Gel permeation chromatography

Gel permeation chromatography S. R. Holding Gel permeation chromatography is the most valuable single technique in the molecular weight characterisation of polymers. The power of this technique lies in its ability to give information on the whole molecular weight distribution. In Britain, the Polymer Supply and Characterisation Centre provides a GPC service for research workers in the polymer field.

The two most fundamental properties of polymers are chemical composition and molecular weight. While the chemical composition can usually be easily by relatively determined the techniques, of a variety determination of polymer molecular weights may not be as straightforward. This is further complicated by the fact that with polymers we are usually confronted with a distribution of proper weights. A molecular understanding of a polymer comes only from considering the molecular weight distribution as a whole. Gel permeation chromatography (GPC) is a chromatographic technique in which the separation obtained is a function of molecular size. The theory of GPC has been reviewed elsewhere [l], but essentially small molecules are retarded in their passage through the separating column by their ability to diffuse into the pores of the porous column packing. Large molecules, not able to diffuse into the pores, travel through the interstitial volume and are eluted first. Hence the elution volume is a function of the size of the solvated molecules and this in turn is related to the molecular weight. permeation term The gel chromatography was first applied by J. C. Moore [2] in 1964. Although the basic mechanism had been used for biopolymers G& (see later unt;; it Aqueous development of macrorectic~~~ (rigid) polymeric packings, by Moore, and their application to characterising synthetic polymers, which led to the rapid advancement of the technique. By the end of the 196Os, suitable instrumentation had been developed

S. R. Holding,

B.A., M.Sc, Ph.D.

Is responsible for the running of the Polymer Supply and Characterisation Centre. This is suooorted bv the Science and Enaineerina Rekkarch Co;ncil and is situated at thi Rubber and Plastics Research Association, Shawbury, Shropshire. It provides GPC services for universities, polytechnics, and industry. Endeavour, New Series, Volume 8. No. 1, 1994. OlSo-9327194 $0~00+50 I@ 1994 Pergamon Press. Printed in Great Britain)

and GPC was established as an important technique in characterising synthetic polymers. At that time, the instrumentation used in GPC was well in advance of other forms of liquid chromatography. In the 1970s gas chromatography workers applied their methods and experiences to working with a liquid mobile phase and high performance (HPLC) liquid chromatography emerged. These general advances in liquid chromatography have now been applied to GPC and this has led to a generation of benchtop new instruments for the fast molecular weight characterisation of polymers. The Polymer Su~~ply and Characterisation Centre

Polymer and The SUPPlY Characterisation Centre (PSCC) was established in Britain in 1969 with the prime function of providing polymer characterisation services to research universities and workers polytechnics.‘ihe past experiences and current practice of the Centre reflect the general development of GPC When the PSCC was first set-up, GPC was only one of a number of techniques used for characterising polymers; however. it soon became the The early dominant technique. instruments used were either Waters 200’s or variations of the same general design. These were large free standing instruments; with the usual arrangement of 4 X 1 metre columns, three to four hours were typical run times. Also, the low efficiency of these systems required the use of a correction for ‘band broadening’ (the increase in peak width caused by diffusion, etc., rather than by separation the chromatographic mechanism itself). Most work was carried out on an instrument using tetrahydrofuran (THF) as solvent; this system could be used for a wide variety of polymers and it operated routinely. Valuable work was also carried out in characterising polyolefins, but many instrumental and design problems were encountered in operating a system at the necessary high temperatures.

Attempts at measuring the GPC of raised at polyamides (also temperature) were not as successful as the work on polyolefins. In 1979, the Centre was re-equipped with modular benchtop GPC instruments. Major advances had by then been made in the general instrumentation, but the most significant changes were in the heart of the chromatographic system-the columns. The marked reduction in the column packing bead size and the minimizing of ‘dead space’ has resulted in shorter, more efficient columns. Typical run times were reduced to 45 minutes, and band broadening corrections were no longer required. Significant changes have also occurred in the manipulation of GPC data. The calculation of average molecular weights from raw GPC data is tedious and the development of computer techniques for handling the data was soon started. Initially, the data were taken manually from the chomatograms and the resulting table of chromatogram heights was processed by a main frame computer. Subsequently, the use of a data logger to acquire the data for the main frame computer was found to be advantageous. More recently, the Centre has changed over to a dedicated mini-computer for data logging and interpretation. A complete datahandling system has been commissioned which automatically acquires the data which later can be processed to give both graphical and numerical results. A further advancement of the GPC technique has been the incorporation of a detec?ar for producing absolute molecular weight data. The Centre has used a low-angle laser light-scattering (LALLS) photometer both in the ‘stand alone’ or static mode and combined with GPC. The value of this system will be discussed later. The Centre currently offers routine services for GPC with THF as the solvent, and also a high-temperature system for polyolefins. A multidetector capability is available for a 17

more complete characterisation of copolymers and ‘absolute’ molecular weights can be determined using the LALLS photometer. Further, the range of polymers which can be characterised is being extended by the -use of a larger range of solvents for GPC work. Current

practice

in GPC

A typical GPC system consists of a valve, pump, sample introduction separating columns, and detectors. As careful control of the flow-rate is required, high quality, constant-volume pumps are usually used. The separating columns may consist of a number of ‘mixed bed’ columns (each containing the full range of column packing pore sizes) or, more traditionally, a bank of columns, each containing a specific pore size packing, selected to cover the required molecular weight range. The column packing will normally consist of lo-micron beads, and typical column dimensions are 25 or 30 cm long by approximately 8 mm internal diameter. Currently, a major effort is being put into a further reduction in the packing bead size to increase the column efficiency. Of the range of detectors available, those based on the differential refractive index (DRI) are the most widely used for GPC. THF is a solvent suitable for a large number of polymer types and is consequently the most useful solvent for GPC. Although THF systems are frequently operated at ambient temperature, control of the will improve the temperature repeatability of the system. Other routine solvents which can be used at ambient temperature include toluene and dichloromethane.

been found that the polystyrene-based column packings used at the Centre are stable for several months at 150°C. There is particular interest in the high end of the molecular weight distribution of polyethylene, and the present ability to characterise this important part of the distribution is questionable. There seems to be some evidence to suggest that the larger bead size packings can be more suitable for lookin+ at very high molecular weights (c. 10 ). Also the extremely large pore size polymeric packings appear to be inherently less stable mechanically. Alternative solutions to this problem are mentioned later. High temperature GPC with chlorobenzene solvents has also been applied to characterising other polymers, including asphalts, pitches, and EPDM. In addition, polyamides and polyesters have been characterised using phenolic solvents at 100°C [4]. Polar polymers

Dimethylformamide (DMF) is probably the most useful solvent for characterising polar polymers which are not soluble in THF. The use of this solvent is reasonably straightforward; the analysis temperature is usually raised to 80°C and the main difficulty encountered is in the calibration. Using DMF as the solvent with polystyrenebased column packings results in polystyrene calibrants being excessively retarded, implying that an undesirable interaction is occurring. At the Centre, we have recently started employing DMF as a solvent.

Poly(ethylene dYCO1) and PolY(ethylene oxide) calibrants have been found to be acceptable. However, whilst these two calibration polymers have the same repeat unit, their respective calibrations have displayed slightly different slopes. Aqueous

Data misinterpretation

GPC theory has been covered elsewhere [l] and a wide range of applications has been reported [6]. However, there appear to be relatively few comments on the frequent and easy misinterpretation of data. With modern bench-top instrumentation, it

log IO MP lO*E2

IO* E3

IO+E4

c

High temperature

GPC

The molecular weight characterisation of polyolefins has always been considered difficult. Although the value of GPC for this application was soon established, many problems have been (and still are) encountered in working at the high temperatures necessary. Polyolefin characterisation is usually carried out with di- or trichlorobenzene as the solvent and at temperatures of 130°C to 150°C. Temperatures of 150°C or higher are desirable when characterising polypropylene; unfortunately, 150°C is the maximum temperature for most commercial GPC instruments. The refractive index detectors normally used for GPC are liable to be unstable when used at temperatures as high as this; this problem can be avoided by using fibre optics or alternatively, infra red detectors can be of value. For stability reasons, silica-based column packings have frequently been used for high-temperature GPC; however, it has 18

GPC

The separation of biopolymers on soft xerogels, using the same mechanism as GPC, has been carried out for many years, and is generally known as ‘gel filtration’. High performance separations of water-soluble polymers have been carried out on both silicabased and polymer-based column packings. Problems have been encountered with respect to column stability [5] and limited molecular weight separation ranges, but the main problem is the correct selection of eluent. Most water-soluble polymers are polyelectrolytes (even apparently neutral polymers, such as dextran show some ionic activity) and the correct selection of ionic strength, pH, and the nature of the buffer are essential considerations in avoiding unwanted interactions with the column packing material.

l2OC )-

l4OC I-

z 8 % f F

l6OC I-

1800

, -

2000

2200

2400 Figure 1 Typical

third order polynomial

calibration.

lO*E5

lO*E6

lOicE

should be possible simply to replace the separation columns in an HPLC system with GPC columns and proceed to carry out the molecular weight characterisation of a wide range of polymers. This is possible providing the perspective of the chromatographer can also be changed. In GPC, the raw data usually consists of a broad peak which is essentially an envelope of a multitude of peaks, each corresponding to a particular molecular weight. A small error in elution volume does not usually confuse the correct identification of peaks in HPLC; in GPC, on the other hand, this small elution volume error can lead to a significant error in the calculated molecular weights. In GPC methodology, instrumation and therefore, precise definition of the flow-rate is essential; this is achieved either by using high-quality pumps or by correcting the assumed flow-rate from the elution position of a reference material. On-line minicomputers, acquiring and processing raw chromatographic data rapidly, can produce ‘average molecular weights’. A small change in an assumed or measured parameter can cause the same raw data to indicate significantly different average molecular weights. GPC is not an absolute technique and interpretation of the data starts with a calibration. The systems are usually calibrated with polymers known to have a narrow molecular weight distribution. In our experience, the calibration curves with the best fit are either linear or a third order polynominal (figure 1). Calibration can also be achieved with a single welldefined, broad molecular weight distribution calibrant (often used for polyethylene). When GPC is used together with the calibration, average molecular weights can be calculated, but these will be expressed as if the sample were of the same polymer type as the calibrants. For’ polymers of a the ‘universal different We 1 calibration procedure’ can often be applied. This essentially involves a mathematical correction dependent upon the Mark-Houwink parameters (determined by dilute solution viscometry) for the sample/solvent/ calibrant combination. Unfortunately, these parameters are usually literature values, the acceptability of which ought to be questioned more seriously. As GPC is a comparative technique, the use of ‘polystyrene equivalent molecular weights’ (when polystyrene is the calibrant) may often be preferable to applying the universal calibration procedure. A problem in final data interpretation is the usual necessity for

Mokcular

wght

Figure 2 The Molecular Weight Distribution of a polyethylene sample-with the internal marker peak. If the dotted baseline is used in the calculation: M, = 422,000, ti, = 14,100. If the baseline is as shown (extended beyond the marker peak); M, = 438,000, fil, = 22,600.

an operator decision on where the chromatogram baseline should be drawn and where the sample peak starts and finishes (figure 2). Many of these interpretation problems can be avoided if the technique is used in a purely comparative manner. Care must be exercised to prevent results from GPC being treated as if they were absolute determinations. GPCILALLS

To avoid the uncertainties associated with column calibration and the need to apply the universal calibration procedure, it is possible to combine GPC with an absolute molecular weight measurement such as LALLS. At the Centre, LALLS has been used on its own and also combined with GPC. In a preliminary collaborative experiment, using LALLS in the ‘stand alone’ or static mode, the measurement was shown to be highly reproducible [7]. We have found the combined technique to be easy in practice, but the results must be handled with care. A comparison of figure 3 and 4 demonstrates how, with our methodology, the number average molecular weight appears to be overestimated by GPCILALLS. In its static mode, LALLS will give an absolute value for the weight average molecular weight. In the GPC/ LALLS mode, the determined weight average molecular weight is a close approximation to the absolute value. We have found that both static LALLS and GPCiLALLS to be valuable techniques; however, the necessary ancillary measurements, such as the

sample/solvent differential refractive index, can be tedious to make and also require a relatively large sample. Future trends

in GPC

It is unlikely that any major changes will occur in the general instrumentation used in GPC. The main activity in development appears to be in producing ever smaller bead size column packings. The new ultrafine packings will be used to reduce the total column length and thus speed up the polymer characterisation. The work towards ultrafine packings appears to be having most successwith small pore size packings, and this is likely to highlight the current difficulties in applying GPC to very high molecular weight polymers. Already, the current 10 micron packings seem to be less suitable at the high end than the earlier, larger packings. Future trends in characterising the high weight polymers are likely to be a return to the larger bead size columns or the development of alternative techniques such as field-flow fractionation [8] or hydrodynamic chromatography [9]. The general trend to speed up polymer characterisation is being assisted by the adoption of dedicated mini computers and the development of appropriate software to allow the rapid processing of data. Unfortunately, the speed and ease of generating numerical results tends to work against a proper appreciation of the significance of the molecular weight distribution as a whole. It is possible 19

90 -

troublesome areas is in the characterisation of water soluble, synthetic polymers. There still appears to be a need for the development of stable column packings with high exclusion limits suitable for aqueous GPC. If this problem is solved, there appears to be sufficient interest in this area, and plenty of experience with natural polymers, for major advances to be made. Gel permeation chromatography is now a well established tool in the molecular weight characterisation of polymers. With the advent of benchmany more top instrumentation, laboratories are involved in the technique. Unfortunately, many new users initially may misunderstand the intricacies of the technique, but when these are fully appreciated, much valuable information becomes readily available from this basically easy and rapid method.

80 -

70 z 2 ; z G

60 -

50 -

z rp “3

40 -

5 3 4

30-

f 20 -

Figure 3 The Molecujar Weight Distribution of a typical determined by GPC: M, = 206,000; M, = 29,000.

polystyrene

sample as

10090

-

80 5 % ; ? TJ z ? g 5 : 5’ s

70-

60-

50

40-

3O-

References [l] Dawkins J. V. and Yeadon G., High Performance Gel Permeation Chromatography In ‘Developments in Polymer Characterisation-1’. (J. V. Dawkins, ed.) Applied Science Publishers, London, 1978. [2] Moore J. C., J. Poly. Sci., Part A, 2 835, 1964. [3] Porath J. and Flodin P., Nature, Lond. 183 1657, 1959. [4] ‘High Temperature Gel Permeation Chromatography-Polymer solubilities, Solvents, Temperatures’ Waters Application Note 1981. [5] Barker P. E., Hatt B. W., and Holding S. R.. .I. Chromatoer. 206. 27. 1981. ]6] Cooper A. R., Analysis of Polymers by Gel Permeation Chromatography In ‘Analvsis of Polvmer Svstems’ (L. S. Bark and N. S. Allen, eds) Applied Science Publishers, London, 1982. [7] Dumelow T., Holding S. R., and Maisey L. J., Polymer Com”

Figure 4 The Molecular Weight Distribution of a polystyrene samplejthe same sample as in Figure 3) as determined by GPCILALLS: M, = 203,000; M, = 60,000.

that there will be a reaction against using the apparent average molecular weights calculated from GPC and that software will be developed for easier comparisons of the raw chromatograms or the normalised molecular weight

20

distributions against an arbitary molecular weight axis. In this way, the true comparative nature of the technique can be used to its maximum advantage. Currently, one of the most

munications, 24, 307, 1983. [8] Yau W. W. and Kirkland J. J., J. Chromaton. 218. 217. 1981. u

[9] Tijssen R., Bleumer J. P. A., and Van Kreveld M. E., J. Chromatogr. 260. 297. 1983.