymers exhibit smaller dimensions than linear polymer of the same molar mass, a reduction in size as compared to the corresponding linear analog is taken as indication of branching and is often used to quantify the degree of branching. However, since SEC separates by molecular size, coelution of species differing in molar mass and topology will occur for heterogeneous polymers, complicating data interpretation. A true separation into homogeneous fractions would thus be of advantage. Fig. 23 shows the separation of linear and hyperbranched polyesters. Two peaks are observed, the gradient volumes of which increase with molar mass until reaching constant 1st dimension retention volumes. In Fig. 6 it was shown that the branched polyester elutes at higher gradient elution volume than the linear one. Therefore the peak at higher 1st dimension elution volume is assigned to the branched polymer. While a good separation is obtained at high molar masses, the peaks merge for the lower ones. The molar mass at which both peaks merge can be interpreted as the molar mass of the first branched species. It is interesting to note that the peak of the linear polymer at high molar mass extends only a slightly along the ordinate. This broadening along the ordinate might be regarded as the contribution of system and column band broadening, since no additional dispersity should exist at a given molar mass. The peak width of the hyperbranched polymer seems to be of similar magnitude. This seems to indicate that at a given molar mass the contribution of heterogeneity with respect to degree of branching is low in the investigated system. Star-shaped polystyrenes were separated from linear ones using a combination of temperature gradient chromatography (TGIC) and SEC [50,49]. A separation of star-shaped polymers in terms of molar mass and number of branches was achieved using combinations of LCCC and SEC or TGIC and LCCC, with the latter approach providing a better resolution [96].

Please cite this article in press as: W. Radke, J. Chromatogr. A (2013), http://dx.doi.org/10.1016/j.chroma.2013.12.010

G Model CHROMA-354956; No. of Pages 18

ARTICLE IN PRESS W. Radke / J. Chromatogr. A xxx (2013) xxx–xxx

17

Fig. 23. Separation of a blend of a linear and heavily branched polyesters. Ordinate: gradient chromatography, abscissa: polystyrene equivalent molar mass [95]. Complete branching was achieved by reacting the end groups of a hyperbranched polymer of degree of branching DB = 0.5 with A2 B monomer.

The above discussion focused on separations by chromatography only. However, with increasing separation capabilities new and maybe unexpected peaks will appear. The structures of the corresponding molecules have to be identified, by suitable spectroscopic and spectrometric techniques. That is why hyphenation of chromatography with chemical sensitive detection methods, like infrared and nuclear magnetic resonance spectroscopy or mass spectrometry mass is of high relevance. However, not only chromatography benefits from spectroscopic or spectrometric methods, but vice versa. Interpretation of the spectra of complex mixtures benefits from “cleaning up” the spectra by suitable separations. Besides, mass spectrometry in itself is a separation technique, the selectivity and speed of which matches the concentration range after chromatographic separations. Therefore the combination of liquid chromatography with mass spectrometry offers additional potential for two-dimensional separations. More detailed discussion can be found in other reviews [97–100]. 4. Conclusions and outlook Elucidating heterogeneity of polymers requires suitable separation techniques. By carefully selecting the conditions, separations with respect to different structural parameters can be achieved. Within the present manuscript possibilities were described how to approach specific separation problems. Although quite a variety of separation problems can be solved using the above approaches, there are still remaining challenges, which might require completely new separation approaches apart from adsorption, solubility and size based separations. One remaining challenge is the separation of long chain branched materials which might be addressed using new separation modes. Small fractions of long chain branches significantly influence rheology. At low degrees of branching, mixtures of slightly branched materials in a matrix of linear molecules are expected, due to the statistical nature of polymerization processes. Separations of linear and lightly branched structures are difficult, if not impossible by the above discusses separation principles. Molecular topology fractionation (MTF) provides a new innovative separation principle based on the differences of

linear and branched molecules to adopt an elongated conformation when subjected to a flow through a channel with dimensions approaching the dimensions of the macromolecules [101]. Despite the potential of MTF, it suffers from the very high molar masses, required to observe the desired effects. Thus, there is still a need for alternative separation strategies. Another challenge of polymer chromatography arises from the multidimensional heterogeneities inherent in many synthetic polymer systems. A separation in terms of chemical composition and molar mass is still not sufficient to fully elucidate copolymer heterogeneity, as the same chemical composition and molar mass even can arise from numerous differently microstructures, even for a linear chain. Therefore, even successful application of two-dimensional chromatography cannot fully resolve all heterogeneities in synthetic polymers. Separations in even more dimensions are required. However, the problems and limitations of two-dimensional chromatography in terms of analysis time, detectability flow rate optimizations, etc. indicate that merely extending the present approach of “simply” coupling two different chromatographic systems will not be applicable for a higher number of dimensions. New solutions need to be developed. References [1] A.H.E. Müller, K.L. Wooley, in: A.H.E. Müller, K.L. Wooley (Eds.), Polymer Science: A Comprehensive Reference, Elsevier, Oxford, 2012, p. 1. [2] T. Macko, D. Hunkeler, Adv. Polym. Sci. 163 (2003) 62. [3] H. Pasch, B. Trathnigg, HPLC of Polymers, Springer, Berlin, 1997. [4] T. Chang, J. Polym. Sci. Part B: Polym. Phys. 43 (2005) 1591. [5] T. Chang, Adv. Polym. Sci. 163 (2003) 1. [6] A. Baumgaertel, E. Altuntas¸, U.S. Schubert, J. Chromatogr. A 1240 (2012) 1. [7] H.J.A. Philipsen, J. Chromatogr. A 1037 (2004) 329. [8] G. Glöckner, Gradient HPLC of Copolymers and Chromatographic Crossfractionation, Springer, Berlin, 1991. [9] A. Gorbunov, B. Trathnigg, J. Chromatogr. A 955 (2002) 9. [10] A.M. Skvortsov, A.A. Gorbunov, J. Chromatogr. 507 (1990) 487. [11] A.A. Gorbunov, A.M. Skvortsov, Adv. Colloid Interface Sci. 62 (1995) 31. [12] A.A. Gorbunov, A.M. Skvortsov, Int. Lab. 25 (1995) 8J. [13] B. Trathnigg, B. Maier, A. Gorbunov, A. Skvortsov, J. Chromatogr. A 791 (1997) 21. [14] B. Trathnigg, A. Gorbunov, J. Chromatogr. A 910 (2001) 207. [15] A.A. Gorbunov, A.V. Vakhrushev, Polymer 45 (2004) 6761.

Please cite this article in press as: W. Radke, J. Chromatogr. A (2013), http://dx.doi.org/10.1016/j.chroma.2013.12.010

G Model CHROMA-354956; No. of Pages 18 18 [16] [17] [18] [19] [20] [21] [22] [23] [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] [49] [50] [51] [52] [53] [54] [55] [56] [57] [58] [59] [60] [61] [62]

ARTICLE IN PRESS W. Radke / J. Chromatogr. A xxx (2013) xxx–xxx

G.J.F.A.M. Skvortsov, Macromolecules 35 (2002) 8609. A. Skvortsov, B. Trathnigg, J. Chromatogr. A 1015 (2003) 31. A.J.P. Martin, Biochem. Soc. Symp. 3 (1949) 4. A. Favier, C. Petit, E. Beaudoin, D. Bertin, e-Polymers 009 (2009). D. Berek, Macromolecules 31 (1998) 8517. Y. Gong, Y. Wang, Macromolecules 35 (2002) 7492. Y. Brun, Y. Alden, J. Chromatogr. A 966 (2002) 25. M.A. Bashir, W. Radke, J. Chromatogr. A 1131 (2006) 130. W. Radke, S. Lee, T. Chang, J. Sep. Sci. 33 (2010) 3578. J. Ryu, S. Park, T. Chang, J. Chromatogr. A 1075 (2005) 145. J. Ryu, T. Chang, Anal. Chem. 77 (2005) 6347. J. Ryu, K. Im, W. Yu, J. Park, T. Chang, K. Lee, N. Choi, Macromolecules 37 (2004) 8805. S. Park, D. Cho, K. Im, T. Chang, D. Uhrig, J.W. Mays, Macromolecules 36 (2003) 5834. D. Cho, S. Park, T. Chang, A. Avgeropoulos, N. Hadjichristidis, Eur. Polym. J. 39 (2003) 2155. X. Jiang, A. van der Horst, J. Peter, Schoenmakers, J. Chromatogr. A 982 (2002) 55. E. Reingruber, F. Bedani, W. Buchberger, P. Schoenmakers, J. Chromatogr. A 1217 (2010) 6595. D. Berek, D. Hunkeler, J. Liq. Chromatogr. Relat. Technol. 22 (19) (1999) 2867. D. Berek, Macromol. Symp. 231 (2006) 134. D. Berek, Macromol. Symp. 258 (2007) 198. M. Schollenberger, W. Radke, J. Chromatogr. A 1218 (2011) 7828. M. Schollenberger, W. Radke, Polymer 52 (2011) 3259. H. Maier, F. Malz, G.n. Reinhold, W. Radke, Macromolecules 46 (2013) 1119. P. Schoenmakers, P. Marriott, J. Beens, LC–GC Eur. June (2003) 1. B. Trathnigg, S. Fraydl, M. Veronik, J. Chromatogr. A 1038 (2004) 43. W. Lee, D. Cho, B.O. Chun, T. Chang, M. Ree, J. Chromatogr. A 910 (2001) 51. T. Biela, A. Duda, K. Rode, H. Pasch, Polymer 44 (2003) 1851. W. Radke, K. Rode, A.V. Gorshkov, T. Biela, Polymer 46 (2005) 5456. V.V. Evreinov, A.V. Gorshkov, T.N. Prudskova, V.V. Gur’yanova, A.V. Pavlov, A.Y. Malkln, S.G. Entelis, Polym. Bull. 14 (1985) 131. M. AlSamman, W. Radke, A. Khalyavina, A. Lederer, Macrmolecules 43 (2010) 3215. H.C. Lee, H. Lee, W. Lee, T. Chang, J. Roovers, Macromolecules 33 (2000) 8119. W. Lee, H. Lee, H.C. Lee, D. Cho, T. Chang, A.A. Gorbunov, J. Roovers, Macromolecules 35 (2002) 529. K. Im, S. Park, D. Cho, T. Chang, K. Lee, N. Choi, Anal. Chem. 76 (2004) 2638. K. Im, H.-W. Park, Y. Kim, S. Ahn, T. Chang, K. Lee, H.-J. Lee, J. Ziebarth, Y. Wang, Macromolecules 41 (2008) 3375. J. Gerber, W. Radke, e-Polymers 045 (2005). J. Gerber, W. Radke, Polymer 46 (2005) 9224. H.C. Lee, W. Lee, T. Chang, J.S. Yoon, D.J. Frater, J.W. Mays, Macromolecules 31 (1998) 4114. H. Pasch, Polymer 34 (1993) 4095. M.A. Bashir, W. Radke, J. Chromatogr. A 1163 (2007) 86. M.A. Bashir, A. Brüll, W. Radke, Polymer 46 (2005) 3223. Y. Brun, J. Liq. Chromatogr. Relat. Technol. 22 (1999) 3027. Y. Brun, J. Liq. Chromatogr. Relat. Technol. 22 (1999) 3067. Y. Brun, P. Foster, J. Sep. Sci. 33 (2010) 3501. H.C. Lee, T. Chang, Macromolecules 29 (1996) 7294. D. Berek, Eur. Polym. J. 45 (2009) 1798. M.A. Bashir, W. Radke, J. Chromatogr. A 1225 (2012) 107. Y. Zhu, T. Macko, J. Ziebarth, Y. Wang, Macromolecules 43 (2010) 5888. I.G. Romero, M.A. Bashir, H. Pasch, e-Polymers (2005) 079.

[63] M. Shakun, T. Heinze, W. Radke, in preparation. [64] H.O. Ghareeb, W. Radke, Polymer 54 (2013) 2632. [65] D. Braun, I. Kramer, H. Pasch, S. Mori, Macromol. Chem. Phys. 200 (1999) 949. [66] S.J. Kok, T. Hankemeier, P.J. Schoenmakers, J. Chromatogr. A 1098 (2005) 104. [67] T. Macko, A. Ginzburg, K. Remerie, R. Bruell, Macromol. Chem. Phys. 213 (2012) 937. [68] L.-C. Heinz, H. Pasch, Polymer 46 (2005) 12040. [69] T. Macko, H. Pasch, Macrmolecules 42 (2009) 6063. [70] R. Brüll, H. Pasch, H.G. Raubenheimer, R. Sanderson, A.J. van Reenen, U.M. Wahner, Macromol. Chem. Phys. 202 (2001) 1281. [71] M. Augenstein, M. Stickler, Macromol. Chem. 191 (1990) 415. [72] I. Park, S. Park, D. Cho, T. Chang, Macrmolecules 36 (2003) 8539. [73] J. Falkenhagen, H. Much, W. Stauf, A.H.E. Müller, Macromolecules 33 (2000) 3687. [74] W. Lee, D. Cho, T. Chang, K.J. Hanley, T.P. Lodge, Macromolecules 34 (2001) 2353. [75] B. Trathnigg, O. Jamelnik, A. Skvortsov, J. Chromatogr. A 1128 (2006) 39. [76] A.M. Skvortsov, J.G. Fleer, Macromolecules 35 (2002) 8609. [77] B. Trathnigg, A. Gorbunov, A. Skvortsov, J. Chromatogr. A 890 (2000) 195. [78] A.M. Skvortsov, A.A. Gorbunov, D. Berek, B. Trathnigg, Polymer 39 (1998) 423. [79] A.A. Gorbunov, A.V. Vakhrushev, Polymer 50 (2009) 2727. [80] C. Rappel, B. Trathnigg, A. Gorbunov, J. Chromatogr. A 984 (2003) 29. [81] X. Yang, Y. Zhu, Y. Wang, Polymer (2013), http://dx.doi.org/10.1016/j. polymer.2013.05.018. [82] B. Trathnigg, M.I. Malik, N. Pircher, S. Hayden, J. Sep. Sci. 33 (2010) 2052. [83] M. Mlynek, W. Radke, J. Chromatogr. A 1284 (2013) 112. [84] D. Berek, J. Sep. Sci. 33 (2010) 3476. [85] D. Berek, Macromol. Chem. Phys. 209 (2008) 695. [86] D. Berek, Macromol. Chem. Phys. 209 (2008) 2213. [87] D. Berek, Polymer 51 (2010) 587. [88] M. Janco, T. Hirano, T. Kitayama, K. Hatada, D. Berek, Macromolecules 33 (2000) 1710. [89] D. Berek, M. Janco, K. Hatada, T. Kitayama, N. Fujimoto, Polym. J. 29 (1997) 1029. [90] W. Hiller, P. Sinha, M. Hehn, H. Pasch, T. Hofe, Macrmolecules 44 (2011) 1311. [91] T. Li, S. Strunz, W. Radke, R. Klein, T. Hofe, Polymer 52 (2011) 40. [92] X. Jiang, A. van der Horst, V.S.P.J. Limac, J. Chromatogr. A 1076 (2005) 51. [93] R.E. Murphy, M.R. Schure, J.P. Foley, Anal. Chem. 70 (1998) 4353. [94] S.G. Roos, A.H.E. Müller, K. Matyjaszewski, in: K. Matyjaszewski (Ed.), Controlled/Living Radical Polymerization, 2000. [95] M.A. Samman, Development of two-dimensional chromatographic methods for the separation and characterization of polyesters of different degrees of branching, TU Darmstadt, 2012 (PhD thesis). [96] K. Im, Y. Kim, T. Chang, K. Lee, N. Choi, J. Chromatogr. A 1103 (2006) 235. [97] J. Falkenhagen, S. Weidner, in: C. Barner-Kowollik, T. Gruendling, J. Falkenhagen, S. Weidner (Eds.), Mass Spectrometry in Polymer Chemistry, Wiley-VCH Verlag GmbH Co. KGaA, Weinheim, Germany, 2012, p. 500. [98] T. Gruendling, S. Weidner, J. Falkenhagen, C. Barner-Kowollik, Polym. Chem. 1 (2010) 599. [99] H. Pasch, W. Schrepp, MALDI-TOF Mass Spectrometry of Synthetic Polymers, Springer, Berlin, Heidelberg, 2003. [100] S. Weidner, J. Falkenhagen, in: L. Li (Ed.), Maldi Mass Spectrometry for Synthetic Polymer Analysis, John Wiley & Sons, Hoboken, NJ, 2010, p. 247. [101] R. Edam, D.M. Meunier, E.P.C. Mes, F.A. Van Damme, P.J. Schoenmakers, J. Chromatogr. A 1201 (2008) 208.

Please cite this article in press as: W. Radke, J. Chromatogr. A (2013), http://dx.doi.org/10.1016/j.chroma.2013.12.010