Columns

Capillary columns for capillary HDC are used for larger particles, generally in the range from 1 to 60 | m. New capillary columns allow separation in the sub-micron range. Packed columns are effective for smaller particles, with diameters of 30-1000 nm. Since its introduction, HDC studies have been mainly devoted to separations with packed columns.

Packed columns Columns may be slurry- or dry-packed in the same way as for liquid or gas chromatography. Packings used include ion exchange resins, cross-linked poly(styrene-divinylbenzene), nonporous glass and silica gel among others. As an example, analysis has been performed with a slurry-packed column (cross-linked PS spheres of uniform diameter 20 | m: 50 cm length, 0.78 cm internal diameter).

The main determining parameter is packing particle size. A general trend in liquid chromatography is an increase in plate number when particle diameter decreases (N is inversely proportional to the square of particle diameter). It has been shown in the initial work on HDC that resolution is increased by using packings of small monodisperse spheres; Figure 7 shows this result. Moreover, resolution is also decreased when size increases (more than a factor of 2 between 20 and 40 |im). Whatever the size of the packing material, the elution region is very limited, defined by a maximum ratio RF of 1.15 in most papers. By using 2 |im nonporous silica gel packing and tetrahydrofuran as eluent, a value of 1.21 is obtained for RF, yet the chromatogram contains only five peaks: four for PS and one for toluene. RF values as high as 1.3 have been obtained.

Figure 7 RF versus packing diameter for five latexes. Open circles, 1000 nm; open squares, 800 nm; open triangles, 600 nm; filled circles, 400 nm; filled squares, 200 nm. (Adapted with permission from results of Small H (1974) Journal of Colloid and Interface Science 48: 147-161.

Large (125-180 |im) but porous particles are still used (at low flow rate: 2 cm min_1, in the presence of 0.01 mmol L"1 NaCl) to take advantage of the pores as capillaries, with an RF value of 1.16 or up to 1.39 in the measurement range 240-1230 nm. The equivalent capillary radius R is 2.8 |im, calculated according to the formula:

1-J2-Rf

By combining HDC and SEC, and using porous particles, the RF may be increased by a factor 1.1-1.2 to a value of 2 or 2.2. The peak broadening decreases or increases, so that resolution is inferior to that obtained on nonporous packing. Moreover, small size porous packings in SEC (2 | m Hypersil or polymeric 3 | m packing with 5 or 15 nm pore radius) allows a high plate number in a 0.45 cm column to be obtained, with an expected peak number of 66. Considering a resolution of unity, constant with V, the peak capacity is:

Figure 7 RF versus packing diameter for five latexes. Open circles, 1000 nm; open squares, 800 nm; open triangles, 600 nm; filled circles, 400 nm; filled squares, 200 nm. (Adapted with permission from results of Small H (1974) Journal of Colloid and Interface Science 48: 147-161.

This leads to p = 37 in this case, and the number obtained is approximately 15, which is an excellent value. The authors of this work also used nonporous monodisperse particles, leading to the theoretical plate height minimum value (5 | m), with no dependence of flow rate in the range 3-7.5 cmmin-1. N is 42 000 for a 15 cm length column, with 1.4-2.7 | m silica particles so that a mixture of styrene polymers in the range of molecular weight 104-107 are well separated in 6 min, but shear degradation occurs for the higher molecular weight polymers.

Capillary columns With open capillary tubular columns, the resolution is poorer than with packed columns, but the Rf may be as high as 1.45, so that the peak capacity may be the same as that obtained with a packed column.

The main parameters are length and diameter. A systematic study of the length has been made with stainless-steel columns of 30, 60 and 120 m, and internal diameters of 0.25 and 0.5 mm. The 30 m column gave results of insufficient quality, so most of the work was done with the 60 m columns and finally optimized with 120 m length columns. By going from 60 to 120 m, the theoretical plate height was found to be unchanged and N increased from 380 to 800, for a 4 |im sample. A way to illustrate this increase in resolution is to consider the calibration as represented in SEC. A lesser slope (Figure 1B) allows a better separation.

Table 2 Characteristics of capillary hydrodynamic chromatography systems

Length

Internal diameter

N

Maximum

(m)

(mm)

(m)

RF max

88-201

250-500

?

1.3

50

180-450

25

1.5

60-200

250-500

10

1.55

15

100

25-200

>1.2

0.45-7

1000

6

1.5

0.15-0.2

1

250

1.1

0.7-3.3

1.2-10

105

1.05

91 -168

250-500

16-100

1.4

12

15 000

?

1.15

30-120

250-500

16-250

1.5

2

4

2000

>1.15

2

6.5

?

1.42

5

7

600

1.63

2.5

10

580

> 1.46

Reproduced with permission from Revillon A etal. (1991) Journal of Applied Polymer Science: Applied Polymer Symposium 48: 243-257.

Reproduced with permission from Revillon A etal. (1991) Journal of Applied Polymer Science: Applied Polymer Symposium 48: 243-257.

The second characteristic of the column is its diameter. Table 2 summarizes conditions of separation and typical results (N, RF). It can be seen that capillary diameter varies considerably, from 1 to 1000 (or even 15 000) |im. Most of the initial work has been done with 250-500 | m internal diameter column. The choice of tube diameter corresponds to the different ranges of sizes to be separated. The limit in Rf shows that over a certain sample size, no separation occurs. This limit of size may be related to the ratio of the average radius of the sample, rp, to the tube radius, R. A third order law, relating rp to R, may have a reasonable approximation in a linear one in agreement with Small's observations:

with f =— 7 and k = 0.1 (R and rp in |im). A column of diameter 500 | m may have a medium range of separation of 18 |im instead of 5 |im for the 250 |im one. Taking into account the published results, the ratio of R to rp is about 100, and for a given diameter of column, the usable rp/R range varies roughly from 10-3 to 10-1. The interest in large diameter columns is because of the decrease of rate of shear, y, but the resolution is higher for narrow tubes.

To reduce extra-column band broadening, optimization of the injection-detection system has been attained with 50-100 |m capillaries judging by results obtained in capillary electrophoresis. With microcolumns, efficient separation has been obtained for PS samples with molecular weight of 103-106Da. The chromatogram was similar to that of SEC, but with a limited RF of 1.1 (instead of 2 in SEC) and a low number of plates (N = 50). Work by Tijssen shows a very high number of plates (N = 105 m-1), but a more limited RF, of 1.05. The increase in N was not accompanied by an increase in Rf, so that the peak capacity remained low (less than 10). More recent work indicates higher values of RF: 1.63 and rather good resolution between latex samples. Even with 4000 plates, the peak capacity is only about 7. Table 3 summarizes the conditions and results observed with capillary and packed column HDC.

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