Practical Copolymer Analysis Using Gpc/Sec And

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el permeation chromatography/size-exclusion chromatography (GPC/SEC) is a powerful analytical technique that provides critical information about polymer performance. Unlike other methods, GPC/ SEC does not simply identify average molar mass; rather, this method determinesG the complete molar mass distribution of macromolecules.

Advances in instrumentation design are creating new opportunities to use GPC/ SEC for more challenging analyses such as copolymers. In this eBook devoted to Practical Copolymer Analysis using GPC/SEC and Related Techniques, sponsored by PSS Polymer Standards Service GmbH, experts discuss some of the challenges inherent in analyzing copolymers with different compositions and molar masses. Readers will learn how several GPC/SEC methods may improve copolymer sample characterization.

First, Peter Kilz, director of PSS, explains the benefits of using multiple concentration detectors in GPC/SEC mode for chemical composition analysis. He highlights an investigation of block copolymers using a dual detector approach as an example.

Kilz then suggests another way to measure the chemical composition of copolymers using liquid adsorption chromatography, also known as polymer- HPLC.

Readers will also learn why a combination of various separation techniques, such as 2D chromatography, can overcome several major challenges inherent in copolymer analysis by allowing samples to be simultaneously separated by composition and molar mass.

Last, Daniela Held of PSS Polymer Standards Service GmbH, explains how fractionation or hyphenation (e.g., with chemical detectors) solves the problem of identifying unknown macromolecules, which is often problematic with GPC/SEC systems.

While numerous analytical methods are available to laboratories, GPC/SEC remains an important and effective option. With the tips presented in this eBook, there are even more applications of the technique available to analysts. Table of Practical Copolymer Analysis Contents Using GPC/SEC and Related Techniques

Dual Detection Compositional Analysis by GPC/ SEC With Multiple Detection Peter Kilz 4

Interaction Chromatography of Polymers Polymer Liquid Adsorption Chromatography 10 Peter Kilz

2D Analysis Two-Dimensional Chromatography of Copolymers Peter Kilz 15

GPC/SEC–MS for Comonomers Comonomer Identification: Strategies and Detection Options 23 Daniela Held 4 | JUNE 20174 |JUNE |LC 4 | JUNE 20174 |JUNE |LC C Peter Kilz, PSS Polymer Standards Service GmbH, Mainz, Germany Mainz, GmbH, Service PeterStandards Kilz,PolymerPSS COM B M to launch launch to webinar C lick here here lick launch launch finder lick to to lick app app Y U

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DETECTION W its variation with molar mass (chain length). chemical composition of copolymers and This approach provides information on the Multiple Concentration Detectors? What Is the Advantage of Using this eBook. two will be discussed in the next two articles in analysis with multiple detection, while the latter This article will discuss the chemical composition chemical • chemical • characterize such samples: may co-elute. different composition and different molar mass with compromised that fractions by the fact are chemically simple or homogenous. informationmass aboutunknownsamples that GPC/SEC analysis reveals amultitude of molar comprehensive • There are different methods on how to However, for results may copolymers be mass simultaneously. to be separated by composition and molar SEC mode by concentration detectors samples with different composition by HPLC based on retention differences of ITH

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These results are obtained from the from a detector calibration for each differences in detector response when component. To determine fi different concentration detectors are calibrated concentrations of a homopolymer i are for the components of a copolymer. This injected. The detector area is then plotted method is also applicable to determine versus the injected mass. Figure 1 shows compositions of polymer blends. such a plot for a two detector set-up (UV For each comonomer in the and RI) measured for one component macromolecule an independent detector using its homopolymer. signal is required: The detector response calibration should • When two components with unknown not be confused with the molar mass composition are present, two detector calibration of a GPC/SEC system where signals are required. the elution volume of a reference material • n components will require n with a known molar mass is measured.1 independent detection signals. Obviously, there is a practical limit (four How Can Copolymer signals have been reported) because of Molar Masses be Measured? detector applicability and selection, and An independent task of copolymer band broadening becomes a significant analysis is the determination of meaningful problem. Many results have been copolymer molar masses. Obviously, results published on chemically heterogeneous based on a single molar mass calibration samples using two detectors. Possible with standards will not give accurate results detector combinations and their benefits because the calibration will depend on are shown in Table 1. the local composition of the species. Its To measure the chemical composition, a hydrodynamic radius will depend on the detector response calibration is required. chemistry of the comonomers and their In each detector cell the sample fraction location (sequence distribution) in the will generate a detector signal that is macromolecule. the superposition of the responses of all The best way to overcome the calibration components present. In a general form dilemma is the use of on-line viscometers this can be described as overall detector that measure the intrinsic viscosity even for signal = c1*f1 + c2*f2 + ... = ∑(ci*fi) with ci complex molecular architectures. Molar concentration component i in copolymer masses are available with the universal and fi response factor component i in calibration concept described by Benoit and copolymer. coworkers.2 Please note that light-scattering The individual concentrations of the detection is not generally applicable components are unknowns, while the because the local scattering contrast (dn/dc) response factors can easily be determined will change with composition.3

5 | JUNE 2017 | LCGC Interaction GPC/SEC–MS for Dual Detection Chromatography of 2D Analysis CoMONOMers Polymers

Figure 1: Detector calibration using a homopolymer and injecting different concentrations.

Since not every lab has an on- block copolymer will hydrodynamically line viscometer there is an empirical behave like a pure homopolymer of method for samples with few hetero- the same chain length. In the case of contacts (e.g., block copolymers, long A and B segments in the AB block graft copolymers) using the multiple copolymer, only the A–B link acts as concentration detector approach a defect position and will not change described above.4 the overall hydrodynamic behavior From a GPC/SEC point-of-view the of the AB block copolymer chain. most simple copolymer is an alternating Consequently, the molar mass of the copolymer (AB)n that can be treated copolymer chain can be approximated exactly like a homopolymer with a by the molar masses of the respective repeating unit (AB). The next simple segments. Similar considerations are copolymer architecture is an AB block true for ABA, ABC and other types copolymer, where a sequence of of block structures and for comb- comonomer A is followed by a block of shaped copolymers with low side-chain B units. The only hetero-contact in this densities. chain is the A–B link that can influence In such cases the copolymer molar the size of the macromolecule. The A mass Mc can be determined from segment and the B segment of the AB the interpolation of homopolymer

6 | JUNE 2017 | LCGC Interaction GPC/SEC–MS for Dual Detection Chromatography of 2D Analysis CoMONOMers Polymers

Figure 2: Molar mass distribution with overlaid chemical composition distribution of a styrene/MMA block copolymer with poor block formation.

calibration curves Mi(V) and the weight more powerful and universal methods 5 fractions wi of the comonomers i. have to be employed, such as 2D separations.

lg Mc(V) = ∑ wi(V) lg Mi(V) [1] Application Example: The calculation of copolymer molar Investigation of a Block Copolymer mass averages (Mn,c, Mw,c etc.) and Block copolymers are an important class copolymer polydispersity (Dc) is of polymers used in many applications performed as in conventional GPC/SEC from thermoplastic elastomers to polymer calculations. blend stabilizers. The synthesis is most In situations where the number of often done by ionic polymerization, which hetero-contacts can no longer be is both costly and sometimes difficult neglected, this simplified reasoning to control. However, block copolymer breaks down and copolymer molar properties strongly depend on the exact masses cannot be measured accurately chemical composition, block molar mass by GPC/SEC alone. This applies to and block yield, for example. These statistical copolymers, polymers with parameters can be evaluated in a single only short comonomer sequences and experiment using copolymer GPC/SEC high side chain densities.6 In such cases with multiple detection.

7 | JUNE 2017 | LCGC Interaction GPC/SEC–MS for Dual Detection Chromatography of 2D Analysis CoMONOMers Polymers

Table 1: Copolymer analysis using multiple concentration detectors.

Detector 1 Detector 2 Benefits Example UV or DAD RI Popular combination of universal (RI) and All copolymers in which one component has (set to a fixed tunable detector (UV); easy to operate and a chromophore wavelength) maintain IR RI IR is a versatile alternative to UV and can be Copolymers without UV differences tuned for functional groups (e.g. aliphatic biodegradable polymers) IR IR Detectors 1 and 2 work at different wave- Only special copolymers (e.g., PE, PP, lengths; good sensitivity, easy to operate and polyolefins) maintain, no additional band-broadening as only single detector is used. ELSD FLD Good sensitivity in the ELSD and fluorescence Copolymers with fluorescent components allow determination of comonomer traces UV or DAD UV or DAD Detectors 1 and 2 work at different wave- Only special copolymers with significant (set to a fixed (set to a fixed lengths; good sensitivity, easy to operate and differences in UV spectra, (e.g., photo- wavelength wavelength maintain with DAD/MWD: no additional band- lithography products) broadening as only single detector is used.

Figure 2 shows the measured molar not producing block structure but that the mass distribution of a styrene/MMA block MMA was added to chains of different copolymer using RI and UV detection. styrene molar mass. The RI responds to the styrene and MMA units whereas the UV tuned to 260 nm 1.References D. Held, The Column, 4(6), (2008). predominantly picks up the presence of 2. H. Benoit et al., J. Chem. Phys., 63, 1507, (1966). styrene in the copolymer. After detector 3. a) H. Benoît and D. Froelich in Light Scattering from Polymer Solutions; M.B. Huglin, Ed., response calibration, the styrene and (Academic Press, London, Chapter 11, 1972). MMA content in each fraction can be b) P. Kratochvíl in Classical Light Scattering from Polymer Solutions; A.D. Jenkins, Ed.; (Polymer Science Library measured. 5; Elsevier: Amsterdam, 1987), pp 203–215. 4. J.R. Runyon et al., J. Appl. Polym. Sci., 13, 2359, (1969). This was used to create a dedicated 5. P. Kilz and H. Pasch; Coupled LC techniques in homopolymer calibration curve from the molecular characterization; in: Encyclopedia of Analytical Chemistry; R.A. Myers (ed.); (Wiley, Chichester 2000). measured PS and PMMA calibration curves 6. F. Gores and P. Kilz, Copolymer Characterization using the MMA content in each fraction. Using Conventional SEC and Molar Mass-Sensitive Detectors, in: T. Provder (Ed.) Chromatography of In Figure 2, the MMA content distribution Polymers (Chapter 10, ACS Symp Ser 521, American (blue solid line) is superimposed to the Chemical Society, Washington, DC, 1993). molar mass distribution (MMD) of the Peter Kilz studied polymer chemistry in product. It is obvious that the MMA Mainz, Germany and Liverpool, UK and content is not constant throughout the is head of the software development MMD, but continuously increases with department. He is also responsible for the molar mass. The trimodal MMD system solutions, client server set-ups and itself only shows the presence of three support. different species, while the additional MMA content information clearly reveals E-mail: [email protected] that the copolymerization process was Website: www.pss-polymer.com

8 | JUNE 2017 | LCGC ThinkingThinking ForwarForward.d. GPC/SEC Theory or

practice? PSSPSS araree woworldrldld leaders llead ders ini mmacromolecularacromolelcular l chcharacteri- haractet rii- zationzation anandd hhaveave tthehe expertise to hhelpelp you witwithh your analysisanalysis requirements. We oofferffer a ranrangege ooff proproductsducts andand services ffromrom instruction courses anandd trainintrainingg If there is one thruthru contract anaanalysis,lysis, consuconsulting,lting, metmethodhod ddevelop-evelop- ment andand qqualiﬁualiﬁ cation services aallll tthehe wawayy up to thing we can supplyingsupplying turnkey GPCGPC/SEC/SEC and LCLC/2D/2D systems. do, it’s both. AllAll this comes with the ppersonalersonal and direct susupportpport of our dedicated team of innovative and pioneeringpioneering specialists. Is there any better way of achievingachieving your analysisanalysis ggoals?oals?

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PhonePhone + 49 66131131 996239062390 You‘ll ﬁ nd the ideal GPC/SEC solution at PSS under: www.pss-polymer.com [email protected] 9 | JUNE 2017 | LCGC 10 2017 |JUNE |LC 10 2017 |JUNE |LC application application application application C Peter Kilz, PSS Polymer Standards Service GmbH, Mainz, Germany.Mainz, GmbH, Service PeterStandards Kilz,PolymerPSS Polymer to view view to to view view to C note C note lick lick lick lick hromatogra C S S p p omposition omposition on on Gradient- G G s s C C o o red red S E TPE C L iq Here, the separation is based on interaction interaction on based is separation the Here, (LAC). chromatography adsorption liquid use to is copolymers of kinds all of composition detectors. different two least at using determined is copolymers block example, of, CCD for the and applied is volume) (hydrodynamic size the on based separation GPC/SEC case, this In eBook. this of chapter first the in discussed was which detection, dual including techniques CCD. the into insight any provide not do detectors these addition, In composition. on the of dependence the from scattering) light- of case (in and GPC/SEC in co-elution potential from suffers copolymers for detectors these of applicability the homopolymers, of MMD the of determination the for detectors (CCD). distribution composition chemical the and (MMD) distribution mass molar the simultaneously: present be can distributions two least at because challenging is analysis Copolymer u An alternative way to measure the chemical chemical the measure to way alternative An several using determined be can CCD The sensitive mass molar are there While id id p hy A dsor p tion tion dn/dc

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100 Column: C8 (4 × 150 mm) Gradient: 100% ACN ➞ 100% THF in 20 min 90 Detection: ELSD

60 Styrene 1% TH F % e e

50 e

40 36% Styren 36% 100% Butadiene 23.5% Styrene 50% Styren 50% 16% Styren 16%

30 100% Styrene

10 11 12 13 14 15 16 17 18 19 20 Elution time (min)

Figure 1: Separation of a polystyrene-polybutadiene copolymer using LAC. The higher the PBd content, the higher the elution time. Data courtesy of Dr. Yefim Brun, DuPont Central Research and Development.

of the sample with the stationary gradient pumps are recommended for phase. This technique is commonly copolymer HPLC. regarded as polymer HPLC, and is In contrast to GPC/SEC (where referred to as gradient polymer elution polymeric phases dominate), silica-based chromatography (GPEC). column packings are the most important stationary phase. Both normal phase What Is Used in LAC/Polymer-HPLC? (NP) and reversed phase (RP) separations In general, the set-up for polymer-HPLC have been described in the literature. A is quite similar to GPC/SEC systems. good summary of different applications However, a few modifications are in copolymer separation is given by needed. Pasch.1 LAC requires the adsorption and Detectors used in gradient LAC are desorption on a stationary phase. In mainly UV/DAD detectors (if possible) most cases, isocratic separation is not and evaporative light-scattering sufficient for copolymers. Therefore, detectors (ELSD), while refractive index gradients with respect to pH value, (RI) and light‑scattering detectors (in ionic strength, eluent composition or solution) are typical detectors in isocratic temperature are applied. The most elution. common approach is to use eluent composition gradients, therefore,

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Figure 2: Calibration curve obtained with the data from Figure 1. In contrast to a typical GPC/SEC calibration curve the content of one of the comonomers is plotted on the y-axis.

What Is Measured? reversed-phase mode on a C8 modified When analyzing copolymers or polymer silica column using ELS detection. blends in LAC mode, the retention The corresponding calibration is time of the sample is indicative of its shown in Figure 2. Please note that this average chemical composition and its separation is optimized for samples peak width is a measure of the CCD. As with high styrene content as can easily copolymer retention depends on the be seen from the calibration curve in eluent and columns used (phase system), Figure 2. the retention axis has to be calibrated Figure 3 shows an example of a either by standards with known chemical polymer-HPLC separation covering composition or multiple detector a wide polymer polarity range from combinations can be employed to PMMA to PBd. Even samples with similar measure the local chemical composition. polarity as PPE and PSt can be separated Figure 1 shows the separation of easily under these conditions. random styrene-butadiene copolymers of different styrene content separated in

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100 PSt PPE PBd 80 PMMA SAN 25%A N SAN 33%A N SAN 19%A N 60

Gradient Composition (%B) 20

0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0

Elution volume (mL)

Figure 3: Separation of different homopolymers and copolymers on a C18 column using a gradient of acetonitrile and THF (both with methylene chloride). Data courtesy of Dr. Yefim Brun, DuPont Central Research and Development.

0.035 Pharmaceutical PEG copolymer

0.030 CCD results G : 4.9378e1 dG : 3.0627e0 0.025 S : -8.027e-1 Vp : 8.6790e0 Gp : 6.0247e1 0.020 A : 1.9261e0 w(C) <20% 11.69 0.015 w% : 73.28 >70% 15.03

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Figure 4: Results of a chemical heterogeneity analysis of a PEG copolymer.

What Are the Results? The average chemical composition of Peak position is not a good measure to a copolymer can be easily described by determine and quantitatively describe the moments of the CCD as measured the chemical composition distribution of in polymer-HPLC experiments. The samples with wide chemical heterogeneity. average composition, G, the width,

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dG, and the skew, S, of the CCD can product control) to visualize small sample easily be calculated using a modern differences that might otherwise be macromolecular chromatography data missed. system (MCDS). The value of the skew Figure 4 shows the results of a parameter is zero, if the composition pharmaceutical PEG copolymer with distribution is symmetrical. broad chemical composition distribution The average composition is giving quantitative and comprehensive described as results on chemcial composition. – G= µ (G) ∑ci ∙ G 1 = i References ∑ci 1. H. Pasch “Chapter 5: Liquid adsorption chromatography” in H. Pasch, B. Trathnigg, HPLC of Polymers, with c the local concentration in the Springer-Verlag Berlin Heidelberg New York (1998). i 2. (a) E. Schröder, G. Müller and K.-F. Arndt, chromatographic fraction. The definition Polymer Characterization, Carl Hanser of dG and S can be found in the Verlag, München Wien New York (1989) (b) User Documentaton PSS WinGPC Unity MCDS. literature.2(a),(b) Small differences between otherwise Peter Kilz studied polymer chemistry in similar samples can be flagged by the Mainz, Germany and Liverpool, UK and determination of the copolymer amount is head of the PSS software development above (or below) a certain reference department. He is also responsible for composition (e.g., how much of my system solutions, client server set-ups sample has chains with a composition and support. higher [lower] than x% component B). It has also proven useful to overlay the E-mail: [email protected] CCDs of similar samples (e.g., from Website: www.pss-polymer.com

14 | JUNE 2017 | LCGC 15 2017 |JUNE |LC Peter Kilz, PSS Polymer Standards Service GmbH, Mainz, Germany Mainz, GmbH, Service PeterStandards Kilz,PolymerPSS of C Tw to view view to view to primer video C C lick lick lick hromatogra o-dimensional o-dimensional C S S 2 p p D on on Polymer 2 G o D s s C o o Primer red red p A nalysis olymers method combinations. They can show sample sample show can They combinations. method dimension to investigate the MMD. or end groups) with GPC/SEC in the second critical conditions (separation according to CCD separation according to CCD) under or LAC approach is to combine (Polymer-HPLC, LAC or copolymers others),graft common the most term. and not as afactor as an additivepeak capacity each dimensionbecause contributes to the total n-dimensional separation is substantially higher this. in The an corresponding peak capacity and cross-fractionation) help can to overcome chromatography, chromatography orthogonal dimensional chromatography (also called 2D techniques into a single experiment (multi- limited chromatographic resolution. technique, is that the method may from suffer that cannot solved be by using any detection distribution (CCD). Aproblem in GPC/SEC, (MMD) and the chemical composition simultaneously: the molar distribution mass present be distributions can at least two Copolymer analysis is challenging because Figure 1 shows the advantages of 2D 2D of advantages the shows 1 Figure For copolymer analysis (e.g., block copolymers, A combination of different separation p hy hy

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Figure 1: Two peaks in GPC/SEC and LAC can be interpreted in different ways when the samples are heterogeneous in composition and molar mass. A few examples for possible solutions are shown.

differences that cannot be detected To perform a 2D analysis, the by analysis in individual separation components (and methods) required for methods. For all four samples, both each single separation technique are separation techniques show no needed. Standard instrumentation, such difference in their chromatograms. as pumps, autosamplers, column ovens Only on-line two‑dimensional and detectors can be used here. chromatography provides users with The pivotal part in any the entire picture. two‑dimensional set-up is the fraction transfer step between the separation What Is Needed for methods, which can be on-line or off- 2D Chromatography? line, manual or automated. Off-line In two-dimensional chromatography, systems require a fraction collection two different separation techniques are device with manual transfer and combined; for example HPLC, LACCC, reinjection of the first dimension GPC/SEC, TREF, IC, CE, GC or others. fractions into the second instrument. In A sample is separated using one on-line 2D systems the fraction transfer technique and the sample fractions is preferentially done automatically. are then analyzed using the second Comprehensive 2D work employs technique. complete transfer of the injected mass

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Figure 2: 3D surface plot for visual inspection and the results for the four identified peaks.

from the first to the second dimension. columns will be used in the second This ensures that the complete sample dimensional separation. However, is analyzed. Heart‑cut approaches are the availability of high throughput not considered comprehensive for and high-speed GPC/SEC columns obvious reasons. allows for much faster 2D results. If the Advantages and limitations of transfer is on-line, for example with the different off-line and on-line a dual loop valve, two-dimensional approaches are summarized in Table 1. chromatography can be fully automated Different set-ups and approaches for and the time to analyze a sample can two‑dimensional analysis are described be decreased to 2–3 hours. in the literature.1 Very important in 2D chromatography Obviously, two-dimensional is data presentation and analysis. separations take more time depending Comprehensive 2D experiments yield on the number of transfer injections a three-dimensional data array (similar from the first into the second to DAD/PDA data sets) of data tuples dimension. Time requirements can in the form of [(dimension 1 property), be substantial if analytical GPC/SEC (dimension 2 property), (concentration)]

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Figure 3: Example for a 2D contour plot with 4 different peaks where LAC shows only 2 peaks and GPC/SEC shows only one broad peak with shoulders.

which can be expressed in multiple possible. The CCD can be determined ways. via an appropriate calibration with 3D surface plots generated from a samples of known composition. The lattice plot by interpolation, visualize MMD can be calculated based on a results, however quantification is not conventional molar mass calibration of possible. They can be regarded as the second dimension.2 virtual 3D landscapes that can be Figure 3 shows a 2D contour plot as well viewed from any observation position as the corresponding first and second in space and allow users to investigate dimension LAC and GPC/SEC traces. the peak shape or trace amounts on the back side of major peaks (see Practical Advice for 2D also Figure 2). Contour plots (or In general, performing a 2D experiment contour maps) are two-dimensional is often easier than expected. The representations of the 3D surface major work is to develop robust plots. Different concentrations are methods for both dimensions. normally shown as different colors. Commercial tools are available for the The chromatographic axis of the first transfer from one dimension to the dimension is plotted on the ordinate, other and the result presentation and while the properties determined in evaluation. the second dimension are shown on The proper sequence of separations the abscissa. Here, quantification is methods is important to achieve highest

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Figure 4: Entire picture of a mixture of polystyrene-polybutadiene block copolymers with different molar masses and structures and different polybutadiene content. Up to 16 different species could be identified and quantified. Peak 1 is a linear PS-PBd copolymer with approx. 20% butadiene content, peak 16 is a 4-arm star polymer with approx. 80% butadiene content.

resolution and accurate determination dimensional experiments. Complete of distributions. It has been shown that miscibility of the mobile phases used in it is best to apply the method with the all dimensions is a stringent necessity. highest selectivity for a property as the Otherwise the separation in the second first dimension. This ensures the highest method is dramatically influenced purity of eluting fractions are transferred and the fraction transfer is restricted into the subsequent separation. or completely hindered. In gradient Obviously, techniques such as GC and systems, this requirement has to be SFC that destroy the mobile phase can verified for the total composition range. only be used as the second dimension. In GPC/SEC dimensions, the transfer For characterization of copolymers, of mixed mobile phases can affect molar mostly LAC or LACCC has been mass calibration. In order to get proper hyphenated with GPC/SEC, but also molar mass results, the calibration curves other combinations have been applied have to be measured using the extremes successfully. of mobile phase composition and tested The compatibility of mobile for changes in elution behaviour and phases that are transferred between pore-size influence in the GPC/SEC chromatographic dimensions is an column packing. important issue in designing two- It has been shown to be beneficial

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Table 1: Comparison of 2-dimensional transfer options

Transfer Mode Advantages Disadvantages Example Manual Off-line • Very simple • Time-consuming Test tubes, manually • Fast set-up • Not for routine work exchanged • Not precise • No correlation of fraction elution to transfer time • Not quantitative Automatic Off-line • Simple • Less precise Fraction collector, • Easy • No correlation of fraction storage valve • Fast set-up elution to transfer time • Not quantitative Automatic On-line • Correct concentrations • Transfer not quantitative Injection valve with single-loop • Correct transfer times • Setup time (with actuation) • Automation Automatic On-line • Correct concentrations • Setup time Dual-loop valve with • Correct transfer times • Special valve required 1. actuated 8-port valve • Quantitative transfer 2. combination of two conven- • Automation tional 6-port injections valves

to use the GPC/SEC eluent as one and stucture.3 Here a mixture of 4 component of the mobile phase in the copolymers with 20%, 40%, 60% and previous dimension to avoid potential 80% butadiene is analyzed. In addition interference and mobile phase to the different composition, there are incompatibility. two linear structures (PS-PBd, PS-PBd- The best detectors for GPC/SEC PbD-PS) and two branched structures, dimensions are DAD/PDA detectors a 3-arm star with PS‑PBd copolymer (if chromophores are available) or ELS arms and a 4-arm star with PS‑PBd detectors, due to the high sensitivity copolymer arms. of these detectors and the baseline This 16-component mixture has been stability. RI detection is possible but analyzed using LAC on a silica phase sometimes these detectors are not 5-µm 60 Å column with a gradient sensitive enough or the chromatograms iso-octane/THF (20% THF linear to show strong solvent peaks at high 100% THF) followed by GPC runs on elution volumes. a SDV styrene-divinylbenzene phase in THF. Figure 4 displays the resulting What Are the Results for 2D contour plot and the results. Both Chromatography of Copolymers? dimensions have been calibrated A famous example showing the so that it is possible to determine high peak capacity and the possible the molar mass distribution and the results with 2D chromatography chemical heterogeneity for each single is the separation of polystyrene- peak. The butadiene content axis polybutadiene block copolymers with and the molar mass axis are shown different composition, molar masses for all peaks. The colour code gives

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additional information about the Peter Kilz studied polymer chemistry concentration. in Mainz, Germany and Liverpool, UK and is head of the PSS software development department. He is also 1.References H. Pasch, Chapter 7: Two-Dimensional Liquid Chromatography, in H. Pasch, B. Trathnigg, responsible for system solutions, client HPLC of Polymers, Springer-Verlag Berlin server set-ups and support. Heidelberg New York, USA (1998). 2. D. Held, The Column, 5(6), 18–21 (2008). 3. P. Kilz et al., ACS Adv. Chem., 247, 223–241 (1995). E-mail: [email protected] Website: www.pss-polymer.com

21 | JUNE 2017 | LCGC Thinking Forward.

The whole is more than the sum of its parts – the value of 2 dimensions by PSS.

True Molar Masses Composition Comonomer distribution End groups 2-dimensional chromatography gives you more answers – by doubling the power of the analysis you quadruple the amount of information and resolution. The PSS 2D Polymer Analyzer with WinGPC UniChrom Software combines separation techniques such as HPLC and GPC/SEC in an automated, easy-to-use and user-friendly solution. Find out what your samples really contain!

22 | JUNE 2017 | LCGC 23 | JUNE 201723 |JUNE |LC and and I Daniela Held, PSS Polymer Standards Service GmbH, Mainz, Germany Mainz, GmbH, Service Standards PolymerPSS Held, Daniela C dentification: dentification: o M G onomer onomer C D etection etection chain also affects performance. performance. affects also chain the along monomers the of distribution the but properties, material’s the affect product final the in present comonomers of percentage properties. product macroscopic the governs also that heterogeneity additional an is composition it. measure to choice of method the is GPC/SEC and heterogeneity, well-known a is distribution mass molar The heterogeneity. is macromolecules of majority vast the of feature common a processes, statistical monomers. are composedof polymers three different is called acopolymer. In addition, terpolymers of monomers, it different types comprises two it is consideredstructure, ahomopolymer. If it of monomer with theone chemical same type a macromolecule is synthesized using only compositions,structures, and end groups. If different lengths (and, therefore, molar masses), polymerized be can to large molecules with monomer units, small building that blocks our daily lives. They are produced from Macromolecules in are found everywhere In the case of copolymers, chemical chemical copolymers, of case the In are polymerizations technical all Since Op S N ot only does the average average the does only ot trategies trategies tions

gettyshutterstock.com/VAlex Images/xxxxxxxx Interaction GPC/SEC-MS for Dual Detection Chromatography of 2D Analysis CoMONOMers Polymers

Figure 1: Examples for copolymer and copolymer structures: the chemical different comonomers red and blue form different structures (e.g., combs and stars) and have different distributions along the chain (e.g., block-like structure or random copolymer).

Figure 1 shows a few examples however, is that they provide no of typical copolymer structures. information about the distribution of the Copolymers can be block copolymers, comonomers along the macromolecule that only have one link between two chain. It is also not possible to determine monomers of different chemistry, if the composition is the same for short alternating polymers (very rare), or and long chains. This justifies fractionation random copolymers. When structural techniques such as GPC/SEC (and also variations are possible or introduced interaction chromatography, liquid on purpose, additional variations are adsorption chromatography [LAC], and also possible. Overall, this makes LAC at critical conditions [LACCC]) are so comprehensive characterization a important; they allow for the separation real challenge, although the number of complex mixtures into less complex of potential monomers is limited, fractions. This enables identification generally. using spectroscopic or spectrometric FTIR spectroscopy (either in techniques and significantly enhances the transmission-IR or attenuated total information collected during the analysis. reflection [ATR]) and nuclear magnetic GPC/SEC systems are, therefore, resonance [NMR] are powerful techniques increasingly equipped with either that can be used to quickly identify traditional fraction collectors or comonomers in products. The hyphenated with information-rich disadvantage of these batch methods, chemical detectors. The hyphenation

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Figure 2: Chromatogram of a four-component mixture separated on a GPC/SEC. The vertical lines indicate the fractions collected by the fraction collector.

itself can either use a full on-line investigated using spectroscopic or approach, where an additional detector spectrometric methods, for example. is directly connected to the GPC/SEC The advantage of this combination of system, or off-line where fractions are techniques is that sample components collected and analyzed later. Here, have been separated before using off-line includes a variety of technical identification methods and often- solutions from stopped-flow techniques disturbing GPC/SEC solvent can be to sophisticated collection modules. easily removed for the fractions. To obtain a significant amount of Traditional Fraction Collection fractionated sample with a reduced Adding a fraction collector to a number of separation runs, it is often GPC/SEC system is easy; even fully useful to have a (semi)-preparative GPC/ automated use is possible. Figure SEC instrument. 2 shows a fractionated sample with Many existing analytical GPC/SEC the collected fractions marked in the instruments can be used for semi- chromatogram. After collecting the preparative fractionations with only a fractions, the solvent can be evaporated few modifications: and the fractions can be further

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Figure 3: Chromatogram of a polymer mixture consisting of a polymer part and several small side products. All slices are collected on a rotating Germanium disc, while the GPC/SEC solvent (THF) is automatically evaporated. After the run has been completed the spots on the Germanium disc are investigated using FTIR detection.

• The pump should be able to run at consuming. In addition, several manual higher flow rates (e.g., 3–10 mL/min). steps are required to prepare the fractions • The injection loop should be replaced for the following detection techniques. or upgraded by a unit that can inject higher volumes (e.g., up to 1 mL). Chemical Detectors • The analytical GPC/SEC columns A few chemical detectors are already should be replaced by semi- available to be hyphenated with GPC/ preparative columns. These columns SEC.1 have a larger internal diameter (20 They are used for different applications mm compared with 7.5–8 mm for and the following summary discusses their analytical columns) and have an general advantages and limitations. increased sample capacity. • A fraction collector should be added IR Detection and Raman Detection to the system. The vast majority of FTIR detection in polymer analysis is off-line detection. A disadvantage of this approach is that On-line detection with an IR detector is it requires a larger amount of solvent, mainly used in high-temperature GPC produces more waste, and is time (HT-GPC) for polyolefin characterization.

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These detectors provide important Raman detection suffers from similar information, such as about short-chain problems as FTIR detection with respect branching.2 to solvent effects. Off-line Raman is also For many other GPC/SEC applications, possible with this approach. However, on-line IR detection cannot be used if a glass disk is used instead of a because typical GPC/SEC solvents, such Germanium disk, it is also possible to as THF, adsorb in the same region as investigate the fractions with hand-held the investigated polymers. Therefore, Raman devices. the strong solvent signals mask the A limitation of this off-line approach much smaller polymer signals. An is that if salts must be added to the automated, universal, and long-term GPC/SEC solvent for interaction-free stable mechanism to suppress the chromatography, these salts must be solvent peaks has not yet been found. volatile (examples have been previously Hence, off-line techniques are more published4). common. Here, the GPC/SEC effluent is directed to a heated nozzle for solvent Mass Spectrometry evaporation followed by the deposit Although the main task in mass of analyte fractions on a Germanium spectrometry (MS) is to determine the disk, which is in an off-line step scanned molar mass, it is also used to identify afterward using a standard FTIR species or to distinguish between detector. This elegant technique allows species when the spectroscopic results one to separate small quantities and to are ambiguous. detect analytes without the influence of Different mass spectrometric methods the solvent. have been used in macromolecular Figure 3 shows a chromatogram of a analysis, in which matrix assisted laser polymer and a sketch of how the off-line desorption ionization-time of flight approach works. After the separation on (MALDI-ToF) and electrospray ionization the GPC/SEC column and the collection (ESI) are the most common techniques step (including solvent evaporation), in combination with GPC/SEC. the comonomers are identified using an FTIR detector. A spectrum can be MALDI-ToF obtained at all points of interest on Nearly the same equipment used for the Germanium disk, and it is also off-line FTIR detection can also be possible to monitor if the comonomer used for MALDI-ToF detection. Here, concentration changes. the Germanium disk is replaced by a This technique is also often used in disk with a MALDI matrix. Detection additive analysis to identify unknown is also off-line. MALDI-ToF is a useful additives (e.g., in master batches).3 technique, especially in the life

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sciences sector, but it suffers from • Fractions with a narrow molar mass the fact that it must be possible to distribution are transferred into the ionize the macromolecules and from ESI-MS after separation, so easy-to- mass discrimination effects and matrix interpret spectra are obtained. selection. • The sensitivity of the mass spectrometric analysis is significantly ESI improved. The molar mass range In contrast to MALDI-ToF, many GPC/ after on-line hyphenation can be SEC-ESI couplings are on-line. The most extended to 10–20 kDa, depending applied and best described MS method on the resolution of the mass for hyphenation with GPC/SEC is ESI. spectrometer. One major advantage of the ESI– MS approach is that these mass A drawback is that GPC/SEC methods spectrometers can be easily connected requiring salts or additives might to a liquid chromatography (LC) system require modification. At the very least, and that the possibility for multiple the use of volatile salts is a prerequisite charged states allows it to go to higher for hyphenation with MS instruments. molar masses. Ideal solvents are those that are easy For quantification, GPC/SEC-ESI-MS to evaporate such as THF, chloroform, requires a concentration detector (e.g., methylene chloride, or toluene. In RI, or UV). Besides LC components, a addition, the interpretation of spectra, simple T-connector that splits the flow especially for copolymers, is quite into ideal flows for the concentration demanding and time consuming, so MS detector and the mass spectrometer has a supporting role in determining the (e.g., 9:1) is the only requirement. chemical heterogeneity of copolymers. This type of GPC/SEC-MS coupling has been applied to a wide variety of NMR Spectroscopy macromolecules (e.g., homopolymers Although on-line and stopped-flow and copolymers); among them are data have been reported, GPC/SEC– polyacrylates, polyesters, polyethers, NMR coupling is still not a standard polyamides, resins, polycarbonates, technique. The use of deuterated proteins, and polystyrene.5 As a rule of solvents or the need to suppress the thumb, macromolecules having at least intensive solvent signals by special one functional group per monomer unit NMR pulse sequences has limited the that can be ionized in solution can be applicability. An additional problem analyzed. is the low sensitivity of NMR in The hyphenation with the fractionating combination with the low concentrations method GPC/SEC has several advantages: used in GPC/SEC. However, more

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progress is needed to adapt this useful 1.References “Chapter 10: Chemical Detectors” in A. Striegel et detection technique. al., Modern Size-Exclusion Liquid Chromatography, John Wiley & Sons, Inc., Hoboken, NJ (2009); P. Kilz and H. Pasch, “Coupled Liquid Chromatographic A Comment on UV Detection Techniques in Molecular Characterization,” in R.A. The use of a specific detector such Meyers, Ed., Encyclopedia of Analytical Chemistry, John Wiley & Sons, Inc., Chichester (2000). as an ultraviolet (UV) detector set 2. P. Montag, “Tips & Tricks: GPC/SEC A Development in High Temperature (HT) GPC/SEC” The Column 4(12), 14–17 (2008) to a specific wavelength or a diode 3. P. Montag, “Old Questions—New Solutions: Modern array detector (DAD) can also help to Ways for Complex Polymer Analysis and Additive Tracing” The Column 2(11), 16–18 (2006). identify monomers or to investigate 4. D. Held, “Tips & Tricks: Evaporative Light Scattering the chemical heterogeneity. UV Detection in GPC/SEC” The Column 9(18), 2–5 (2013). 5. T. Gründling, M. Guilhaus, C. Barner-Kowollik, detectors are used in GPC/SEC, Anal. Chem. 80, 6915 (2008). when the investigated polymers have chromophoric groups. If the Daniela Held studied polymer chemistry UV wavelength is specific for the in Mainz (Germany) and is working in the repetition unit, concentration signals are PSS software and instrument department. measured. An example is the detection She is also responsible for education and of polystyrene with a UV detector set to customer training. 254 nm. The number of macromolecules where UV detection can be used is E-mail: [email protected] limited, however. In addition, DAD Website: www.pss-polymer.com spectra are useful only for a very limited number of applications and their use for identification is negligible. UV detectors are useful in combination with RI detectors to measure the distribution of comonomers along the chain. Dual detection with concentration detectors is a useful tool to monitor varying chemical heterogeneity.

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