Introduction to Modern Liquid Chromatography, Third Edition part 70 ppt

13.10 SYNTHETIC POLYMERS Separations of synthetic polymers are usually carried out for one of two purposes: 1 determination of the molecular-weight distribution of a sample Section 13.10

646 BIOCHEMICAL AND SYNTHETIC POLYMER SEPARATIONS

side stream

Column volumes

25

20

15 % acetonitrile

C8 (150× 9.4-mm i.d.); load, 153 mg rh-insulin from proinsulin process; gradient, 17–29%

acetonitrile in 0.25M acetic acid in six column volumes; flow rate, 0.3 mL/min Fractions from 3.3 to 4.3 column volumes were pooled (mainstream) Fractions 3.2–3.3 and 4.4–5.4, plus the protein eluted during column regeneration (not shown), were combined as the side stream Adapted from [162]

and 4.4–5.4 C-Vs plus recovered solvent from column regeneration) contained an additional 15% of the product (for re-separation)—for an overall insulin recovery of 97% The initial purity of the sample for RPC separation was 91.5% This example illustrates a common property of preparative separations: in contrast to analytical chromatography, prep-LC chromatograms may suggest poor separation of product from impurities, but when fractions are collected and analyzed, the results often are acceptable

Scale-up experiments were carried out next, using six, successively larger, axial-compression columns, with the bed-volume increased from 10 mL to 80L,

as summarized in Tables 13.9 and 13.10 In each separation the weight of insulin applied to the column was 14 to 15 g per L of column-volume (C-V), the flow rate was 1.5 C-V/h, and the gradient slopes were 2%/C-V Minor changes in gradient slope were necessary to maintain column performance, as measured by product purity and recovery Flow rates were increased in proportion to the volume of the column as the process moved from lab to pilot plant to production Purity was 98.5, 98.6, and 98.6% at lab scale, pilot scale, and production scale, respectively, while mainstream yields were 82, 79, and 83% It may seem remarkable that mainstream purities, recoveries, and elution volumes remained consistent from lab scale to production scale over a 10,000-fold range of column volumes, but this should be true of scale-up if carried out properly (Section 15.1.2.1)

Table 13.9

Column Sizes Used for rh-Insulin Scale-up Studies

Source: Data from [162].

Table 13.10

Summary of rh-Insulin Scale-up Studies

Flow Rate Gradient Load Purity Yield (C-V/h)a (%B/C-V)b (mg/mL)

Source: Data from [162].

aC-V is empty column-volume.

bChange in %B per column-volume (C-V) of mobile phase; proportional to gradient steepness.

The purification of rh-insulin on a production scale was next carried out for both two-chain insulin and proinsulin Separation was more challenging for the two-chain process because of a 20%-higher concentration of structurally related impurities

and a gradient of 17–30% acetonitrile over 6 C-V at a flow rate of 1.4 C-V/h (0.8 L/min) The purity of the charge was 80%, and the mainstream purity was 98.5% For insulin derived from the proinsulin process, the purity of the feedstock was higher (91%), which resulted in purified product of higher purity (99.1%)

comparable results

The product fractions from high-performance RPC were subjected to an additional purification step by SEC Two lots of rh-insulin from each process were then compared to insulin purified by conventional chromatography that did not use high-performance RPC The results showed that purification by RPC results in higher purity levels, equivalent biopotency, and comparable low levels of contamination by

648 BIOCHEMICAL AND SYNTHETIC POLYMER SEPARATIONS

endotoxin or host cell protein No siloxanes (potential breakdown products from the stationary phase) were detected

13.9.3 General Requirements for Prep-LC Separations of Proteins

Targets for yield and purity are needed, as well as methods (usually RPC) to measure yield and purity Estimates of product weight (e.g., tons/year) and targets for the expected delivery time and purification cost are also required A defined starting material is necessary, as well as an expected range of purity for the starting material

A standard for the purified product is helpful, but the standard can be produced

as part of process development Mass spectrometry and other physical methods can be useful for confirming the identity and purity of purified product Additional development requirements may be imposed if the separation is intended to produce pharmaceutical products under current Good Manufacturing Practices (cGMP; [165, 166]) or ISO 9000 guidelines [167]

For protein products, a primary requirement is product stability during sep-aration While, in principle, proteins can be denatured during RPC and renatured afterward [168], this approach has generally not been favored for purifications by means of RPC Not only must the product retain its biological activity, there should also be no detectable chemical changes such as oxidation, deamidation, or cleavage

of peptide bonds Mass spectrometry methods greatly simplify the task of detecting the latter modifications Methods for assessing the stability of proteins under various conditions are well established It is often preferable to measure the stability profile

of a protein product before chromatography development, so that time is not wasted exploring modes or mobile phases that are incompatible with the product

13.10 SYNTHETIC POLYMERS

Separations of synthetic polymers are usually carried out for one of two purposes: (1) determination of the molecular-weight distribution of a sample (Section 13.10.3.1), and/or (2) determination of different compound types or classes in the sample (Section 13.10.3.2) These applications differ fundamentally from other HPLC separations covered in this book For this and other reasons the present section represents only an introduction to separations of synthetic polymers

13.10.1 Background

Synthetic polymers are large, man-made molecules; in all cases they are formed from one or more different monomers, which occur in the molecule many times If a single

monomer is polymerized, the result is a homopolymer (Fig 13.46a); short-chain members of such a sample are referred to as oligomers An example is ethylene as

for the reaction of p ethylene molecules to form a polymer molecule) If two

(or more) different monomers are used to create a synthetic polymer, we have a

copolymer (Fig 13.46f ) Homopolymers can differ in length, as in Figure 13.46a,

c, e Molecular length is expressed either as the degree of polymerization p (i.e.,

where p is the number of monomeric units) or the molecular weight of the polymer.

(b)

(f )

(i )

(k)

(c)

(e)

(h)

(d )

(g)

( j )

(l)

homopoly-mers of varying length; (b), (c), and (d) are linear homopolyhomopoly-mers with different end-groups; (g), functional groups have been introduced along the chain; (h), a cyclic homopolymer; (f ),

a random copolymer, (i), a block copolymer; (k), a graft copolymer; (j) and (l) are branched

homopolymers, with short-chain branching and long-chain branching, respectively

Finally, homopolymers can differ in molecular shape or topology Linear molecules

exist (Fig 13.46a– f ), as well as cyclic (small) oligomers (Fig 13.46h) and related

polymers Depending on the synthetic process, branches may be deliberately or

accidentally introduced (Fig 13.46g, j–l) Individual molecules can differ in the

number of branches, their length, and their position in the molecules In the case of copolymers the sequence of the monomers is relevant If this sequence is determined

by a purely statistical process, random copolymers are formed The opposite extreme

is that of block copolymers (Fig 13.46i), where long sequences of a single monomer occur within the molecule Finally, there are graft copolymers (Fig 13.46k), where

chains formed from a different monomer are attached to the primary polymer backbone

Polymer properties are affected by structural features, which are therefore important A higher molecular weight generally leads to a stronger polymer End-groups and functional groups are critically important for polymers used in reactive formulations such as adhesives, sealants, and coatings Branching usually affects the processing properties of polymers Block copolymers can have very dif-ferent properties compared to random copolymers One structural property that is not depicted in Figure 13.47 is the degree of stereoregularity of the chain, usually

called tacticity In an atactic polymer, the monomeric units are oriented in a random

fashion; in an isotactic (or syndiotactic) polymer, all monomers are positioned in the same (or alternating) direction Stereoregular polymers usually exhibit a much higher degree of crystallinity As a result atactic polypropene (or ‘‘polypropylene,’’

as it used to be called) is a soft plastic, whereas isotactic polypropene is hard and strong

650 BIOCHEMICAL AND SYNTHETIC POLYMER SEPARATIONS

4 3 2 1 0 –1

–2

ln k

%B

p= 5

p =

15 25 100

(min)

(min)

(min)

(a)

(b)

(c)

(d )

reten-tion behavior of a homopolymeric series; (b) hypothetical illustrareten-tion of the elureten-tion of a low-molecular-weight homopolymer with a 35-minute gradient; (c) similar separation as in (b) for a gradient time of 10 minutes; (d) hypothetical illustration of the separation of a polymeric

blend by interactive liquid chromatography (i-LC)

Just as the properties of a synthetic polymer are affected by its molecular struc-ture, so is polymer chromatographic behavior This allows us to separate polymers based on molecular weight, chemical composition (functionality), stereoregularity, degree of branching, and so forth, as summarized in Table 13.11 for different separation modes

Many physical properties of synthetic polymers are important in relation to their chromatographic behavior, none more so than their solubility For any kind of material to be separated by liquid chromatography, it must be dissolved completely (any agglomerates or particles are detrimental to chromatographic separation)

Table 13.11

Effect of the Molecular Structure of Synthetic Polymers on their Chromatographic Behavior

Molecular End Chemical Stereo- Branching Section Weight Groups Composition Regularity

Size-exclusion

Interactive liquid

Temperature-gradient

interaction

chromatography

Note:•, major effect;◦, minor effect; —, no significant effect.

aA significant adverse effect may be observed if end groups or functional groups show strong interactions with the stationary phase.

bEffect is strong in isocratic (‘‘critical’’) chromatography of polymers In gradient-elution LC it is usually overshadowed by the effects of molecular weight and, especially, chemical composition.

cA significant effect may be observed if branching introduces different or additional functional groups.

dEffect may be strong, but the technique is not usually applied for the separation of copolymers.

For many polymers, complete dissolution can be difficult; polyolefins (polyethene, polypropene), for example, require elevated temperatures and a high-boiling solvent such as trichlorobenzene Many polar polymers (polyesters, polyamides, polyke-tones) require special solvents such as hexafluoroisopropanol Polymers can also require a long time to dissolve because of the strong interactions between chains (including ‘‘entanglements’’) and the slow diffusion of large molecules

Another important consideration is the HPLC detection of synthetic polymers Many important types of polymers (poly-olefins, poly-acrylates, poly-alkoxides) lack

UV chromophores Consequently the RI detector is mainly used for isocratic sepa-rations of polymers (notably by SEC) Occasionally, infrared-absorption detectors (operating at a fixed wavelength) can be advantageous In gradient separations, the evaporative light-scattering detector has been commonly used, but quantitation can

be a problem; the charged-aerosol detector is a promising alternative Although the characterization of (polar) polymers by mass spectrometry has greatly improved since the mid-1990s, the use of LC-MS for this purpose is still uncommon Some common synthetic polymers, typical solvents used in size-exclusion chromatogra-phy, and the most commonly used detection methods are listed in Table 13.12 For additional information on possible detectors for polymer separation, see Chapter 3, Chapter 9 of [169], and [170–172]

13.10.2 Techniques for Polymer Analysis

Synthetic polymers are not amenable to the resolution of individual molecules,

a difference that sets them apart from other samples for HPLC separation and

analysis Instead, polymer molecules come in a range of sizes or a molecular-weight

distribution (MWD) While many techniques can be used to determine an average

molecular weight for the sample, chromatographic methods such as size-exclusion

(polyethylene), poly

:•

aFor

bRequires

cMore

dCharged-aerosol

652

chromatography (SEC) are able to determine the MWD, as well as number-average

or weight-average molecular weights [169] In addition to a MWD, copoly-mers exhibit a chemical-composition distribution Spectroscopic techniques such

as Fourier-transform infrared (FTIR) spectroscopy and (especially) nuclear-magnetic resonance (NMR) spectroscopy can provide detailed information on chemical com-position, while interactive liquid chromatography can separate different chemical types and provide a chemical-composition distribution (CCD) A nonexhaustive overview of a number of important techniques is provided in Table 13.13 It is seen that NMR is highly useful in the middle column (averages), whereas the right-side column (distributions) is dominated by chromatographic techniques

13.10.3 Liquid-Chromatography Modes for Polymer Analysis

Size-exclusion chromatography (SEC) is reviewed in Section 13.8 and in [169]; its application for the determination of molecular weight or molecular-weight distribution (MWD) is similar for both synthetic polymers and biopolymers There are two main differences between these two applications of SEC For synthetic polymers, SEC is used to determine a molecular-weight distribution [169], whereas for biopolymers the goal is the estimation of molecular weight for individual compounds Likewise the solvent used as mobile phase is often different; usually aqueous mobile phases are used for biopolymers (gel filtration), and organic solvents for synthetic polymers (gel permeation)

In the case of homopolymers, SEC can be coupled to other polymer-characterization methods, notably light-scattering and viscometry (for copolymers it

is difficult to accurately correlate the resulting data [170]) Static light-scattering can

be used to obtain accurate information on the (weight-average) molecular weight of polymer in the SEC effluent, provided that (1) we know how refractive index varies

as a function of polymer concentration, and (2) the detector is properly calibrated Also the concentration of the polymer in the effluent fraction must be accurately known, for example, by using a RI detector in conjunction with light-scattering

In SEC, conditions are selected to suppress interactions between the analyte and the stationary phase as much as possible In interactive liquid chromatography (i-LC), these interactions are used to separate molecules by chemical type or functionality While i-LC separations of polymers are, in many ways, similar to the separation of small molecules by HPLC, there are two overriding differences: (1) the molecular-weight range of polymers (large number of individual species that differ in molecular weight), and (2) a systematic change in analyte retention as the size of the solute molecule increases High-molecular-weight analytes typically exhibit larger

changes in k for a given change in %B, as seem in the examples of Figure 13.11

for several peptides and proteins A similar example for synthetic polymers is

illustrated in Figure 13.47a, which illustrates schematically how retention varies

with composition for oligomers and polymers that differ in their size or degree of

polymerization p (number of monomers) For the oligomers of Figure 13.47a, p equals 5–15; for the polymers, p equals 25 and 100 The curves for larger molecules (larger p) are increasingly steep, to the extent that for large polymers there is only a

654 BIOCHEMICAL AND SYNTHETIC POLYMER SEPARATIONS

Table 13.13

Summary of Techniques for Determining Average Molecular Structures and Molecular Distributions of Synthetic Polymers

Property Determining Averages Complete Distributions

Molecular weight Osmometry light

scattering

Size-exclusion chromatography Hydrodynamic chromatography Sedimentation Ultracentrigugation Chemical composition NMR FTIR pyrolysis

GC-MS

Interactive liquid chromatography (mainly gradient elution)

Functionality (end groups

or functional groups)

NMR titration Interactive liquid chromatography (mainly

isocratic) Chain regularity NMR Temperature-rising elution Fractionation Degree of branching NMRa Molecular-topology fractionation

aAppropriate for polymers with a relatively low molecular weight Also NMR is generally considered appro-priate only for determining short-chain branching, not long-chain branching.

very narrow range of mobile-phase composition (%B) for which the polymer can be eluted isocratically For this reason gradient elution is usually the method of choice for separations by i-LC

Gradient elution is usually carried out with linear gradients, corresponding

different polymeric species Under these conditions retention times for each peak in

a polymer sample will correspond to the intersection of plots as in Figure 13.47a

retention time for adjacent peaks (and therefore better resolution), compared to separations with a shorter gradient Finally, for a sufficiently fast gradient (and

as a single peak The latter behavior for the separation of a low-molecular-weight

polymer with long and short gradients is illustrated in Figure 13.47b, c, respectively.

In the long gradient of Figure 13.47b, the retention of individual oligomers differs

enough so that there is a partial separation of the sample In the short gradient of

Figure 13.47c, this is no longer true, so a single peak is observed—corresponding to

referred to as pseudocritical chromatography, as opposed to (isocratic)

chromatog-raphy under critical conditions, as described in Section 13.10.3.4 Pseudocritical i-LC (i.e., with gradient-elution) is particularly useful for the separation of polymers

according to chemical composition This is illustrated in Figure 13.47d, which shows

the separation of two different kinds of polymers Retention is seen to be a function

of chemical composition in this example, but not of molecular weight Such pseud-ocritical conditions can be approached more closely for (1) higher molecular-weight polymers and (2) shorter gradient times As a result gradient-elution i-LC is well

suited for the determination of chemical-composition distributions Figure 13.48b

120

100

80

60

i-LC (min)

i-LC

SEC (sec)

(a)

(b)

(c)

copolymeric binder produced by two-stage emulsion polymerization of styrene and methyl

methacrylate (a) Second-dimension separation: SEC; (b) first-dimension separation, RPC lin-ear gradient from 65 to 100% in 170 minutes; (c) 2D separation UV detection at 214 nm For

further details, see [170]

provides a practical illustration where a high-molecular-weight copolymer of styrene and methyl methacrylate is very well separated by chemical type (note three peaks in the chromatogram, for three chemical types in the sample) For a further discussion

of Figure 13.38, see Section 13.10.4

Figure 13.47a suggests that there is a ‘‘critical’’ mobile-phase composition for which

all oligomers co-elute The potential benefit of working at or near critical conditions

is that the effect of the homopolymeric chain on retention can be minimized, so that polymers with different structural elements (e.g., end-groups) can be separated as in

Figure 13.47d — regardless of their MWD This implies that critical conditions can

be used to determine differences in polymer functionality [174] NPC separations are especially suited for this kind of separation because of the large effect that a (polar) functional group can have on retention

Some other HPLC procedures for polymer separation are noted in Table 13.13 Very large polymers can be separated by field-flow fractionation (FFF) and by hydrodynamic chromatography (HDC), techniques that are outside the scope of the present book