The behaviour of sub-micrometre particles in a centrifugal field

233 Powder Technology, 12 (1975) 233-238 @ Elsevier Sequoia .%A., Lausanne - Printed in The Netherlands

The Behaviour of Sub-Micrometre

Particles in a Centrifugal Field

M.J. GROVES and H.S. YALABIK Pharmacy

Department,

Chelsea

College

(University

of London).

Manresa

Rd., London

S.W.3

(Gt. Britain)

(Received April 3, 1975)

SUMMARY

A centrifugal photosedimentometer using a continuous helium-neon laser light source has been employed for the investigation of the behaviour of the fat particles in a commercial intravenous emulsion system under centrifugal conditions. When the particles were dispersed in water or dilute saline solutions the optical density of the centrifuged suspension failed to return to base-line conditions, indicating that t.he smaller particles present remained suspended. The use of data-logging equipment enabled the point at which centrifugal and diffusional forces were balanced to be determined with precision, allowing a calculation of the “apparent limiting relative mass”. For the emulsion system under investigation this showed a sharp decrease at around 0.15 Al sodium chloride solution, equivalent to physiological saiine, followed by a rapid increase and a further decline as the concentration of the sodium chloride was increased. This may be explained by the effect of the electrolyte on the hydration of the particles, which affects the overall density of the particles, although for calculation purposes the density is assumed to be that of the oil phase. A complete particle size distribution of the system may only be obtained when base-line conditions are achieved, either by increasing the centrifugal field or, in this case, by increasing the electrolyte. However, at the higher concentrations of sodium chloride different size distributions were obtained according to the particular concentration employed, and also according to the analytical radius used on the centrifuge. The first effect may be adequately explained by the suppression of the hydrated layer around the particles, and by as-

signing arbitrary values for this thickness of this layer the distributions of size could be brought into close agreement with those obtained by electron microscopy. The effect of the different analytical radii is due to the different centrifugal field at each point, since the smaller particles in a system are more readily affected by the electroviscous resistance. A simple extrapolation technique is described which enables t.he distribution under zero gravitational field to be estimated, and it is this distribution which correlates with that obtained by other methods. The results are discussed in relation to the use of this low-speed centrifuge for the particle size analysis of finely divided particulate materials, irrespective of their densities.

INTRODUCTION During an investigation of the formation of emulsions by the self-emulsifying action of

solutions of surfactanta in oil [l - 51, it became desirable to measure the stability of the systems formed under controlled conditions, manifested as changes in the particle size distribution. However, a preliminary investigation (Yalabik, unpr.blished) suggested that the emulsion particles formed by these systems under some conditions had mean diameters well below a micrometre. The problems associated with the size ahalysis of such finely divided material were discussed by Groves and Freshwater [S] and attention was turned to a prototype centrifugal photosedimentometer using a continuous helium-neon laser light source [7] _ Since the stability of these experimental materials was an unknown entity, a

commercially available preformed emulsion system was taken for the initial investigation in order to determine the suitability of this equipment. This product, “Intralipid”, was claimed to be an example of an emulsion system containing sub-micrometre diameter oil particles which were relatively stable over a pericd of time under optimum storage conditions [ 81. _Anomalous results were obtained according to the conditions of the experiment, the system either failing to reach base-line values of optical density during centrifugation, or different particle size distributions being obtained according to the analytical point along the radius of the centrifuge or to the concentration of electrolyte used to disperse the emulsion. The present communication is a continuation of some work described elsewhere [ 91 in which it was pointed out that the apparent density of the oil phase and the viscous drag around the particles are both assumed to be constant when applying Stokes’ equation, whereas electrolyte will affect the hydrated layer around the particles which influences both these factors. Continued investigation of these anomalies has enabled us to demonstrate how this low-speed centrifugal photosedimentometer may be used for other finely divided particulate materials, not necessarily emulsions.

aseptic technique with sterile disposable hypodermic syringes and needles. Since most of the present investigation was carried out using 0.5 ml volumes, the majority of the experiments were carried out on the contents of a single bottle, but replicate results obtained on samples taken from other containers gave substantially similar results. Dilutions were prepared by adding 0.5 ml “Intralipid 10%” to 450 ml of the appropriate diluent in a measuring cylinder and mixing by repeated gentle inversion. Analyses were initiated within a few minutes of preparing the dispersion in order to avoid the possibility of changes being brought about in the primary particles of the system by microbial contamination or temperature fluctuation. The centrifuge runs at a temperature slightly above ambient and all measurements were related to isothermal conditions of 25°C. The density of the oil phase was assumed to be that of soyabean oil, 0.92 [lo], although this must be an approximation since the oil is fractionated and there is no means of allowing for the lecithin which may be dissolved in the oil phase proper, or exist at the oil-water interface. Viscosities and densities of the sodium chloride solutions were determined at 25” as required. The presence of the glycerol was ignored.

Apparatus EXPERIMENTAL

Materials The emulsion system investigated was “Intralipid 10%” (Vitrum A-G., Stockholm, supplied by Paines and Byrne Ltd., Greenford, Middlesex, through KabiVitrum Ltd., Ealing, London, W-5) batch no. 191306. Water, glass distilled; sodium chloride, AnalaR (Hopkin and Williams Ltd). “Intralipid” is a sterile emulsion used for parenteral nutrition by intravenous administration, and is prepared to the following formula: fractionated soyabean oil 10.0 g fractionated egg-yolk phosphatides (lecithin) 1.2 g glycerol 2.5 g water for injections to 100 ml. The product is supplied in 500 ml rubbercapped bcttles and was stored between 2 and 5°C. Samples for analysis were withdrawn using

The centrifugal photosedimentometer using a continuous helium-neon laser has been described in detail elsewhere [ 71, and consists of a rotating hollow transparent disc holding approximately 600 mi. However, the disc is supported in a metal flywheel so that only a limited area of the transparent surface may be utilised. For this reason the analytical point along the disc radius, i.e. the point at which the photodetector was situated underneath the disc, could only be varied between 95 and 110 mm according to the requirements of the experiment, and a volume of 450 ml liquid was sufficient to cover the innermost analytical point. Since the emulsion particles have a density below that of the liquid continuum, it was necessary to employ the centrifuge in the homogeneous mode [ 61, which requires the use of computer facilities to calculate the results according to the method of Kamack [ 11 J_ The optical density of the sedimenting centrifuge

23.5 density of the suspension failed to return to the base-line conditions, i.e. optically clear dispersant, and particle size distributions could not be obtained. However, with the higher electrolyte concentrations and the same rotational velocity, base-line conditions could be obtained, suggesting that complete sedimentation of the system had been achieved. The apparent particle size distributions obtained were different, according to either the electrolyte concentration or the point along the centrifuge disc radius at which the optical density was determined.

Fig. 1. Transmission electron photomicrograph of “Intralipid 10%” (uranyl acetate stain). The scale is 1.0 pm. The grouping of the smaller particles is an artefact caused by migration dgring the drying process

disc contents was recorded as a function of tin]le with the data-logging device described by Tempel and Groves [12]. Electron microscopy dilutions of the “Intralipid 10%” were prepared by adding 1 ml of the emulsion to an equal volume of water, followed by 4 ml of 2% aqueous m-any1 acetate solution. The mixture was agitated gently, sprayed onto carbon grids and allowed to dry before examination in an A-E.1 EMGB electron microscope. Photographs were taken and the particle size distribution determined by a counting procedure, a total of 6504 particles being classified in 16 fields after calibration using latex particles of known size [13]. A typical field is shown in Fig. 1 and confirms the presence of substantial quantities of sub-micrometre diameter particles.

(a) Limiting optical densities The failure of some of the centrifuging suspensions to return to base-line conditions suggested that the centrifugal force experienced at the sampling point was insufficient to allow smaller particles present in a system to move away. On this assumption it seemed possible that the limiting particle size could be calculated for which diffusional and centrifugal forces were exactly balanced, particles smaller than this limit remaining suspended. By recording the centrifugation time with the data-logging system and printing out the voltage--time output on a teletype machine, the point at which the sedimentation process stopped could be estimated to within 2 1s. This allowed calculation of the limiting particle diameter by the use of the centrifugal analogue of Stokes’ equation, assuming that the particles consisted of oil alone: dSt = Stokes’ =

diameter

(pm)

1s . 1067-j In (R,/R,) __~-L (Pl - P2wt

“’ I

(1)

where t (s) is the time for a particle of diameter dSt and density p1 to move from radius R2 to RI through a liquid continuum of density p2 and viscosity 77,in a centrifuge rotating with an angular velocity of o radians s-l_ Similarly, the apparent limiting relative mass M is calculated [14] as:

M = RT ln (RdRd

D(p,-

Lkh2t

(2)

RESULTS

where R is the Gas Constant, T absolute temperature and D is the diffusion coefficient given by

When dispersed in either water or the more dilute of the electrolyte solutions the optical

D = 3N7L,,

236

Fig. 2. Apparent limiting mass anti particle diameter of Intralipid particles at 3000 r.p.m. and a radiusof

11.0 cm.

where AT is the Avogadro Number. Both limiting mass and diameter are shown in Fig. 2 for Intralipid particles dispersed in dilute saline. Similar results were observed for some polyethylene latices intended for use as calibration materials of the inst.rument, and this clearly imposes a practical lower limitation of the use of the instrument under certain circumstances, i.e. insufficient centrifugal force

r141. Under some conditions of either sodium chloride concentration or the analytical radius selected, the optical density eventually reached the base-line conditions, suggesting complete sedimentation and allowing an estimation of the complete particle size distribution. However, different distributions were obtained according to the conditions of the experiment. That different results might be obtained at

different radii of analysis was recognised as long ago as 1923 by Svedberg [14] since the influence of particle shape and the effect of electroviscous drag depend on the sedimentation rate. The percentage P, of particles greater than a selected size grows with decreasing sedimentation rate since the frictional effect on the particles becomes smaller. The frictional factor B,. (=P,, = ,/P,) includes all the retarding influences on the particle but is effectively the particle shape factor, expressing the extent to which the particle deviates from the ideal behaviour of spherical particles. Svedberg [ 143 suggested that the true size distribution could be obtained by repeating the experiment under different conditions, and extrapolating to zero concentration. Jelinek [ 151 pointed out that the value of P, = O could be determined by comparing values of P determined with a series of decreasing rates but with the same sedimentation constant, s, the term [ln(Rr/Rz)/ a2t)] common to both equations (1) and (2). Since the velocity of sedimentation at. the point of analysis (RI) is proportional to w2, Jelinek considered it sufficient to plot o_?’ against P,, and obtain the value of P,, = O by estrapolation to zero. This suggestion appeared feasible since our instrument could be used at different speeds of rotation, but lacked conviction since it does involve extrapolation of the squares of large numbers to zero and this must inevitably be imprecise. Because the analytical radius on the centrifuge used here czn be changed with ease, extrapolations were attempted using the values of P, for different values of ln(R1/Ra). Results for one of the systems examined are shown in Fig. 3, and this appears to be more realistic TABLE

1

Particle size distributions of “Intralipid 10%” obtained on samples dispersed in different concentrations of sodium chloride, extrapolated to conditions of zero centrifugal field. The distributions could be approximated to log-normal, here reported as the mean diameter (at 50%) and the width of the distribution as given by the slope (standard deviation) 20

’ I

L

. . 2 0.99

0.97

0.95

0.93

0.91

Dispersant (M NaCI)

Mean Stokes’ diameter dst(50) (km)

Slope 0

0.35 0.43 0.51 0.59

0.175 0.225 0.285 0.320

1.13 1.22 1.14 1.19

b/R,

Fig. 3. Extrapolation of data to zero sedimentation rate (log Rl/RZ = 0). “Intralipid 10%" dispersed in 0.59 &I sodium chloride at 0.05 (o), 0.07 (@), 0.09 (“1 and 0.10 (A) pm diameters, centrifuge rotating at 3000 r.p.m.

“3i TABLE

2

Particle rection density

size distributions shown in Table for a hydrated layer, of thickness 1.00

1 after cord(r) and

Dispersant (Al NaCl)

d(r) (nm)

dst(5o) (pm)

o

0.35 0.43 0.51 0.59

150 125 80 75

0.45 0.45 O.-l5 0.46

1.14 1.29 1.16 1.21

0.46

1.42

Surface-volume diameter by electron microscope

as an extrapolation technique. &coidingly emulsion systems which came to the base-line were repeated, analysis of optical density being carried out at a number of different radii under otherwise identical conditions. The results were extrapolated to conditions of zero sedimentation rate and are shown in Table 1. The dependence of the results upon the concentration of the sodium chloride is clearly due to the use of swamping electrolyte which is progressively decreasing the electrostatic viscous drag on the emulsion particles. It was suggested [9] that this may be due to a suppression of the hydrated layer which may exist around the particles. This hydrated layer would not only increase the drag around the particles, affecting the viscosity term of eqn. (l), but would also affect the apparent density of the oil phase. Both these factors are ignored when calculating the particlc diameter corresponding to a given time of sedimentation. It was therefore of interest to attempt to correct for this postulated hydrated layer around each particle, of thickness d(r). Values for d(r) were calculated, the hydrated layer being assumed to have a density of 1.00, i.e. slightly above that of water at the temperature of 25°C. The recalculated results are shown in Table 2. DISCUSSION

The observation of the fact that under some conditions the optical densit;r-time curve occasionally fails to return to base-line conditions must be regarded as a limitation to the method of size analysis employed here. Svedberg [14] overcame this difficulty by increasing the spzsd of his centrifuges, leading to the development of the ultra-centrifuge instruments used for the determination of the molecular weight of molecular

dispersions. As discussed elsewhere [IS], the size analyst is not strictly concerned with this type of material but rather with particles which have clearly defined boundaries. The results shown in Fig. 2 can be interpreted qualitat.ively on the assumption that the particles constituting the sysiem ucder investigation have a definable solid-liquid interface, clearly shown on the electron photomicrograph of Fig. 1, to which is adhering a layer of material with properties different from either the fat disperse phase 0; the aqueous continuum. The identity of this layer requires further investigaticn, but it should be pointed out that the properties are compatible with the known characteristics of the phosphatide egg lecithin used as a stabilising agent in the emulsion. For example, lecithin is soluble in oil but, being polar, is most likely to be situated at the oil-water interface. In addition, lecithin swells in water but dissolves in sodium chloride solutions [lo]. This would account for the results shown in Fig. 2 and Table 2, since the effect of the increasing level of electrolyte is to progressively decrease the viscous drag. If this is due to an adhering layer of hydrated lecithin, this would not. only have an effect on the apparent limiting particle diameter (Fig. 2) but also affect the particle density and hence the apparent limiting relative mass of the particle. The depression of the thickness of this layer at a concentration of electrolyte ccrresponding to physiological saline shown indirectiy in Fig. 2 is therefore intrinsicaliy interesting and may be related to the application of this particular emulsion system under physiological conditions. An alternative explanation for the observed effect of electrolyte on the particle size distribution of the emulsion particles may be that of flocculation or aggregation. Experimentally, however, flocculation of the diluted emulsions was not observed prior to carrying out the analysis. In addition, the fact that the distributions can be normalised (Table 2) to a single distribution, itself in close agreement with the result obtained by electron microscc~y, would also tend to militate against this explanation. Although the present investigation has been carried out with an emulsion system with particles of a density below that of the liquid continuum, it was pointed out that the homogeneous mode of operation is not confined

23s to this type of system [12]. The calculation of results is less straightforward and requires computer treatment, but this is less of a problem when the instrument is combined with data-logging facilities. For this reason the lowspeed centrifugal technique can be applied to any material irrespective of the solid density provided that the material is small enough to move under streamline conditions and large enough to move under the centrifugal field, and can be detected by the detection system attached to the apparatus. Using the detection system at a single radius does not provide a true size distribution unless the material under examination has a narrow size range but, as noted earlier [17], does enable the instrument to be employed for comparative purposes. The relationship between the time of sedimentation and the optical density is a characteristic feature of the sedimenting material and can be used for quality control purposes [9]. Nevertheless, the arbitrary assignment of a value of unity for the extinction coefficient in this present work for particles of around and below the wavelength of the illuminating radiation would be expected to produce aberrant results, and it is surprising that the experimental results from the centrifuge could be normalised to agree closely with those results obtained by electron microscopy. The nature of the intense monochromatic and coherent light from the laser source has been suggested [ 71 as one possible reason for this deviation from expectation, and this is an area which clearly requires further investigation. However, the method of extrapolation suggested here for determining the size distribution under conditions of zero sedimentation rate would appear to be a valid means of obtaining the correct size distribution of a sedimenting suspension, requiring analysis of the optical density-time trace at at least two different radii. This may be achieved, as in the present work, by carrying out replicate experiments. Ideally the analyses should be carried out simultaneously on the same suspension_ This suggests a direction Ear improvement of the present instrument and is the subject of patent applications 7476/75 and 7703/75. ACKNOWLEDGEMENTS

M.J.G. wishes to acknowledge the grant received from the Science Research Council for

building the present centrifuge, and the kindness of Messrs. Paines and Byrne Ltd. for the generous provision of samples of “Intralipid 10%“. H.S.Y. is in receipt of a Turkish Government Scholarship and this work forms part of a thesis to be submitted to the University of London. We are both grateful to Mr. M. Wineberg for assistance with the electron microscopy. REFERENCES 1 M.S. Groves. R.M.A. Mustafa and J.E. Carless, Some properties of a water-soluble phosphated nonylphenolethoxylate, J. Pharm. Pharmacol., 24 (19i2) 104P. 2 M.J. Groves, R.M.A. Mustafa and J.E. Carless, Some properties of an oilsoluble fatty alcohol ethoxylate, J. Pharm. Pharmacol., 25 (1973) 736. 3 M.J. Groves, R.M.A. Mustafa and J.E. Carless, Phase studies of mixed phosphated surfactants, n-hexane and water, J. Pharm. Pharmacol., 26 (1974) 616. 4 M.J. Groves, R.M.A. Mustafa and J.E. Carless, A note on the interaction between two phosphated surfactants, J. Pharm. Pharmacol., 26 (19i4) 624. 5 M.J. Groves and R.M.A. Mustafa, Measurement of the “spontaneity” of self-emulsifiable oil, J. Pharm. Pharmacol., 26 (1974) 671. 6 M.J. Groves and D.C. Freshwater, Particle size analysis of emulsion systems, J. Pharm. Sci., 57 (1968) 1273. 7 M.J. Groves, H.S. Yalabik and J.A. Tempel, Development stlldies of a centrifugal photosedimentometer using laser light, Powder Technol., 11 (1975) 245. 8 M.J. Groves, Parenteral Products, Heinemann, London, 1973. 9 M.J. Groves and H.S. Yalabik, Size analysis of sub-micron particles by centrifugal photosedimentometer, J. Pharm. Pharmacol., 26 (1974) 77P. 10 Merck Index, Merck, Rahway, N-J., 7th edn., 1960. 11 H.H.J. Kamack, Particle size determination by centrifugal pipe and sedimentation, Anal. Chem., 23 (1951) 844. 12 J.A. Tempel and M.J. Groves, A data-logging system for a centrifugal photosedimentometer, Powder Technol., 9 (1974) 147. Electron 13 E.B. Bradford and J.W. Vanderhoff, microscopy of monodisperse latexes, J. Appl. Phys., 26 (1955) 864. 14 T. Svedberg and K.O. Pedersen, The Ultracentrifuge, Oxford Univ. Press, London, 1940. Wiley, 15 Z.K. Jelinek. Particle Size Analysis, _ _. New York, 1974: 16 M. J. Groves, Particle size analysis, past, present and future, Analyst, 99 (1974) 959. 17 M.J. Groves, B.H. Kaye and B. Scarlet& The size analysis of sub-sieve powders using a centrifugal photosedimentometer; Br. Chem. Eng., 9 (1964) 742.