Chromatographic Cross-fractionation

16 Chromatographic Cross-fractionation GOTTFRIED GLOCKNER Dresden University of Technology, GDR 16.1

INTRODUCTION

314

16.2 BASIC CONCEPTS OF CHROMATOGRAPHIC CROSS-FRACTIONATION 16.2.1 Orthogonal Size-exclusion Chromatography in Different Solvents 16.2.2 Utilization of Different Modes of Adsorption Chromatography 16.2.2.1 General remarks on adsorption chromatography and gradient elution 16.2.2.2 Solubility effects . 16.2.2.3 Molecular weight effects in adsorption chromatography 16.2.3 Prefractionation by Adsorption Chromatography and Subsequent Investigation of the Fractions by Size-exclusion Chromatography 16.2.4 Prefractionation by Size-exclusion Chromatography and Subsequent Investigation of the Fractions by Adsorption Chromatography OUTLINE OF CHROMATOGRAPHIC CROSS-FRACTIONATION BY HPLC INVESTIGATION OF SIZE-EXCLUSION CHROMATOGRAPHY FRACTIONS 16.3.1 Detection 16.3.2 The Eluent for Size-exclusion Chromatography Separation 16.3.3 On-line or Off-line Operation 16.3.4 The Eluent for HPLC Separation 163.5 Column Dimensions and Activity 16.3.6 Calibration 16.3.6.1 Dependence of size-exclusion chromatography elution volume on molecular weight and composition 16.3.6.2 Dependence of the detector signal on polymer amount and composition 16.3.6.3 Dependence of the retention in nonexclusion HPLC on molecular weight and composition 16.3.7 Evaluation of Experimental CCF Results of Binary Copolymers 16.3.7.1 Determination of molecular weight and polymer amount in each size-exclusion chromatography fraction 16.3.7.2 Correction of the M effect in HPLC 16.3.7.3 Calculation of copolymer composition at any measured HPLC retention time 16.3.7.4 Correction of the UV signal 16.3.7.5 Evaluation of the two-dimensional distribution of the sample

314 314 315 315 315 316 316 316

16.3

16.4 CROSS-FRACTIONATION OF SYNTHETIC POLYMERS 16.4.1 Partially Branched Polymers 16.4.1.1 Branched polystyrenes 16.4.1.2 Branched polyethylenes 16.4.2 Copolymers from Ethylene and Propylene 16.4.3 Statistical Copolymers from Styrene and Acrylonitrile 16.4.4 Copolymers from Styrene and Methyl Methacrylate 16.4.4.1 Block copolymers 16.4.4.2 Statistical copolymers 16.4.5 Statistical Copolymers from Styrene and Methyl Aery late 16.4.6 Statistical Copolymers from Styrene and n-Butyl Methacrylate 16.4.7 Statistical Copolymers from Styrene and Ethyl Methacrylate 16.4.8 Statistical Copolymers from Styrene and Methoxyethyl Methacrylate 16.4.9 Copolymers from Styrene and Acrylic Acid

317 317 317 317 318 318 319 319 319 320 320 320 320 321 321 321 321 321 321 323 324 325 328 328 329 330 332 332 335 335

16.5 CONCLUDING REMARKS

335

16.6

336

REFERENCES

313

314 16.1

Separation Methods INTRODUCTION

In 1952 a report was given on the influence of the precipitant upon the fractionation of cellulose acetate from solutions in acetone: when n-heptane was used, the fraction of highest viscosity had the lowest acetyl value, whereas with water as a precipitant, the fraction of highest viscosity had the highest acetyl content. 1 The authors recognized that the solubility of cellulose acetate is determined both by chain length and composition, and that even the most painstaking fractionation in only one solvent/nonsolvent system could not yield fractions with narrow distributions in chemical com­ position (CCD) and in molecular weight (MMD). They coined the term 'cross-fractionation' for a scheme that included the fractionation of the raw polymer through the addition of one kind of precipitant (water), and the subfractionation of redissolved fractions through the addition of a different one (n-pentane). Rosenthal and White proved the narrowness of the final fractions by repeating the precipitation with water. The authors also suggested cross-fractionation as a general approach in copolymer analysis. In 1962, Topciev et al. derived equation (1) for the volume fraction (j)DPyX of a copolymer with degree of polymerization DP and composition x in the dilute sol phase and the gel phase, 0' and (/>", respectively.2 Teramachi and Nagasawa showed that the values of o and K are linked to the Huggins' constants of interaction between the copolymer constituents and the solvent components. For a binary A/B copolymer and a binary solvent consisting of the liquids 1 and 2, equation (2) holds. Cross-fractionation by solubility effects requires a solvent/nonsolvent system in which XA > XB a n ^ another one in which XA < XB holds.3 4>"DP, J'Dp,

x

= exp[£>P(<7 + K ■ x)]

K = (; - $'[) (XIA - * 1 B ) + (2 - "l) (*2A - X2B)

(1) (2)

The literature dealing with the separation of copolymers has been excellently represented in reviews on polymer fractionation, 4,5 but the list of papers concerned with cross-fractionation is rather short even in a specialized compilation of the literature on fractionation of copolymers.6 Irrespective of its importance, cross-fractionation has been performed only seldom. A principal obstacle is often the difficulty of finding the solvent systems which fulfil the above mentioned conditions for XA a n < l ZB- The application of the method for analytical purposes might also be hampered by the effort which is required when classical solubility fractionation is used: about 8-12 weeks of hard labour are necessary for the evaluation of the two-dimensional distribution in size and composition of each copolymer specimen. The aim of this chapter is a survey of chromatographic (and related) techniques which can facilitate the separations required in cross-fractionation via the participation of a stationary phase, and enhance the speed of data acquisition. The contribution is also intended to be a review of the applications of chromatographic cross-fractionation (CCF) known at present.

16.2 BASIC CONCEPTS OF CHROMATOGRAPHIC CROSS-FRACTIONATION CCF of a binary copolymer requires two chromatographic techniques which must separate the copolymer according to different properties. In the ideal case, one method should yield fractionation by molecular weight and the other by composition. This section deals with suitable combinations of chromatographic techniques. 16.2.1

Orthogonal Size-exclusion Chromatography in Different Solvents

Size-exclusion chromography (SEC) separates polymers by their hydrodynamic volume, given in equation (3), where M is the molecular weight and [r\\ the intrinsic viscosity of a component with the hydrodynamic volume Vh. Substituting the Kuhn-Mark-Houwink equation (4), equation (3') results. Kv and a are constant for a given polymer in a given system. In a different solvent, the same polymer will have different values of the constants Kv and a. The exponent a increases with the thermodynamic quality of the solvent. Vh = M-M

(3)

M - K vM a

(4)

Vh =

KvM1+a

(3')

Chromatographic Cross-fractionation

315

In 1980, coupling two GPC devices and running each with a different mobile phase was suggested.7 Both devices utilize steric exclusion columns, but the eluent in GPC(2) is thermodynamically poorer than that in GPC(l). Hence, an eluate portion (a 'slice') from GPC(l) will undergo further separation in GPC(2), which will possibly be enhanced by partition or adsorption interactions of the copolymer on the column packing material. The authors proposed the term 'orthogonal chromatography' for the method which they used for the separation of copolymers from styrene and n-butyl methacrylate (see Section 16.4.6). 16.2.2 16.2.2.1

Utilization of Different Modes of Adsorption Chromatography General remarks on adsorption chromatography and gradient elution

Adsorption chromatography (AC) is chromatographic separation due to energetic interactions between the solute and the surface of the porous packings. If separation by composition is aimed at, the column should retain one kind of structural unit more strongly than the others. The chance of obtaining these interactions must not depend on the size of the solute if disturbance of the AC separation by SEC effects is to be avoided. Hence, the pores of the packings must be either large enough to give access to all kinds of solute molecules or so small that all of them are excluded. Most of the AC results presented in Section 16.4 have been obtained with small-pore packings. Chromatographers distinguish between normal-phase and reversed-phase AC. The former utilizes a polar stationary phase which retains the solutes according to their polarity. If gradient elution is performed the polarity of the eluent mixture increases in the course of the experiment. In reversedphase chromatography (RPC), the stationary phase is less polar than the eluent. An example of a RPC system is a C18 column (i.e. packed with porous silica with a bonded layer of octadecyl hydrocarbon chains) in combination with a gradient decreasing in polarity. According to experience, nonexclusion HPLC of synthetic polymers usually requires gradient elution.8 Water is a very popular eluent component in low-molecular chromatography. Unfortu­ nately, it is a strong precipitant for most synthetic polymers. Therefore we shall concentrate on the discussion of gradients from nonaqueous solvents. A normal-phase gradient can be formed, e.g. from CC14 through the addition of THF or acetonitrile, or a RP gradient from MeOH through the addition of THF or dichloromethane (DCM). In RPC, separation is primarily governed by solvophobic retention.9 With polymers, the measures taken in order to make the mobile phase an unpleasant environment for the solute can easily interfere with the boundaries of the narrow solubility window. 16.2.2.2

Solubility effects

The dissolution of any material requires a negative change in Gibbs free energy. The increase in entropy, AS, is rather large in the dissolution of low-molecular species but much smaller with polymers. Here, the change in enthalpy, AH, must be small if positive (or, better, large negative) to ensure solubility. In terms of Hildebrand's concept this means that a polymer will be soluble only in liquids whose solubility parameters are close to that of the polymer under consideration. With low-molecular solutes, the entropy contribution is so large that a broader diversity in the values of the solubility parameters can be tolerated. Hence, the variety of potential eluent components is much greater in low-molecular-liquid chromatography than in polymer chromatography. In the extreme case, solubility is the predominant factor in polymer chromatography. Examples used for CCF are the temperature-rising elution fractionation of branched polyalkenes10 or the high-pressure-precipitation-liquid chromatography (HPPLC) of copoly(styrene/acrylonitrile) specimens (see Sections 16.4.1.2 and 16.4.3, respectively). 1112 Both examples were separations by composition. The supplementary fractionations by molecular weight were performed by SEC. For solubility-based fractionation by composition a nonsolvent must be chosen with a strong and unambiguous dependence of the cloud point on polymer composition. Alkane hydrocarbons act in this way for copoly(styrene/acrylonitrile)13 and related copolymers with units of sufficiently differing polarity. 14 A precipitant for separation by molecular weight should yield cloud points independent of copolymer composition in an adequate range around the mean value. It was, for instance, possible to fractionate copoly(styrene/acrylonitrile) samples with about 17-34 mass-% acrylonitrile according to molecular weight through methanol as a precipitant. 13

316

Separation Methods

From experience gathered so far it can be inferred that the combination of a solvent with a nonsolvent, of suitable chromatographic strength, seems to be a promising general concept for designing the eluent system in nonexclusion polymer chromatography.

16.2.2.3

Molecular weight effects in adsorption chromatography

The adsorption of polymers on a solid surface reaches a plateau value at a rather low con­ centration of the solute. On alumina, silica or charcoal, the molecular weight dependence of ma, the amount adsorbed per gram adsorbent, is approximately given by equation (5). ma = constant Me

(5)

At very high values of M the exponent e approaches zero. High values (e « 0.3) have been found for M < 100000 and thermodynamically poor solvents. In good solvents, the influence of M on adsorption is less pronounced. 15 A similar effect of solvent quality has been observed in thin-layer chromatography (TLC) investigations. Separations by composition were achieved with good solvents of a suitable polarity whereas separations by molecular weight were reached preferably with thermodynamically poor eluents. 16 Thus it does not seem impossible to perform both tasks of chromatographic cross-fractionation by the combination of two suitable techniques of AC, especially if solubility effects are utilized.

16.2.3 Prefractionation by Adsorption Chromatography and Subsequent Investigation of the Fractions by Size-exclusion Chromatography At the present state, SEC is certainly the most versatile method for separation by molecular size. Therefore promising CCF procedures are most likely to include SEC. It has been suggested that SEC should be used in the second stage after previous separation by composition in order to avoid the disturbance of SEC by chemical heterogeneity via changes in a (cf. equation 3') and also any possible distortion of the shape of the SEC peak. Using thin-layer chromatography (TLC) for the first separation and SEC for the second one, partially branched polystyrene samples17 and m'Woc/c-copoly(methyl methacrylate/styrene/methyl methacrylate)18 have been investigated. By the same sequence of methods, but with the AC stage performed in a column with dried silica, a di7?/oc/c-copoly(styrene/methyl methacrylate) sample has been analyzed. 19 ' 20 The definite advantage of using SEC at the last stage is the fact that the elution in SEC is always isocratic. Thus, additional dual detection is possible which provides information on whether the preceeding separation by composition has really met its goal. In the most favourable case this sequence of methods, (i) AC, (ii) dual detection SEC, enables quantitative analyses to be performed even without the need for a set of calibrating copolymers. SEC with Fourier transform IR detection has been successfully employed for the investigation of copoly(styrene/acrylic acid) specimens which had been prefractionated by gradient HPLC on a normal-phase AC column (see Section 16.4.9).21

16.2.4

Prefractionation by Size-exclusion Chromatography and Subsequent Investigation of the Fractions by Adsorption Chromatography

The advantages of using SEC for the first separation are: (i) In SEC the distribution constants for all sample components are restricted to the range 0-1. Thus, the chromatograms are relatively short, and even the last eluting portions will not be diluted too much for direct injection during further chromatographic investigations, (ii) SEC is performed isocratically. Hence, all fractions are obtained in the same solvent. The SEC eluent can be a main component of the eluting system in the second separation, (hi) Copolymers, with their tremendous number of constituents differing in molecular weight and composition, are extremely complex mixtures with respect to chromatography. The mutual interference of different species is less dramatic in SEC than in separation mechanisms where the sample constituents compete for active sites on the surface of the column packing. The drawback of SEC of the complex initial sample is that a fraction of a given hydrodynamic volume may consist of constituents differing in composition and consequently also in molecular

Chromatographic Cross-fractionation

317

weight, as seen in equation (3'). The importance of this effect can be judged by the Kv and a values for the polymers under investigation. From the Kuhn-Mark-Houwink constants given in the literature 22 for THF as a solvent, it can be concluded that in favourable cases the composition effect are only small. This holds true for copolymers of styrene and acrylonitrile (<24 mass-% AN) or methyl methacrylate. (Sometimes, the data for a given system from different sources differ by almost the same degree.) Thus, prefractionation by SEC is a tolerable compromise between the practi­ cability of the experimental procedure and precision. The subsequent analysis will reveal the chemical composition distribution in each SEC slice. This can be done by utilizing solubility or adsorption effects and employing TLC or HPLC equipment. (The possibility of performing another SEC in a poor solvent was already dealt with in Section 16.2.1). The prime costs are much lower for TLC than for HPLC, but the latter requires less labour and can be automated. In addition, quantitative evaluation is more difficult with TLC than with HPLC. The combination of SEC and TLC has been used for CCF of stat-copoly(styrene/methyl acrylate)23 and sta£-copoly(styrene/ethyl methacrylate) specimens.24

16.3 16.3.1

OUTLINE OF CHROMATOGRAPHIC CROSS-FRACTIONATION BY HPLC INVESTIGATION OF SIZE-EXCLUSION CHROMATOGRAPHY FRACTIONS Detection

The detection of the polymer after the second separation is the central question of how to design a CCF procedure. The detection must be sensitive because the two-fold chromatography causes a high dilution, and it must be capable of monitoring the polymer in a multicomponent solvent whose composition varies according to the gradient programme. UV detection is suitable with respect to sensitivity, but what is required are absorbing structural units in the polymers, and eluents which are transparent at the selected wavelength. Copolymers with styrene units can be monitored by UV around 254 nm in eluents composed of alkane hydrocarbons and DCM or THF. Examples are given in Sections 16.4.3, 16.4.4, 16.4.5, 16.4.7 and 16.4.8. For copoly(styrene/acrylonitrile) in 2,2,4trimethylpentane/THF, the highest sensitivity was observed at 259 nm. The drawback of UV detection is its restriction to transparent solvents and solutes carrying chromophoric groups. The ideal detection in polymer HPLC should be capable of monitoring any polymer in any solvent without disturbance by the optical properties of the latter and the gradient programme. Evaporating the solvent and measuring the nonvolatile polymer residue is a straight­ forward approach. It had been put into practice with the moving-wire detector 2 5 - 2 7 and is again utilized in the evaporative light-scattering detector 2 8 - 3 0 and the rotating disc LC/FID detector. 3 1 - 3 3 These instruments have been used successfully for monitoring polymers in gradient elution. 34 ' 35

16.3.2

The Eluent for Size-exclusion Chromatography Separation

The direct injection of SEC fractions into the HPLC apparatus requires a sample volume of about 100 jA, i.e. the HPLC column will be overloaded with respect to the SEC eluent. The first consequence is that the SEC eluent must not have a strong UV absorption if UV detection is used for the HPLC analyses, otherwise, a great part of the HPLC chromatogram may be hidden under the absorption caused by the SEC eluent. The second conclusion is that the disturbance of the chromatographic process in the second column can be minimized if the SEC eluent is, at the same time, a main component of the HPLC eluent system. THF is a rather common eluent in SEC and fortunately well suited for a direct combination with gradient HPLC. The THF for SEC can even be used with an antioxidant (mostly butylated hydroxytoluene) because this additive is easily separated in the HPLC from the polymer species of interest.

16.3.3

On-line or Off-line Operation

If the two devices are on-line the flow in the first must be stopped during the investigation of every eluate slice by HPLC. The latter takes about half an hour per sample. In practice, about ten SEC

318

Separation Methods

fractions should be analyzed. Thus, on-line coupling implies in total a rather long standby time of the SEC. The apparatus can be used more efficiently in off-line configuration where the fractions of interest are collected from an uninterrupted SEC elution. Such a configuration would enable an SEC apparatus to serve several HPLC machines or to perform independent SEC analyses. Off-line operation with the help of an autoinjection system and an autosampler is just as convenient as on-line operation, but it has the consequence that certain SEC fractions are, at any rate, investigated some hours after being delivered. In this respect, the stabilizer in the SEC eluent has additional importance in preventing the dissolved fractions from being spoiled by reactions with oxygen.

16.3.4 The Eluent for HPLC Separation With UV detection, the HPLC eluents must be transparent. This condition is especially stringent in gradient elution because the changing concentration of an absorbing solvent would lead to a dramatic shift of the baseline in the course of the programme. In gradient HPLC with UV detection around 254 nm, the common antioxidants for THF may cause trouble due to their absorption in this region. Nonstabilized THF is required which, however, readily reacts with oxygen forming hazardous peroxides. Therefore, THF for HPLC must be handled with care. The atmosphere in vessels to be filled or the vapour space above the liquid should always be oxygen-free. Purging the THF container in the HPLC apparatus continuously with N 2 or He is indispensible for long-time use of the THF because even the slightest contact with oxygen can yield UV-absorbing compounds or complexes. In line with general recommendations for HPLC, a cover of inert gas should also be applied to the complementary components in the gradient because dissolved oxygen from these eluents can cause an extra UV absorption of THF in the HPLC apparatus. This has been observed with the combination of THF and isooctane.12 Apart from these difficulties, gradients based on THF combined with SEC using THF eluent offer the advantage that the solvent of the SEC fractions matches with a main constituent of the gradient HPLC. The starting eluent must be miscible with the sample solvent and should be a poor solvent of low elution strength. If the starting solvent is too strong the polymer will not be retained properly (see Section 16.3.6). In this case, the solvent power of the starting eluent must be diminished either by increasing the nonsolvent concentration, by choosing a different solvent/nonsolvent combination, or by increasing the difference in polarity between sample and eluent. In normal-phase chromatography, a column of higher activity will also help to gain better retention.

16.3.5

Column Dimensions and Activity

The investigation of SEC fractions without pretreatment has the advantage of saving labour and enabling straightforward automation. The price to pay is the rather large injection volume in gradient HPLC. This volume must be small in comparison with the pore volume of the column. According to experience gathered so far, the injection volume should not exceed 5% of the available column volume even if the polymer sample is easily retained, as is e.g. stat-copoly(styrene/ acrylonitrile). Sfa£-copoly(styrene/ethyl methacrylate) specimens are less easily retained; here the injection volume is better restricted to 2% of the available volume. If the volume ratio is small and the starting eluent poor enough, the polymer injected will be properly retained, and will elute after the required increase in eluent strength. Another essential factor influencing retention is the activity of the column. In order to restore the initial value a thorough flushing is necessary after each run. This general rule is of special importance in polymer chromatography where traces of the sample may not be completely eluted from the column during the gradient programme. These would lead to drifting retention data, memory effects (i.e. elution in subsequent gradient cycles) and reduced column life. In the literature there are numerous reports of such phenomena. 3 6 - 3 8 It is good practice to use the return gradient for restoring the activity of the column. This requires the use of liquids which are on the one hand good solvents for the polymers investigated and on the other hand chromatographically powerful eluents. THF is quite suitable. Rinsing the column with its ten-fold volume of pure THF has proved successful.39 Good results have been obtained with cleaning cycles from 100% THF to 100% methanol and back again to 100% THF before returning to the initial eluent composition.

Chromatographic Cross-fractionation

319

The time required for flushing the column can be reduced by increasing the flow rate. Favourable results have been obtained with 2 ml min~ * in the rinsing period and 0.5 ml min" * analytical flow rate on 60 x 4 mm columns packed with 5 jtxm particles.

16.3.6

Calibration

Quantitative evaluation of chromatograms requires proper retention and complete elution of the sample. If the starting eluent is too strong, the injection volume too large for a given column, or the column not active enough, separating the sample polymer from the injected solvent will become difficult. Some of the polymer may elute together with the injected solvent. With optical detection, this portion is difficult to quantify and may even elute unnoticed. Unretained portions are more easily detected if they elute outside the solvent plug. The 'normal' elution of unretained polymer occurs in the interstitial volume of the column. On small-pore packings, a peak appearing at approximately half the elution volume of sample solvent is a distinct signal of insufficient retention. Elution in this position was sometimes observed with staf-copoly (styrene/acrylonitrile) samples. Unretained portions of copolymers with methacrylate units tended to elute in a peak which followed the solvent peak. Occasionally two peaks appeared: i.e. elution of unretained polymer partly in the interstitial volume and partly immediately after the solvent. From a quantitative point of view, incomplete retention is as bad as incomplete elution during the gradient. On columns with small-pore packings, this is likely to occur with polymers of rather lowmolecular weight. Repeated injections of a given sample with comparison of peak areas obtained in such a series may help to detect incompleteness of elution: increasing areas approaching a limit indicate saturation of the column. Another test involves changing the column activity. This can be done with columns of identical geometry but chemically different packings. In normal-phase chromatography the exchange of a silica column, e.g. for a CN column, will cause larger peaks if elution from the silica column is incomplete. Another and even simpler means is reducing column activity by the addition of a chromatographically strong component to the eluent. This can be done as long as retention is complete (vide supra). If such an addition, e.g. of methanol to a hydrocarbon eluent, causes increasing peak areas the assumption of incomplete elution is verified. With optical detectors, correction must be made for solvatochromic effects. In the worst case, the system to be investigated and the limitations of chromatographic possi­ bilities will not permit complete retention and complete elution. Then one has to make sure carefully that incompleteness acts upon all sample constituents to the same degree.71 Only if this is proved may one dare to judge the composition of the whole sample on the basis of results obtained from the properly eluted portion. The following passages deal with the relations needed for quantitative evaluation of proper chromatograms.

16.3.6.1

Dependence of size-exclusion chromatography elution volume on molecular weight and composition

The composition effect can be judged by the values of the Kuhn-Mark-Houwink constants for suitably graded copolymers and the parent homopolymers of the system under investigation. (The latter requirement cannot be met in every case because of insolubility or other basic obstacles.)

16.3.6.2

Dependence of the detector signal on polymer amount and composition

This information is required for all detectors employed.40 The detector response is rather straightforward for the refractive index (RI) detector which is commonly used as a nonspecific mass detector in SEC. The influence of polymer composition on the RI signal is smallest if the refractive indices of the structural units are close together but sufficiently apart from that of the solvent. The composition dependence of the UV signal is much more complicated because of: (a) the difference in absorptivity for the different constituting units, (b) the effect of solvent composition in gradient elution, and (c) the hypochromic effect of adjacent groups. The latter has been found

Separation Methods

320

to be significant with stat-copoly(styrene/acrylonitrile)41'42 methacrylate). 43 ' 44

16.3.6.3

and

stat-copoly(styrene/methyl

Dependence of the retention in nonexclusion HPLC on molecular weight and composition

The retention in gradient HPLC has been repeatedly claimed to be independent of M. In gradient HPLC with sol vent/nonsol vent systems, a molecular-weight effect was always observed. Figures t 6, 7, 11 and 16-18 show decreasing retention with increasing SEC fraction number. Of course, this could be due to a composition shift in the model copolymers but it is improbable that this shift should always be an increase in styrene content with decreasing molecular weight in more than 20 specimens which represent four different copolymer systems and which have been polymerized in three different laboratories — a decrease in styrene irrespective of whether the sample composition is above the azeotropic composition, below or just equal to it. Thus, the faster HPLC elution of the later SEC fractions indicates decreasing retention with decreasing molecular weight. From the retention time, the gradient programme and the gradient delay the eluent composition at peak position can be calculated. For the systems investigated so far, equation (6) gives the volume fraction of the nonsolvent, (/>*, in the eluent portion which delivers a copolymer with molecular weight M . 4 5 - 5 0 Equation (6) had been found previously, relating turbidimetrically measured 0* data and molecular weight.51 0* = A + B/VM (6) The M dependence is small in comparison with the composition dependence. For copoly(styrene/ acrylonitrile), a 1 % change in composition has been found to cause a greater effect on retention than a 20% change in M. 45 The calibration for both M and composition can be performed most efficiently by CCF of model mixtures. Figures 7, 11 and 16-18 contain the respective information for the systems copoly(styrene/acrylonitrile), copoly(styrene/methyl methacrylate), copoly(styrene/ethyl methacrylate) or copoly(styrene/methoxyethyl methacrylate). For each system, the basic data had been collected within one day. Repeated checking of the composition calibration by injecting a suitable mixture of model copolymers is recommended because changes in eluent quality will have a pronounced influence on copolymer retention in gradient HPLC. If stock solutions are used one should be aware of incompatibility effects even between specimens of the same system but different composition. Checking for possible precipitation is recommended.

16.3.7

Evaluation of Experimental CCF Results of Binary Copolymers

This section deals with experience in the investigation of copolymers containing one kind of UV-absorbing unit by SEC and gradient HPLC with UV detection. The immediate results of the experiments are (i) the SEC curve with the indication of the cuts between subsequent fractions and (ii) the UV records from the gradient HPLC.

16.3.7.1

Determination of molecular weight and polymer amount in each size-exclusion chromatography fraction

Provided there is knowledge of both the actual SEC calibration and the detector sensitivity, the M value and the portion of each SEC fraction can be estimated from the area and shape of each slice and its elution volume. Summation of these data should yield the average M of the whole sample.

16.3.7.2

Correction of the M effect in HPLC

Investigation of sta£-copoly(styrene/acrylonitrile) by SEC and gradient HPLC in isooctane/THF yielded the actual values of the constants in equation (6). From the results shown in Figure 7 we t Figures can be found in Section 16.4

Chromatographic Cross-fractwnation

321

obtained for the slope factor B — 17.16 + 0.204w. The quantity w indicates the AN content (mass-%) of the specimen. For the sample in Figure 9 the composition effect on B is within the limits B = 21.9 (for 23% AN) and 23.5 (for 3 1 % AN), with 5 = 22.55 for the main portion (26.4% AN). This last value was used in a first approximation of the M correction. We referred all data to M = 140000 which was near to the number average of that sample, i.e. we calculated (/>* f from the individual (pfj obtained with SEC fractions with a molecular weight M: *, w = *f w + 22.55(140000"° 5 M" 0 - 5 ). In a binary gradient with the solvent component B, *f can be found from B% by *f = 1 -(B%)/100. In the S/AN system mentioned we had(B%) ref = 39.015 + 1.178w, i.e. the higher the AN content was, the more THF was required for eluting that sample portion. From this relation, the composition w was obtained through w= — 33.124 + 0.849(B%) ref .The correlation is indicated by r2 = 0.9869. (Unfortunately, in ref. 49, equation (2), the sign was not properly given).

16.3.7.3

Calculation of copolymer composition at any measured HPLC retention time

From the calibration with model mixtures (see Section 16.3.6.3) the composition dependence of the HPLC retention time can be found for M ref by interpolation. With the help of this relation, the composition w can be easily derived from the 0* f data of each point of the HPLC elution curve.

16.3.7.4

Correction of the UV signal

The height of the UV signal in a HPLC chromatogram, /zw, is determined by the amount of copolymer present and its content in the UV absorbing unit (e.g. styrene). Knowing the latter from the previous evaluation and the detector calibration, it is possible to correct the signal /iw which gives the value h representing the actual amount of copolymer present at this point.

16.3.7.5

Evaluation of the two-dimensional distribution of the sample

If there is a strong effect of composition on hydrodynamic volume or on the quantity B in equation (6) the steps 1 to 4 of the previous evaluation must be repeated on the basis of the knowledge gained so far. Finally, the corrected h data of each HPLC are added up, and also the sums of all fractions. The portion of the starting material contained in a given SEC fraction must be reflected by the ratio of the h sum for that fraction over the total sum. This proves that exact aliquots had been injected into the HPLC apparatus and no concentration change (e.g. by solvent evaporation) had occurred. In the case of deviations, the h values must be corrected according to the ratio of the SEC slices to the total area of the SEC curve. The final h data represent the amount of copolymer having the composition w and the molecular weight M. In order to obtain the contour map of the sample, corresponding values of h must be entered and connected in a plot of composition vs. M.

16.4

CROSS-FRACTIONATION OF SYNTHETIC POLYMERS

16.4.1

Partially Branched Polymers

Although branching often occurs as a consequence of chain transfer reactions the final products can be considered as pseudocopolymers of bi- and multi-functional monomers. Linear low-density polyethylenes (LLDPE), also dealt with in this chapter, are actually real copolymers from ethylene and a-alkenes such as butene-1, hexene-1 or octene-1, where the incorporation of the a-alkenes causes short-chain branching.

16.4.1.1

Branched polystyrenes

The investigation of partially branched polystyrene was performed by SEC and TLC. 17 A sample of 2 g was fractionated by SEC on a set of columns packed with polystyrene gels. The 12 fractions

322

Separation Methods

obtained were subsequently investigated by TLC on silica with a mixed eluent containing cyclohexane, benzene and acetone (12:4:i;). The amount of acetone was not specified but from the report on calibrating experiments it can be concluded that v was in the range 0.6-0.8. These eluent mixtures separated the fast running linear polystyrene from the branched portions which were more strongly retained. Figure 1 shows the densitograms of TLC traces from the starting sample and its SEC fractions. Five species can be distinguished which differ in degree of branching. This was concluded from the hydrodynamic radii derived from the SEC results (see Figure 2) and the effect of the acetone content

* f

Figure 1 TLC of a partially branched polystyrene and its GPC fractions 3-12: (a) densitogram of the thin-layer chromatogram obtained from the nonfractionated sample with indication of five constituents, cf. Table 1, (3-12) densitograms of the fractions, M decreasing with increasing number. (Reproduced by permission of Elsevier Scientific Publishing Company, Amsterdam, from J. Chromatogr., 1977, 141, 72)

Figure 2 Distribution of hydrodynamic radii Rs of the constituents of a partially branched PS sample, cf. Figure 1 (dashed curve: nonfractionated sample). (Reproduced by permission of Elsevier Scientific Publishing Company, Amsterdam, from J. Chromatogr. 1977, 141, 74)

Chromatographic Cross-fractionation

323

Table 1 Constituents of the Polystyrene Sample shown in Figures 1 and 2 Component total 1 2 3 4 5

10500 10500 19500 10500 5500

Molecular weight* backbone

Relative amount3 (%) branches

5000 5000 (19500) 10500 5500

1400 1900

23 15 16 42 4

B. G. Belenkij and E. S. Gankina, J. Chromatogr., 1977, 141, 13.

in the TLC eluent mixture on the R{ values of these five components. The curves obtained were compared with those of linear polystyrene standards graded in molecular weight. Results are listed in Table 1.

16.4.1.2

Branched polyethylenes

The crystallizability of polyethylene is determined by branching. Temperature-raising elution fractionation (TREF) 10 is a suitable technique for separating polyethylene according to the degree of short-chain branching. In analytical TREF, 50 /ig sample material was deposited onto the inert packing material in a thermostated column by slow cooling (1.5 K h" 1 ) of a 0.5% solution in xylene. Subsequently, the polymer was eluted with 1,2,4-trichlorobenzene while the column tempera­ ture was steadily increased. The method was calibrated with the help of standards whose -Me content had been measured through IR spectroscopy. Thus, the amount of-Me per 1000 C atoms could be estimated from the elution temperature. This procedure was scaled up by using 4 g sample material and a 500 x 127 mm TREF column. Thus it was possible to perform CCF by further investigation of the fractions through SEC. 52 The SEC was carried out by using a column set which included a pair of bimodal columns, 10 nm plus 100 nm, and a 400 nm column. The apparatus was equipped with an IR detector and run at a flow rate of 0.7 ml m i n - 1 . The CCF method was applied to low-density polyethylene (LDPE) and LLDPE whose short-chain branching is mainly due to inserted a-alkenic comonomers. Figure 3

Figure 3 Distribution in short-chain branching and molecular weight of (a) linear low-density polyethylene and (b) PE radically polymerized under high pressure (LDPE). (Reproduced by permission of the American Chemical Society from Polym. Prepr., Am. Chem. Soc, Div. Polym. Chem., 1982, 23, 134)

Separation Methods

324

shows the frequency distribution for a sample of both LLDPE and LDPE as the function of -Me content and molecular weight. Nakano and Goto also recognized the potential of TREF for CCF of crystallizable polymers and designed an automated apparatus for the investigation of branched polyalkenes.53 Stepwise they increased the temperature of the column whose glass-bead packing had been coated with the sample under investigation, and directed the eluate of this column on-line to the SEC apparatus. For this reason the column for the fractionation by crystallizability was only 150 x 8 mm in size. The void volume of this column was 2.5 ml, which also was the sample volume for the subsequent SEC performed with o-dichlorobenzene (o-DCB) at 140 °C and a column packed with mixed polystyrene gels. The temperature steps for the TREF column were 2 K in the middle of the operating range (40-140 °C) and larger (10 K) at its edges. The last step was even greater (from 110 to 140 °C). Figures 4 and 5 show computer-aided drawings of the contour map and the perspective representation of the results from a 1:1 mixture of linear or highly branched polyethylene with 2.0 or 29.5 -Me groups per 1000 C atoms.

16.4.2

Copolymers from Ethylene and Propylene

The separation by composition was first performed in a glass column (1000 x 80 mm) packed with Celite 545 on which the copolymer sample had been deposited from solution. Fractionation was carried out at (or slightly above) the melting point of the sample under investigation, i.e. at a

5

Log M

Figure 4 Contour map of the mixture of two polyethylene samples. The sample eluted at about 100 °C contained 2 -Me groups per 1000 C atoms, the other one had 29.5 -Me per 1000 C atoms. (Reproduced by permission of John Wiley & Sons, Inc., New York, from J. Appl. Polym. Sci., 1981, 26, 4230)

Log M* Figure 5

Perspective picture of the two-component mixture: see Figure 4

Chromatographic Cross-fractionation

325

temperature in the range between between 117 and 123 °C. An exponential gradient was im­ plemented using butyl cellosolve and xylene. Since only the latter is a solvent for the specimens investigated, the gradient was one of thermodynamic quality. SEC was performed in o-DCB at 135 °C on a set of four columns packed with PS gels. From the estimated MMD of each fraction and its ethylene content a diagram was constructed which showed the range of chemical composition and molar mass of the starting sample. It indicated the outline of the distribution in molar mass and composition but did not contain the contour lines necessary for full information on MMD and CCD. 5 4

16.4.3 Statistical Copolymers from Styrene and Acrylonitrile Azeotropic copolymers of this system (sta£-copoly(S/AN); rx = 0.41; r 2 = 0.04) contain 24 mass-% AN or 61.9mol-% S. Products of about this composition are commercially available. Samples with an AN content below 50 mass-% are soluble in common organic solvents whereas copolymers richer in AN require DMF or DMSO which are solvents even for polyacrylonitrile (PAN). Ogawa and Sakai reported on the investigation of two model copolymers with 27.8 or 51.3 mass-% AN. 55 About 5-6 g sample material had been loaded from a solution onto a Celite support just before the latter was packed into the column (700 x 27 mm). The first step was separation by composition through elution with a linear gradient (toluene:propanol 1:1)/DMF. The n-propanol was added in order to reduce the precipitating power of the nonsolvent toluene. The eluate was collected in 250 ml portions from which the polymer was isolated by precipitation. The AN content of the fractions was estimated from nitrogen analyses. Fractions of nearly equal composition were used for cloud-point measurements in DMF/ (n-propanol: toluene). The data were plotted vs. molecular weight, using the AN content as a parameter. The curves showed that the influence of composition exceeded that of M. Thus, the first fractionation was mainly a separation by composition. The size distribution of each fraction was measured by SEC. The results from both sets of experiments were used for the construction of contour maps. The corresponding diagram of the sample with 51.3% AN showed a broad chemical composition distribution with two maxima at about 36 and 68 mass-% AN. The composition distribution of the sample with 27.8% AN was in the limits of 24.5 and 29.6 mass-% with two maxima at about 26 and 29%. A slight increase in AN content with increasing M was observed.55 Efficient CCF of copoly(S/AN) has been performed by SEC and HPPLC with 2,2,4trimethylpentane (isooctane)/THF. 4 5 ' 4 7 , 4 9 ' 5 0 , 5 6 The isooctane precipitates according to AN con­ tent. THF is a solvent. In most experiments it contained about 10 vol-% methanol. This admixture brought the refractive index close to that of isooctane. A mixture of two S/AN copolymers (I with 16.1 and II with 30 mass-% AN) was fractionated by SEC with THF as an eluent on a set of five ^-Styragel columns at a flow rate of 1 ml rnin"1. After the injection of 0.87 mg sample mixture with 59.3% S/AN I, eluate fractions of 0.5 ml each were collected. These SEC slices were investigated by HPPLC on a C18 column (150x4.6 mm; dv= 10 //m) without any additional treatment. The multilinear gradient programme started with 10% B (THF + 10% methanol). Within three minutes the B concentration was raised to 60%. The separation was performed by slowly increasing the concentration of B from 60 to 90% within 12 minutes. Figure 6 shows the result of this experiment, which was the first published example of CCF using HPLC equipment for the separation both by size and composition.56 Then a mixture of five S/AN copolymers was separated by SEC and HPPLC of the eluate slices. Table 2 gives a compilation of the sample components and their characteristics. A CN bonded-phase column (150x4.6 mm; dp = 5 fim) allowed almost baseline separation of the copolymers (see Figure 7). The column was operated at 50 °C and the flow rate 1 mlmin" 1 . The gradient was isooctane/THF (50-100% B in lOmin). The CCF required in total about seven hours experimental work. It was performed on 1.04 mg starting material. 49 Columns packed with RP C18 or bare silica of either small or large pores yielded about the same separating power for S/AN. 11 ' 57 Commercial S/AN copolymers have been cross-fractionated by SEC and HPPLC on a reversedphase C18 column (150 x 4.6 mm; dp = 5 jum; dQ = 6 nm) 47 and also on the CN column mentioned above. 49 The average AN content of each SEC fraction was calculated from the first moments of the CCD curves measured by HPPLC. For comparison, the average composition of each SEC fraction was independently measured by pyrolysis gas chromatography (PGC) and by dual detection SEC.

UV , R I

35

40

209

45.6 x I0 3

t tmin)

Figure 6 Chromatographic cross-fractionation by HPPLC of SEC fractions. Analysis of the mixture (59.3:40.7) of two statcopoly(styrene/acrylonitrile) samples/The first HPPLC peak is due to the copolymer with 16.1 mass-% AN, M n 325000, the second to that with 30 mass-% AN, Mn 71000. (Reproduced by permission of Huthig & Wepf Verlag from Makromol. Chem., Rapid Commun., 1983, 4, 529)

t (min)

Figure 7 Chromatographic cross-fractionation of stat-copoly(S/AN) by HPPLC of SEC fractions. The five model copolymers {cf. Table 2) are eluted in a sequence I-V. The molecular weight of the SEC fractions (M x 10" 3 ) is indicated at the HPPLC traces. (Reproduced by permission of Elsevier Applied Science Publishers, from 'Integration of Fundamental Polymer Science and Technology', ed. L. A. Kleintjens and P. J. Lemstra, 1986, p. 91)

Chromatographic Cross-fractionation

327

Table 2 Characteristics of the sra£-Copoly(styrene/acrylonitrile) Specimens used for the CCF Separation shown in Figure 7 I 16.1 72.6 325a 11.5

Sample No. Acrylonitrile (mass-%) Styrene (mol-%) Molecular weight x 10" 3 Relative amount in the mixture injected (%) a

III 29.1 55.4 510a 17.7

II 23.0 63.0 480a 22.5

V 42.7 40.6 340a 22.2

IV 36.4 47.1 380a 26.1

Measured by osmosis.

40 r—

g 30 <#

20

4.5

*■„

5.5

5.0 Log M

6.5

Figure 8 Average AN content of SEC fractions: comparison of CCF results with dual detection SEC (■), pyrolysis gas chromatography (O), CCF on a CN column (cf. Figure 9) (x) and CCF on a C18 column (•). (Reproduced by permission of Elsevier Applied Science Publishers, from 'Integration of Fundamental Polymer Science and Technology', ed. L. A. Kleintjens and P. J. Lemstra, 1986, p. 94) Fraction no. 6

32

5

•

>.

30

28

2 26 J

24

•

22

4.5

5.0

■

5.5

6.0

Log M

Figure 9 Contour map of a commercial staf-copoly(S/AN) sample as obtained through CCF by HPPLC of 10 SEC fractions. HPPLC conditions as in the calibrating experiments shown in Figure 7. (Reproduced by permission of Elsevier Applied Science Publishers, from 'Integration of Fundamental Polymer Science and Technology', ed. L. A. Kleintjens and P. J. Lemstra, 1986, p. 92)

Separation Methods

328

Figure 8 shows results from this investigation, demonstrating reasonable agreement in the highmolecular region. Whereas dual detection SEC and PGC can only reveal the average composition of an SEC fraction, HPPLC has the additional capacity of evaluating the CCD in each fraction. Figure 9 shows the contour map obtained as the result of CCF of a commercial S/AN copolymer sample.

16.4.4

Copolymers from Styrene and Methyl Methacrylate

16.4.4.1

Block copolymers

Belenkij et al. reported in 1975 on the investigation of a m'Woc/c-copoly(MMA/S/MMA) by preparative SEC of 40 g sample material and subsequent TLC of the fractions using chloroform: methanol mixtures. It was possible to separate poly(methyl methacrylate) homopolymer (PMMA) from block copolymers of the same molecular weight which remained at the start (R{ = 0) whereas the PMMA reached R{ values of about 0.5-0.7. The ratio of PMMA to block copolymer in a SEC fraction was estimated from data obtained by pyrolysis gas chromatography of the acetone extracts from the TLC zones of both components. 18 The complete CCF of a dtf?/oc/e-copoly(S/MMA) sample (47:53 mass-%, 739000 M n ) was achieved by AC and SEC. 19 The AC yielded separation by composition and was performed in a column (150 x 50 mm) packed with activated dry silica. The chromatographic bed was subdivided by filter paper into nine compartments. The sample (300 mg) was loaded on a pile of filter paper (about 30 sheets) in the lower part of the column shown in Figure 10. The development was performed by soaking an ethyl acetate:benzene mixture (72.5:27.5 vol-%) into the vertically orientated column. It took about five hours until the eluent front reached the last but one compartment at the top of the column. Then the column was immediately dismantled and the polymer extracted from each compartment by THF. The same was done with the paper block where

Cylindrical column

Adsorbent (silica gel)

Spacer (filter paper)"

Magnetic stirrer

Figure 10

Sketch of the adsorption column used for CCF of d/fr/oc/c-copoly(styrene/methyl methacrylate). (Reproduced by permission of Marcel Dekker Inc., from J. Macromol ScL, Phys., 1980, B17, 218)

Chromatographic Cross-fractionation

329

the immobile remainder of the sample was found. As a whole, about 93% of the initial material was recovered. The MM A content in the fractions ranged from 61.1 mass-% (in the lowest compartment, F8) to 15.1% in F2 at the top of the column. The rest of the sample block contained 79.0% methyl methacrylate. In 1982, Inagaki et al.20 showed that the position of the SEC curves of all these fractions was almost the same (see Figure 11). This explained why the SEC investigation of the unfractionated sample by dual detection technique could reveal scarcely any chemical heterogeneity.19 Every siphon volume contained all sample constituents in a roughly constant ratio. The authors stated that 'no variation of the SEC point-by-point composition does not necessarily guarantee uniformity in composition for a sample copolymer'.20 This careful experimental work is an excellent demonstration for the necessity of crossfractionation.

26 28 l/e (ml)

I

2

1 I

I

30

I

I

I

I065 2 I0 5 5 2xl0 4 Molecular weight (PMMA)

Figure 11 Chromatographic cross-fractionation of
16.4.4.2

Statistical copolymers

Azeotropic copolymers of this system (sta£-copoly(S/MMA); rt = 0.53, r2 = 0.49) contain 47 mass-% MMA or 52.0mol-% S. The copolymers are soluble in most organic solvents. Alkane hydrocarbons and lower alcohols act as precipitants. Separation by composition was achieved by gradient HPLC with dichloroethane/THF (expo­ nential from 3 to 20%) on a silica column (250 x 6 mm, d0 = 6nm; dp = 9/im) 58 as well as with isooctane/(THF+ 10% MeOH) on a silica column (150 x 4.6 mm; dQ = 6nm; dp = 5/rni)/ CCF has been performed by SEC and gradient HPLC. A sample of about 1 mg was fractionated first by hydrodynamic volume through SEC. The column was a set of two Toyo Soda Mixed Gel Columns GMH6 (2 x 600 x 7.8 mm). The injection volume was VQ = 0.2 ml, the sample solvent and the SEC eluent THF at a flow rate of 1 m l m m - 1 . The sample was the mixture of seven model copolymers. Their characteristics and the sample composition are given in Table 3. The SEC refractive index signal is shown in the left part of Figure 12. The curve does not indicate the components present because the differences in molecular weight are too small.

Separation Methods

330

Table 3 Characteristics of the srat-Copoly(styrene/methyl methacrylate) Specimens used for the CCF Separation shown in Figure 12 Sample No. Methyl methacrylate (mass-%) Styrene (mol-%) Molecular weight x 10" 3 Relative amount in the mixture injected (%) 1

I 11.4 88.2 160a 12.7

II 23.8 75.5 250a 14.0

III 37.0 62.1 150a 12.1

IV 49.5 49.5 185a 12.7

V 64.0 35.1 235a 12.7

VI 76.2 23.1 220a 17.2

VII 88.5 11.1 220a 18.5

Measured by light scattering.

Eluate portions of 1 ml each were used for subsequent gradient HPLC on a column (60 x 4 mm; dQ = 5 nm; dp = 5 /xm) packed with silica Nucleosil 50. The elution was performed at 0.5 ml min~ 1 flow rate with a gradient isooctane/THF (10-80% B in 14 minutes). Figure 12 shows that the seven copolymers had been sufficiently separated in the main SEC fractions 3, 4 and 5. The sample appearing first is the specimen I. The peak sequence is determined by the increase in MMA content. The samples V, VI and VII have comparatively high values of M and, in accordance with this, are scarcely detectable in the low-molecular SEC fractions 6 and 7. I E I I II I

ll

I

l/e

Figure 12 Chromatographic cross-fractionation of staf-copoly(S/MMA) by gradient HPLC of SEC fractions having M x 10" 3 values 300 (fr. 2), 190 (3), 125 (4), 80 (5), 50 (6) and 30 (fr. 7).

CCF was also attempted by AC and subsequent SEC. 60 About 100/ig staf-copoly(S/MMA) were eluted from a silica column (50 x 4.6 mm; dQ = 3 nm; dp = 5 /mi) by stepwise increasing the chloro­ form content in the mixture with dichloroethane. The two main fractions (obtained with DCE:CHC1 3 80:20 or 70:30) from ten repeated elutions were combined and analyzed by SEC. 16.4.5

Statistical Copolymers from Styrene and Methyl Aery late

Azeotropic copolymers of this system (sta£-copoly(S/MA); r 1 =0.75; r 2 = 0.18) contain 20.1 mass-% MA or 76.6 mol-% S. A high-conversion sample containing 51.7mass- % MA units was investigated by CCF. Because of the high degree of conversion (92%) and the deviation from the azeotropic point a broad CCD towards higher MA content was expected.23 At first, SEC separation according to molecular weight was performed. The column (600 x 25 mm) was packed with TSK gel and was capable of fractionating 10 mg per injection. The separation into eleven slices was repeated a hundred times. The corresponding ones were united, yielding eleven fractions sufficiently large for further investigation.

Chromatographic Cross-fractionation

331

The CCD of the fractions was evaluated by TLC on silica through gradient elution with CCl 4 /methyl acetate (the latter linearily increasing from 6 to 43 vol-%). Standard samples of known composition were used as markers in each TLC run. They enabled the chromatographic traces to be evaluated in terms of composition. Figure 13 shows the result obtained with the help of a TLC scanner, and Figure 14 the contour map of the high-conversion sample. The authors discussed their own work very critically (Tn general, the CCD determined by the TLC method may not be sufficiently accurate') 23 but in a thorough examination the results proved sufficiently reliable. The coupling of high MA contents with high-molecular weight values is understood as the consequence of the gel effect. At a high degree of conversion, the monomer mixture is richer in MA than at the beginning. Hence, the very large macromolecules growing after the start of the Schulz-Trommsdorf effect contain distinctly more MA units. This is clearly seen in Figure 14.

MA (mol. %)

Figure 13 Chromatographic cross-fractionation of stat-copoly(S/M A) by TLC of SEC fractions. (Reproduced by permission of the American Chemical Society from Macromolecules, 1983, 16, 544)

40

60

80

100

MA (mol %)

Figure 14 Contour map of a high-conversion stat-copoly(S/MA) sample with 56.4 mol-% MA as obtained by CCF through SEC and TLC of the fractions. (Reproduced by permission of the American Chemical Society from Macromolecules, 1983,16, 544)

Separation Methods

332

16.4.6 Statistical Copolymers from Styrene and w-Butyl Methacrylate Azeotropic copolymers of this system (sta£-copoly(S/tt-BMA); rx = 0.63; r 2 = 0.64) contain 58.4 mass-% BMA or 49.3 mol-% S units. Report was given that a copolymer with 33 mol-% S obtained by free-radical polymerization could be separated from admixed PS by 'orthogonal chromatography'.7 This term refers to coupled-column GPC with different eluents in each device. The column in GPC(l) was packed with /i-Styragel whereas in GPC(2) a Bondagel column was used. The separation reported was performed with THF in GPC(l) and a n-heptane:THF mixture in GPC(2). The content of heptane proved decisive. Best results were obtained with 63.8 vol-%. This binary eluent enabled an azeotropic copoly(S/n-BMA) to be separated from both parent homopolymers. 61 ' 62 At 60% n-heptane, the copolymer peak merged with the peak of poly(n-butyl methacrylate) 61 (see Figure 15) and at 65% with the PS peak. 62 The two GPC devices were on-line coupled, but GPC(l) was stopped while GPC(2) was working. -AB-

63.8%

60.0%

57.0%

50.0%

900

700

500

Ms) Figure 15 Orthogonal chromatography of the mixture of PS (-AA-), poly(n-butyl methacrylate) (-BB-) and azeotropic copoly(S/n-BMA) (-AB-). The n-heptane content in the eluent of GPC(2) is indicated at the chromatograms. (Reproduced by permission of Marcel Dekker Inc., from Sep. Purif. Methods, 1982, 11, 17)

Most of the results and experimental details were reported in the paper from 1983 but again no example of real cross-fractionation was given. The reason might have been that 'mixed mechanisms in GPC(2) and the coupling of composition with sequence length effects cause calibration referencing retention time to be very difficult'.63 UV scanning of the peaks from GPC(2) was the main source of information concerning the composition of any constituent. The advantage of coupling two GPC devices is the possibility of compensating for an adverse influence of molecular weight by adjusting the exclusion potency of the two columns. On the other hand, the separation power for fractionation by composition is comparatively low. To enhance it, one should employ a column with an active packing and perform gradient elution (see Sections 16.2.4 and 16.3).

16.4.7

Statistical Copolymers from Styrene and Ethyl Methacrylate

Azeotropic copolymers of this system (r1 =0.49; r2 = 0.40) contain 48.2 mass-% EMA or 54.1 mol-% S units. CCF of sta£-copoly(S/EMA) samples was performed through the combination of

Chromatographic Cross-fractionation

333

SEC and TLC. The TLC was carried out using 'Chromarods'. These are quartz rods (152 x 0.9 mm) coated with a sinter-fused layer of silica. The samples were spotted near to the lower end and developed along the vertically positioned rods. This was done with a gradient toluene/acetone. After the development, the Chromarods were dried and then slowly moved through the hydrogen flame of an ionization detector. The detector signal provided information on the amount of organic material resting at any given position of the rod. This information and the data from SEC were used for computer-aided plotting of the contour maps of samples polymerized to various degrees of conversion. 24 Sta£-copoly(S/EMA) samples have also been cross-fractionated by SEC and column HPLC with a variety of gradients and stationary phases. The SEC was carried out with 1 mg sample in 200 pt\ THF. Two Toyo Soda Mixed Gel Columns GMH6 (2 x 600 x 7.8 mm) and THF eluent at a flow rate 1 ml m i n - 1 were used. Figure 16 shows an example obtained with a silica column and a gradient isooctane/THF (30-70% B in eight minutes). The injections were performed into isooctane: MeOH (99:1). Then the eluent composition was changed gradually to 69:30:1 isooctane:THF:MeOH where the gradient started. 1 % MeOH was kept constant during the whole analysis. Such a polar additive is essential for suppressing disturbances in gradient HPLC on silica. The sample was the mixture of five model copolymers whose characteristics are listed in Table 4.

Figure 16 Chromatographic cross-fractionation of sta£-copoly(S/EMA) by gradient HPLC of SEC fractions on a silica column. The SEC fractions had the following M x 1(T 3 values: 290 (fr. 1), 135 (2), 85 (3), 55 (4), 35 (5), 22 (6) and 14 (fr. 7)

Table 4

Characteristics of the sta£-Copoly(styrene/ethyl methacrylate) Specimens used for the CCF Separations shown in Figures 16 and 17

Sample No. Ethyl methacrylate (mass-%) Styrene (mol-%) Molecular weight x 10 " 3 Relative amount in the mixture injected (%)

I 4.7 95.7 29.5a 19.6

II 32.2 69.8 35.8a 19.8

III 54.6 47.7 40.8a 20.5

IV 68.0 34.0 46.0a 19.6

V 92.5 8.2 36.7a 20.5

' By SEC measurements based on a PS calibration.

Figure 17 shows the chromatograms obtained from the same mixture on a CN-bonded-phase column. The HPLC separation had been carried out on the same SEC fractions as in the investigation shown in Figure 16. The gradient and injection conditions were the same in both analyses. The detector signal at 259 nm reveals the five specimens present in the mixture, whereas the RI curve from the SEC does not give much information. The higher the EM A content of a given sample the more THF was needed for elution, both on the silica and CN column. 64

334

Separation Methods 11 ir

v.

in iff I

2

WHM te (min)

Figure 17 Chromatographic cross-fractionation of sta£-copoly(S/EMA) by gradient HPLC fractions on a CN bonded-phase column

Figure 18 Chromatographic cross-fractionation of staf-copoly(S/MEMA) by gradient HPLC of SEC fractions having M x 1(T 3 values 500 (fr. 1), 200 (2), 150 (3), 100 (4), 65 (5) and 40 (fr. 6)

16.4.8

Statistical Copolymers from Styrene and Methoxyethyl Methacrylate

Azeotropic copolymers of this system (sta£-copoly(S/MEMA); r1 =0.41, r2 = 0.48) contain 61.1 mass-% MEMA or 46.85 mol-% S units. Chromatographic cross-fractionation was performed through the combination of SEC and gradient HPLC. 65 Figure 18 shows the results obtained with the mixture of four model copolymers. The characteristics of these specimens are compiled in Table 5. The SEC was carried out in THF eluent at 1 ml min" x flow rate. A set of two Toyo Soda Mixed Gel Columns GMH6 was used (2 x 600 x 7.8 mm). The injection volume was 200 /d, the sample amount 1.12 mg. The gradient HPLC was performed on a CN column (60 x 4 mm; dp = 5 fim) at 0.5 JJ\ min~ * flow rate. The gradient was an increase of the methanol content from 2 to 52% within ten minutes. The other eluent components were isooctane and THF. The elution of the sample constituents occurred in the order of increasing MEMA content. The results correspond with expectations based on the

335

Chromatographic Cross-fractionation Table 5

Characteristics of the sraf-Copoly(styrene/methoxyethyl methacrylate) Specimens used for the CCF Separation shown in Figure 18

Sample No. Methoxyethyl methacrylate (mass-%) Styrene (mol-%) Molecular weight x 10" 3 Relative amount in the mixture injected (%)

I 25.9 79.8 96 32.9

II 49.0 59.0 137 26.7

III 62.4 45.5 173 23.0

IV 87.4 16.6 306 17.4

molecular weight of the copolymers: IV with the highest M was preferably contained in the highmolecular SEC fractions 1-3, whereas copolymer I with a low-molecular weight was mainly present in the fractions 5 and 6.

16.4.9

Copolymers from Styrene and Acrylic Acid

Azeotropic copolymers of this system (sta£-copoly(S/AA); r1 = 0.15; r 2 = 0.25) contain 44 mass-% A A or 46.9 mol-% S. Samples with A A contents graded from 30 to 70% were investigated by HPPLC and subsequent SEC. 21 The first stage was gradient elution on a set of two silica columns (each 250 x 4.6 mm; d0 = 6 or do = 30nm). The gradient was started with cyclohexane:THF:MeOH = 95:2.5:2.5 and reached THF:MeOH = 50:50 within 75 min. Cyclohexane is a nonsolvent for the copolymers under investigation. Thus, the mechanism of the separation was considered to be precipitation of the samples followed by selective redissolution. The separation by composition was strongly influenced by the molecular weight of the samples which were rather low. This is easily understood by a closer look at equation (6): the M ~0*5 term is especially effective at low values of M. The difficulties involved with the HPLC separation of nonprefractionated and thus rather complex mixtures were overcome by a strict regime of column operation, and certainly also by the flat gradient. About ten fractions were investigated by fast SEC with elution times of about three minutes. The high speed of the second separation made on-line operation possible. Thus, the acquisition of the data was accomplished within 75 minutes. Another advantage of this configuration was that the final isocratic separation enabled UV and FTIR detection. Thus, additional information was obtained concerning the actual composition of the fractions investigated. The system also saved any trouble with aging solutions or handling a lot of sample vials. In many respects it seems to be very well suited for automated CCF in routine process control.

16.5

CONCLUDING REMARKS

Chromatographic cross-fractionation offers at least two advantages. One of them is the acceler­ ation of data acquisition. With classic fractionation techniques, the investigation of a sample requires about two to three months of skillful labour. CCF is capable of subfractionating 10-12 fractions in about six hours. Since these fractions can be obtained from another apparatus in even shorter time, about four samples per day can be investigated with an autoinjection device and a continuously running apparatus. The equipment mentioned in Section 16.4.9 is even more efficient: with this configuration almost 20 samples could be cross-fractionated within a 24-hour period. The second advantage is the effect of the stationary phase which facilitates the separation by composition, and the reliability of SEC for the separation by molecular size. Thus, the restricting basic conditions for the interactions between the polymer constituents and the separating solvent systems are partly raised. In some cases, still another point may be favourable: CCF can be carried out with a tiny amount of material. Several examples mentioned in the previous sections had been performed on 1 mg of starting material. Classic cross-fractionation requires about 10 g. It can be concluded that CCF is a potential tool in copolymer analysis. It is comparatively new in polymer laboratories, but the basic principle has long been known among chromatographers: in 1978, coupled-column liquid chromatography was applied to the separation of complex mixtures.66

336

Separation Methods

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