Instrument

The instrument is similar to a liquid chromatograph: solvent source, pump, injection valve and column. Optical detection is spectrometry, refractometry, tur-bidimetry or light scattering.

Conditions to be satisfied are accurate flow rate control and high detection sensitivity. The first condition is due to the narrow elution domain and the second to the small injected volume of a very dilute sample.

Optical detection is more difficult since particle dimensions are in the range of the wavelength involved, X. With a UV spectrometer, sensitivity is so high that as few as 25 particles in the detector cell give a noticeable signal. Sensitivity varies with wavelength, sample type and with sample size since the signal depends on absorption and scattering, which vary with sample diameter to the power a. The result is that quantitative interpretation needs preliminary signal calibration for each species.

A general formula in liquid chromatography for the instantaneous detector response H at each elution volume V is:

where Kl is the extinction coefficient for particles of diameter Di and a = 2 in the Mie scattering regime for a turbidity detector. In the Rayleigh regime, valid for X/Dp < 0.3, a = 6 for turbidity and 3 for refrac-tometry. Hamielec observed a factor 6 in tur-bidimetry and a linear increase of signal with particle size in refractometry: there was a three times increase between 100 and 500 nm for polystyrene (PS) collo ids. For the same given weight of PS colloids, the UV signal is three times higher for 354 nm than for 88 nm particles. This also explains the effect of wavelength on signal shape and intensity. The peak separation is better with a refractometer, which may appear less sensitive than the turbidity detector. Generally, X = 254 nm is chosen, but differences in relative sensitivity at X = 380, 254 and 220 nm for PS colloids of 38 nm and 176 nm diameter have been found; this variation for larger PS colloids, 0.84 |im and 4 |im diameter, was also observed. A continuous and moderate increase in specific extinction coefficient at 254 nm and a higher and nonmonotonous increase at 220 nm have been measured. A low signal obtained for small particles may be enhanced by working at lower wavelength so that the apparent separation is increased. Difference in absorption may allow the apparent separation of PS/PVC. Intermediate values of a may correspond to a combination of a and K(V). The value a = 1 holds for refrac-tometry and spectrophotometry of polymers in SEC, so that the range 1-6 must be considered for a general data treatment.

Assuming the formulas for number and weight average diameters:

and the simplified general relationship H proportional to NDn, (K and a are independent of V), then:

and:

Figure 3 Effect of n value on average diameters: number (circles) and weight (squares) for standard PS latex 106 nm. (Reproduced with permission from Revillon A and Guilland JF (1990) Software for data acquisition and treatment: hydrodynamic chromatography (HDC) analysis. Journal of Applied Polymer Science: Applied Polymer Symposium 45: 125-137.

For all the investigated PS latexes, average diameters of the number and weight greatly decrease when n is increased (Figure 3). The best agreement with values measured with transmission electron microscopy is obtained for n = 3-4, which is in agreement with theory since their diameters are in the range of the wavelength of detection (254 nm). Except for n = 2, the polydispersity index P = Dw/Dn is not affected by n, and is the same for all samples. Nevertheless, these distributions do not correspond to true ones. The high polydispersity values compared to those determined by transmission electron microscopy (1.02) clearly show the necessity of a band-broadening correction.

A number of workers have built apparatus for HDC by putting together high performance liquid

Figure 3 Effect of n value on average diameters: number (circles) and weight (squares) for standard PS latex 106 nm. (Reproduced with permission from Revillon A and Guilland JF (1990) Software for data acquisition and treatment: hydrodynamic chromatography (HDC) analysis. Journal of Applied Polymer Science: Applied Polymer Symposium 45: 125-137.

chromatography components. Matec Applied Scientific (Hopkington, MA, USA) currently markets a high resolution instrument CHDF-1100 with UV detection. Separation is operated in a narrow capillary (2.5 |imx10m) enclosed in a cartridge. Careful choice of eluent components avoids blocking the column for long-term use. Control of temperature allows accurate flow rate. The signal resulting from absorption and scattering is treated with the help of Mie theory. A mixture of small size latexes is well separated in about 10 min. Length of column may be increased, and diameter may be changed to cover different particle sizes. The apparatus may be used in process control.

Improvements to enhance the accuracy of RF and PSD, resolution, sensitivity of detection and rapidity of analysis include the following:

1. The injection valve may be replaced by a 12-port valve and a delay column, with two injection loops (Valco Europe, Vici, Switzerland). This allows the injection of two (identical or different) samples on one or two consecutive columns. One of the advantages is the accurate determination of RF by the help of an internal standard, avoiding sample mixing or reaction, and peak interference. Moreover, the comparison of the signals of the sample passing through active and inert columns allows determination of percentage particle recovery.

2. An additional fine metering valve (Vernier Handle, 18/21Y, Hoke, Creskill, NJ, USA) may be placed before the packed column to divide the main flow into two parts, one entering directly into the column and the other through the injection valve. This reduces peak asymmetry from 5 to 1.5 and increases plate number by a factor of two. The optimum effect is obtained when the flow is divided in two equal parts; the main flow is less diluted and the side flow entering the column, close to the injection point, prevents broadening of the liquid jet containing the sample.

3. Effort has been put into the reduction of void volume, by direct column injection, design of a detector with reduced connection and cell volumes, on-column detection, improved sensitivity and the use of micro-equipment similar to that used in liquid chromatography and SEC.

4. Recycling, as used in SEC, may improve resolution or RF determination without changing or increasing the RF value.

5. A diode array UV-visible spectrometer allows simultaneous determination of the signal at several wavelengths, which means either signal enhancement or decrease (masking of a component).

6. Laser light scattering is of high sensitivity and may also be used, combining photon correlation spec-troscopy with integration of the signal, at one angle, during a low counting time (a few seconds). It may give directly both the instantaneous size and concentration of the sample. Low angle laser light scattering is the detector used to determine the molecular weight of polyacrylamide. Variants of this include the evaporative light-scattering detection and condensation nucleation light-scattering detection. To overcome a calibration problem and use of standards, online viscosity has been proposed as an alternative, but this method awaits the development of improved pressure transducers. Fluorometry has been suggested as a high sensitivity detector for tagged polymers. The approach in the author's laboratory is discontinuous measurements of sample size on eluted fractions by transmission electron microscopy, photo correlation spectroscopy and sedimentometry.

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