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# Colloid-polymer mixtures in the protein limit

## Abstract

We computed the phase-separation behavior and effective interactions of colloid-polymer mixtures in the “protein limit”, where the polymer radius of gyration is much larger than the colloid radius. For ideal polymers, the critical colloidal packing fraction tends to zero, whereas for interacting polymers in a good solvent the behavior is governed by a universal binodal, implying a constant critical colloid packing fraction. In both systems the depletion interaction is not well described by effective pair potentials but requires the incorporation of many-body contributions.

Adding polymers to suspensions of micro- and nano-particles induces depletion interactions that profoundly affect their physical properties. This phenomenon has important scientific and (bio-)technological applications. Polymers such as polyethylene glycol are routinely added to protein solutions to enable protein crystallization Kulk99 ; Tana02 , a poorly understood process and of great importance in structural biology Durb96 . In cell biology depletion interactions are key in the process of macro-molecular crowding Zimm93 . Food and paint production are among the industrial sectors where depletion phenomena play a role.

In this Letter we focus on mixtures of hard sphere (HS) colloids with a radius $`R_c`$ and non-adsorbing polymers with a radius of gyration $`R_g`$, in the regime where $`q=R_g/R_c>1`$. This is often called the nano-particle or “protein limit”, because in practice small particles such as proteins or micelles are needed to achieve large size-ratios $`q`$. Whereas the opposite “colloid limit” ($`q1`$) has been well studied, the physics in the protein limit is less established. This imbalance is partially due to the lack of well characterized experimental model systems for the protein limit and partially to a poor theoretical understanding. The colloid limit can be well described within the framework of effective depletion pair potentials Asak54 ; Poon02 , in contrast to the protein limit, where the interactions cannot be reduced to a pairwise form Odij97 ; Sear01 . Nevertheless, for biological and industrial applications, this regime is at least as important as the colloid limit.

One of the first theoretical treatments of colloid-polymer mixtures in the protein limit was by de Gennes deGe79a , who showed that the insertion free-energy $`F_c^{(1)}`$ of a single hard, non-adsorbing sphere into an athermal polymer solution scales as

$$\beta F_c^{(1)}(R_c/\xi )^{31/\nu }$$
(1)
when $`R_c<\xi `$, with the polymer correlation length $`\xi (\varphi _p)R_g\varphi _p^{\nu /(3\nu 1)}R_g\varphi _p^{0.77}`$ deGe79 . Here, $`\beta =1/k_BT`$ is the reciprocal temperature, $`\nu 0.59`$ is the Flory exponent and $`\varphi _p=\rho _p\frac{4}{3}\pi R_g^3`$ is the polymer volume fraction for a polymer number density $`\rho _p`$, so that $`\varphi _p1`$ at the crossover from a dilute to a semi-dilute solution deGe79 . The prefactors can be calculated by the renormalization group (RG) theory Eise00 , yielding: $`\beta F_c^{(1)}4.39\varphi _pq^{1.3}`$ which has been verified by computer simulations for small $`q`$ Loui02a . Based on this description of $`F_c^{(1)}`$, de Gennes deGe79  and Odijk Odij97  predicted extensive miscibility for colloid-polymer mixtures in the large $`q`$ limit if $`R_c<\xi `$. However, it is well known that protein-polymer mixtures do phase-separate Doub00 ; Tuin00 . Recently, Odijk et al. Wang01  suggested that a poor solvent could facilitate phase-separation. Sear Sear97  altered the form of $`F_c^{(1)}`$ to include effects when $`R_c\xi `$, and also predicted phase-separation with a truncated virial theory. The same author recently proposed an alternative theory Sear02  where the colloids induce depletion attractions between the polymers, leading to a poorer effective solvent and eventually phase-separation. Mean-field cell model calculations also predict demixing Scha97 . Another promising approach uses integral equation techniques Fuch00  to predict spinodal curves and critical points. However, all these theories suffer from several uncontrolled approximations leading to different predictions for the causes and properties of the phase-separation. To clarify this situation, we performed computer simulations with as few simplifying assumptions as possible, on which we report in this Letter.

We have recently used a coarse-graining technique Loui00  to study the colloid limit, and found quantitative agreement with experimental fluid-fluid binodals Bolh02 , and significant qualitative differences between interacting (IP) and non-interacting (NIP) polymers. Here, we study the same athermal model of HS colloids and non-adsorbing polymers in the protein limit, and calculate, for the first time, the full fluid-fluid binodals by direct simulation. The results for the IP and NIP show even larger qualitative differences, and many-body depletion interactions must be invoked to understand the phase-behavior.

The simulation model consists of polymers on a simple cubic lattice mixed with HS colloids. The interacting polymers in a good solvent are modeled as self avoiding walks (SAW) of length $`L`$, which have a radius of gyration $`R_gL^\nu `$. The non-interacting polymers are modeled as random-walks, for which $`R_gL^{0.5}`$. In both models there is an excluded-volume interaction between the colloidal HS and the polymer segments.

The simulations were performed on a $`D^3`$ lattice with periodic boundary conditions, where $`D=48`$ and $`D=100`$ for the NIP and IP system, respectively. Throughout this Letter, we use the lattice spacing as the unit of length. For the NIP the colloidal HS diameter was $`\sigma _c=5.5`$ and the polymer length was $`L=50,100,200`$ and $`500`$, corresponding to $`q=1.03,1.45,2.05`$ and $`3.2`$, respectively. For the IP $`L=2000`$, and $`\sigma _c=10,14`$ and $`20`$, yielding $`q=3.86,5.58`$ and $`7.78`$, respectively. Colloidal positions have continuous values, but when we calculated the interaction between colloid and polymer the colloids were shifted such that they occupied a constant number of lattice sites to prevent spurious attractive positions for single colloids (other lattice effects, although unavoidable, are expected to be small.) Thermodynamic state points were calculated in the grand-canonical ensemble, i.e. at fixed volume $`V`$, colloid chemical potential $`\mu _c`$ and polymer chemical potential $`\mu _p`$ using Monte Carlo (MC) techniques. The NIP were sampled using an (exact) lattice propagation method Fren90 ; Meij94 , while the IP configurations were generated using translation, pivot moves and configurational bias MC frenkelbook  in an expanded ensemble to facilitate insertion of long chains Yan00 . Typical simulations lengths are $`10^9`$ Monte Carlo moves per state point. In order to determine the liquid-liquid binodals we first estimated the coexistence line by scanning a series of $`\mu _c`$ for several values of $`\mu _p`$ and locate the $`\mu _c`$ for which a sudden density change occurred. Subsequently 8-10 $`(\mu _p`$, $`\mu _c)`$ coexistence state points were simulated simultaneously using parallel tempering Yan00 . When the estimated coexistence points are sufficiently close to the true binodal and to each other, and near the critical point, this scheme results in proper ergodic sampling of both phases. If necessary, the chemical potentials were adjusted towards coexistence. We used the multiple histogram reweighting Ferr87 ; Yan00  technique to determine the precise location of the ($`\mu _c,\mu _p`$) coexistence line, and the phase boundaries in the ($`\varphi _c,\varphi _p`$) plane, where $`\varphi _c=\rho _c\frac{4}{3}\pi R_c^3`$ is the colloid volume fraction, with $`\rho _c`$ the colloid number density.

Figs. 1 and 2 contrast the calculated phase diagrams for NIP and IP for several size-ratios $`q`$. Firstly, we note that both models show extensive immiscibility, in agreement with experiment Doub00 . Secondly, the two systems exhibit striking differences: for the NIP, the critical colloid volume fraction $`\varphi _c^{crit}`$ tends to zero with increasing size ratio $`q`$, while the IP exhibit a nearly constant value of $`\varphi _c^{crit}`$. For both systems the critical polymer concentration $`\varphi _p^{crit}`$ increases with increasing $`q`$. The phase-separation occurs well into the semi-dilute regime for the IP, again in qualitative agreement with experiment Tuin00 . Properties of semi-dilute polymer solutions are independent of polymer length, being instead determined by the correlation length $`\xi `$, which is a function of the monomer density $`c=L\rho _p`$. The phase behavior of the polymer-HS mixture should therefore only be a function of the ratio $`R_c/\xi `$ Sear97 . Indeed, when the phase lines in Fig. 2 are rescaled with an accurate prescription for $`\xi (\rho _p)`$ Loui02a , the binodals nearly collapse onto a “universal binodal”, as shown in the inset of Fig. 2. This explains why the critical colloid packing fraction is nearly constant in the simulations. Similarly, $`\varphi _p^{crit}`$ scales as $`\varphi _p^{crit}q^{(3\nu 1)/\nu }q^{1.3}`$. For comparison, we have also included results for $`q1`$ from Ref. Bolh02  in Fig. 2. These results do not exhibit the same scaling behavior, since they are not in the semi-dilute regime.

The differences between NIP and IP phase behavior can be rationalized with some simple theories. Consider a Helmholtz free-energy $`F`$ of the form $`\beta F/V=f=f_c^{HS}+f_p+f_{cp}`$. Here, the HS free energy $`f_c^{HS}`$ is given by the accurate Carnahan-Starling expression Hans86 , and the polymer free energy $`f_p`$ for either IP or NIP solutions is well understood deGe79 . The contribution due to the HS-polymer interactions $`f_{cp}`$ is non-trivial. A first approximation truncates after the second cross-virial coefficient, yielding $`f_{cp}\rho _cF_c^{(1)}`$. For NIP the insertion free-energy $`F_c^{(1)}`$ is exactly known Eise00 , so that $`f_{cp}`$ takes the form $`f_{cp}^{id}=\rho _p\varphi _c\left(1+\frac{6q}{\sqrt{\pi }}+3q^2\right)\rho _p\varphi _c\widehat{b}_{cp}`$, which defines the reduced cross-virial coefficient $`\widehat{b}_{cp}`$. Since $`f_{cp}^{id}`$ grows with increasing $`q`$, immiscibility sets in at lower colloid packing fraction $`\varphi _c`$. The theory can be improved by realizing that the polymers only exist in the free volume left by the colloids Lekk92 . Simply taking this free volume to be $`1\varphi _c`$ is an adequate first approximation for the protein limit. The trends for the binodal lines calculated from this simple theory, shown in Fig. 1, agree qualitatively with the simulations. For example, the critical point shifts to smaller $`\varphi _c`$ and larger $`\varphi _p`$ for increasing $`q`$, and the binodal lines cross at a low $`\varphi _c`$. For computational reasons the simulations only go up to $`q=3.2`$ and we expect better quantitative agreement for larger $`q`$ since $`\varphi _c^{crit}`$ decreases so that the second-virial theory should become more accurate. In the $`q\mathrm{}`$ limit, this theory yields $`\varphi _c^{crit}1/\widehat{b}_{cp}1/(3q^2)`$, and $`\varphi _p^{crit}=q^3/\widehat{b}_{cp}q/3`$. Note that in the same limit, the penetrable sphere or Asakura-Oosawa model Lekk92  scales somewhat differently: $`\varphi _c^{crit}1/q^3`$ and $`\varphi _p^{crit}1`$. Sear Sear01  already pointed out the $`\varphi _c^{crit}0`$ behavior using a slightly different prescription for the free volume than we employ here. Here we claim that the limiting results are a general feature of free-volume theories.

In the IP case, $`f_{cp}`$ is more difficult to estimate, even for a second cross-virial theory. The $`R_c\xi `$ limit is given by Eq. (1) with the prefactors from RG theory. For $`R_c\xi `$ we have previously shown that $`F_c^{(1)}`$ is given by $`F_c^{(1)}=\frac{4}{3}\pi R_c^3\mathrm{\Pi }+6\pi R_c^2\gamma _s`$ Loui02a , where the polymer osmotic pressure $`\mathrm{\Pi }\xi ^3`$ is well known deGe79 . However, since Eq. (1) is essentially a surface (depletion layer) contribution, we use a simple approximate second cross-virial term $`f_{cp}=\rho _c\left(\beta \mathrm{\Pi }(\rho )\frac{4}{3}\pi R_c^3+4.39\varphi _pq^{1.3}\right)`$, which reduces to the correct form in both the $`R_c\xi `$ and the $`R_c\xi `$ limit 111Obviously this approach, which resembles the more schematic theory of Sear Sear97 , could be improved.. As with our treatment of NIP, we take the effect of the colloid excluded volume into account by computing the polymer densities in the free volume fraction $`1\varphi _c`$ (see Ref. Aart02  for a complimentary approach). The theoretical binodals were calculated using accurate expressions for $`\xi `$ and $`\mathrm{\Pi }`$ Loui02a  and are compared with the simulation results in Fig. 2, in the $`R_c/\xi `$ versus $`\varphi _c`$ plane. The qualitative agreement suggests that we can also use this theory to estimate the effect of a poorer solvent on the binodals. Following Ref. Wang01 , we alter the scaling of $`\xi `$ to $`\xi \varphi _p^{\delta /3}`$ so that $`\mathrm{\Pi }\xi ^3\rho _p\varphi _p^{\delta 1}`$. Interestingly, Fig. 2 shows that using $`\delta 1.5`$ instead of the appropriate exponent for polymers in a good solvent ($`\delta 2.3`$), does not result in important differences in the binodals. Of course, the differences will appear larger in the ($`\varphi _c,\varphi _p`$) plane due to the different scaling of $`\xi `$. One must keep in mind, however, that these predictions follow from a simple scaling theory and qualitatively different behavior may emerge when one approaches the $`\theta `$-point (where $`\delta =1`$).

To illustrate the many-body nature of the depletion interaction we estimated the phase behavior by approximating the system by colloids interacting via pairwise effective potentials. We computed the effective pair interaction $`v(r)`$ between two colloids in a bath of IP’s, by $`\beta v(r)=\mathrm{ln}g(r)`$ for $`\rho _c0`$. The colloid radial distribution function $`g(r)`$ was estimated by measuring the insertion probability of a HS at a distance $`r`$ from a second fixed HS in a SAW polymer solution using the above MC techniques. Results for a single size-ratio $`q=7.78`$ as a function of $`\varphi _p`$ are shown in Fig. 3. Several features are similar to the colloid limit Loui02b : the range shortens and the well-depth increases with increasing $`\varphi _p`$. Interestingly, our simple depletion potential Loui02b , derived for the colloidal limit, also works semi-quantitatively in this regime. A good measure for the attractive strength of an effective pair potentials is given by the second osmotic virial coefficient Hans86 , shown in Fig. 3. The saturation of $`B_2^{}`$ for larger $`q`$ is an interesting qualitative feature: apparently the shortening of the range compensates the deepening of the attraction, so that the total cohesion does not increase with increasing $`\varphi _p`$, something also found in RG Eise00  and integral equation calculations Fuch00 . For pairwise interacting systems, phase-separation typically sets in when $`B_2^{}1.5`$ Vlie00 . Here, the saturation of $`B_2^{}`$ suggests that for large $`q`$ the pair interactions do not provide enough cohesion to explain the phase-separation. We arrive at the same conclusions with simple mean-field theories Hans86 , which should be relatively trustworthy given the long range of the pair potentials. Obviously, for $`q1`$ a pair-level description is not sufficient, and many-body interactions must be invoked.

For the NIP, one might also expect many-body interactions to be important for large $`q`$. A good approximation to the pair-potentials exists Meij94 ; Loui02b , from which the second virial coefficients at the calculated critical points follow: $`B_2^{}(q=1.03)13.1`$; $`B_2^{}(q=1.45)16.4`$; $`B_2^{}(q=2.05)22.7`$. Even though the actual critical $`\varphi _c`$’s are very low, so that a second-virial description might be thought to be sufficient, the analysis above shows that for NIP the pair-interactions provide too much cohesion, opposite to the IP case. Clearly, many-body interactions must also be invoked to describe the phase-behavior correctly, as suggested by other authors Gast83 ; Meij94 ; Odij97 ; Fuch00 ; Sear01 .

In conclusion, we have shown by computer simulations that a mixture of polymers and non-adsorbing HS colloids shows extensive immiscibility in the protein limit, where the polymer-colloid size-ratio $`q1`$. For IP the phase-behavior is dictated by a universal binodal in the semi-dilute regime. For NIP, the colloid packing fraction tends to zero for increasing polymer length. In contrast to the better studied colloid limit, pair interactions are not sufficient to rationalize the phase behavior. We hope that future experiments on HS colloids with non-adsorbing polymer will test these predictions. Future work might include extensions to non-spherical particles, poor solvents, and adsorbing systems.

###### Acknowledgements.

A.A.L. acknowledges the Isaac Newton Trust for financial support. E.J.M. acknowledges the Royal Netherlands Academy of Art and Sciences for financial support. We acknowledge support from the Stichting Nationale Computerfaciliteiten (NCF) and the Nederlandse Organisatie voor Wetenschappelijk Onderzoek (NWO) for the use of supercomputer facilities. We thank M. Fuchs, R. Sear and R. Tuinier for helpful discussions.