micro lc on cellulose triacetate (cta): influence of particle diameter and column temperature

Micro LC on Cellulose Triacetate (CTA) : Influence of Particle Diameter and Column Temperature*

Jian Chen, Dirk Steenackers, and Pat Sandra Department of Organic Chemistry, University of Gent, Krijgslaan 281 (S4), B-9000 Gent,

Belgium

Abstract. Fused silica columns (320 p m i.d.) were packed with 10 pm, 15-25 pm, and 25-40 p m microcrystalline cellulose triacetate (CTA) and the influence of the particle diameter on column efficiency was investigated. The dependence of retention, selectivity, resolution, and analysis time on column temperature was studied for the separation of several racemates on microcolumns packed with 15-25 p m CTA. 0 1995 John Wiley & Sons, Inc.

Key words: micro LC, cellulose tnacetate (CTA), separation of enantiomers, particle diameter, column temperature

INTRODUCTION Swollen microcrystalline cellulose triac-

etate (CTA) has been widely used as enantiose- lective stationary phase in liquid chromatography [l-191. High enantioselectivity for several classes of racemates and high loadability, allow- ing an easy upscale from analytical to prepara- tive work, are the main characteristics of this material [12,14,17-191. The main disadvantage is the high dispersion for most types of racemates, resulting in low column efficiency and long analysis times.

Band broadening and retention mecha- nisms have been studied [e.g., 2,5,8,121, the most complete and systematic study performed by A.M. Rizzi [20-221. The main contribution to the HETP arises from slow mass transfer kinetics on the swollen microcrystalline CTA surface [20]. Two types of adsorption sites have been elucidated, namely the “quick” and the “slow”- type which differ in adsorption/desorption rates. The “slow”-type is predominant in chiral recognition [20,21].

In conventional LC on silica or polymeric particles, the most direct way to improve column efficiency is the use of smaller particles. In LC on CTA, separations are normally performed on 15-25 or 25-40 p m particles as those are the ones which are commercially

available. Although some authors reported on LC enantioseparations on 5-10 p m CTA [4,13,15], the influence of particle diameter on column efficiency has, to our knowledge, not been investigated yet. Increasing the column temperature is another way to accelerate the mass transfer thus reducing the HETP. In this study, the influence of temperature on retention, efficiency, and selectivity has been differ- entiated.

In the framework of this temperature study, to the well-known features of micro FSOT columns such as low solvent consumption, in- creased mass sensitivity, etc., the much lower thermal capacities and flow rates compared to conventional columns, resulting in fast thermal equilibration, should be added.

In this paper, we report on our observa- tions concerning the influence of particle diameter and column temperature on several enantioseparations.

EXPERIMENTAL, Instrumentation. The micro LC system con-

sisted of a Varian 5000 pump with a T-piece flow-split device installed before the injection valve. A LC column 10 cm L X 4.6 mm i.d. served as pressure restrictor. Injection was performed via a Valco C14W valve with 60 nL

* Presented at the 15th International Symposium on Capillary Chromatography, Riva del Garda, Italy, 1993.

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259 CCC 1040-7685/95/030259-05

260 J. Microcolumn Separations, Vol. I , No. 3, 1995

internal loop, and detection via a Varian 2050 UV detector modified with a home made flow- cell (pathlength 320 pm) working at 210 nm. The micropacked column was directly con- nected to the injector valve through a Nylon 66 membrane filter (pore size 0.45 pm) installed in the valve and was placed in a Varian 3700 GC oven. The injector was kept at room temperature. Unless otherwise stated the mobile phase was 96:4 ethano1:water.

Column preparation and materials. Micro- crystalline cellulose triacetate with particle diameters of 10, 15-25, and 25-40 p m (Merck, Darmstadt, Germany) was swollen by boiling in absolute ethanol during 2, 4, and 7 hours, respectively. The swollen CTA was slurry-packed into 320 p m i.d. fused silica tubing at a maximum pressure of 20 bar. 2-Phenylcyclo- hexanone (PCH), 4-phenyl-1,3-dioxane (PD), 1-(9-anthryl)-2,2,2-trifluoroethanol (TFAE), and 1,3,5-tert.butylbenzene (TTBB) were obtained from Aldrich (Bornem, Belgium). Methyl-5-(p- methyl-benzyloxy)-3,3-pentadienoate (MMBP) is a synthetic product from our department.

RESULTS AND DISCUSSION Influence of particle diameter on plate height.

In the enantioseparation of racemates on swollen microcrystalline CTA, dispersion is controlled by a “slow”-type adsorption; this is contrary to silica and polymeric particles (“quick”-type) [20]. For the latter materials, the HETP value decreases linearly with decreasing particle diameter. Due to the “slow”-type mechanism on CTA, the van Deemter plot as well as the dependence of HETP on particle diameter might be different.

To investigate the influence of particle diameter on column efficiency, three 25 cm L X 0.32 mm i.d. columns were packed with CTA particles of, respectively, 10, 15-25, and 25-40 pm, swollen under the same conditions. Micro- scopic evaluation indicated that the particles, after swelling, become larger with 20-40%. For 10 p m CTA, the mean diameter increases to about 12 pm; for 15-25 and 25-40 p m CTA the increase could not be precisely measured, since the particle size distribution is quite high, and was estimated to be in the order of 40%.

Figure 1 shows the H-u curves for 1,3,5- tert.buty1benzene (dead volume marker) and for the two 4-pheny1-l73-dioxane enantiomers on the three different columns at 25°C together with the values of the C term of the three plots. It can be deduced from the figure that the plate

J

3 3 - I

I1 C = 2.3 s g 2

1 -. . 111 C = 0.45 s 111 C = 0.45 s

0- 0.0 0.2 0.4 0.6 0.8 1.0

u (mrnlsec)

Figure 1. HETP plots for TTBB and PD enantiomers at 25°C. Columns: 25 cm L x 320 p m i.d., packed with, respectively, 10 p m ( A )) 15-25 p m (o), and 25-40 p m ( * ) CTA. I: I-phenyl- I,3-dioxane (last eluted enaniomer), 11: 4-phenyl- 1,3-dioxane (first eluted enaniomer), 111: 1,3,5- tert. butylbenzene.

height is little affected by the particle diameter of CTA. The HETP value for the first eluted enantiomer of 4-phenyl-1,3-dioxane is roughly 1.2 mm at 0.5 mm/s on all columns and thus independent on the particle diameter, whereas at 0.5 mm/s HETP values of 0.023 mm (reduced plate height 2.3) and of 0.010 (reduced plate height 2) have been measured for ODS particles of, respectively, 10 and 5 pm. More- over, the C terms in Figure l range from 0.45 to 4.5 s, while for ODS particles the C term is in the order of 0.15 s [23].

Obviously, on CTA the “slow” adsorption- desorption process is dominating band broadening. Guiochon et al. studied the band broadening mechanism on cellulose triacetate using Troger’s base as test compound [24]. Study of the influence of temperature and flow-rate on the band broadening and measuring of adsorption isotherms via frontal analysis [25] revealed that the contribution of the kinetics of the adsorption-desorption mechanism to the C term is more important than the contribution of mass-transfer kinetics by intraparticle diffu- sion. Our observation that the C term is independent of the particle diameter provides evi- dence for the findings of Guiochon et al. that the particle size dependent, mass transfer contribution to the C term is negligible compared to the adsorption-desorption contribution.

Also, after swelling and packing the non- rigid CTA particles, the differences in packing structure for the three different diameters may get smaller compared to the packing of rigid

J. Microcolumn Separations, Vol. 7, NO. 3, 1995 261

silica particles. It is important to mention that no substantial change in retention factors or in selectivity was observed for the different particle diameters.

Resolution as function of column temperature. The separations obtained for PCH, TFAE, PD, and MMBP at ambient temperature on a 22.5 cm L x 320 p m i.d. column packed with 15-25 p m CTA particles are shown in Figure 2. In order to evaluate the influence of temperature on resolution (R& the dependences of retention factor (k), column efficiency (H), and the separation or selectivity factor (a> on temperature were studied.

Retention factor. In Figure 3, the decrease of retention factor k, (second eluted enantiomer) of the four racemates under investiga- tion, as function of the temperature is shown. The reduction of k between 20 and 60°C ranges from 2 to 5 times.

Column eficiency. At ambient temperature, the HETP value for PCH and PD are much lower than for TFAE and MMBP as deduced from Figure 4, showing the dependence of the HETP values on temperature measured for k, of the racemates. According to ref. [21] the efficiencies can be considered as “high” for PCH and PD and as “low” for TFAE and MMBP. The plate height decreases fast upon increasing the temperature but the most significant reduction is observed for the “low efficient” solutes, TFAE and MMBP, with a 4 time increase in column efficiency between 20 and 60°C. The “slow”-type adsorption

C

II D

mm mm mrn

Figure 2. Enantioseparation of PCH, TFAE, PO, and MMBP at ambient temperature. Column: 22.5 em L X 320 p m i.d., 15-25 p m CTA. Flowrate: 1.6 p L / min. A: 2-phenylcyclo- hexanone, B: 1- (9-anthryl)-2,2,2-tnfluoroethanol, C: 4-phenyl-1,3-dioxane, D: methyC5-(p-methyl- benzyloxy)-3,3-pentadienoate.

- 0 Ju 20 30 40 SO 60 70

T (“C)

Figure 3. Retention factors of the last eluted enantiomers as function of the temperature. For conditions see Figure 2.

sites, which create the main contribution to dispersion of “low efficient” solutes, have a very pronounced temperature effect, resulting from a substantial increase of the apparent adsorption-desorption coefficients [24].

Selectivity factor. As expected, for all racemates with the exception of TFAE, the selectivity decreases with increasing temperature (Fig- ure 5). The slight increase for TFAE has been explained by the difference in the temperature dependence of enthalpy of adsorption for the two enantiomers at different bonding sites [21]. For all racemates investigated, selectivity factors remain high, even at temperatures of 60°C.

Resolution. In Figure 6, the resolution is plotted as function of temperature. For PD and MMBP, maximum resolution is obtained at 40”C, temperature at which the loss in selectivity is largely compensated by the improved efficiency. At higher temperatures, the selectivity

0.04 . I . I . I . I . I 20 30 40 50 60 70

T (“C)

Figure 4. HETP for the last eluted enantiomers as function of the temperature. For conditions see Figure 2.

262 J. Microcolumn Separations, Vol. 7, No. 3, 1995

I \ n

-b 1 , I . I . I . I . I . I

20 30 40 50 60 70

T ("C)

Figure 5. temperature. For conditions see Figure 2.

Selectivity factors as function of the

loss no longer can be compensated by the higher plate number at higher temperatures. For PCH no maximum is observed. On the other hand, the higher the temperature, the better is the separation for TFAE as both column efficiency and selectivity increase with temperature.

Analysis time. The high selectivity factors TFAE, MMBP, and PD, even at high temperatures, allow for their analysis to be performed at 50-60"C, with a 2 to 8 times reduced analysis time. Working at room temperature is neces- sary to obtain baseline separation only for PCH. As an example, the separation of 4-phenyl-1,3- dioxane at different temperatures is shown in Figure 7. The overresolution (R, = 3.6) at ambient temperature is reduced to R, 2.03 by increasing the temperature to 60"C, resulting in an analysis time shortening from 70 min to only 10 min.

Racernization of methyl-5- (p-rnethylbenzyl- oq~)-3,3-pentadienoate (allene). When using pure ethanol as mobile phase, racemization was

" I . I . I . I . I . I

20 30 40 50 60 70

T ("(3

Figure 6. ture. For conditions see Figure 2.

Resolution as function of the tempera-

50 "C I

40 "C

Figure 7. Separation of I-phenyl-l,3-dioxane at different temperatures. Column: 22.5 ern L X 320 p m i.d., 15-25 pm CTA. Flowrate: 1.6 p L /min.

found to occur during analysis at temperatures at or above 50°C with methyl-5-(p-methyl- benzyloxy)-3,3-pentadienoate as analyte. Figure 8 shows a typical example of this phenomenon. The racemization of MMBP is believed to fol- low a free-radical mechanism, as shown in Fig- ure 9. It is clear that the degree of racemization will increase, the higher the temperature and the lower the flow rate are. We found, however, that racemization was reduced after adding water, methanol, or isopropanol to the mobile phase (C in Figure 9). It is known that the addition of water and methanol has a strong

Figure 8. Separation of methyl-5- (p-rnethyl-benzyloxy)-3,3-pentadienoate at different temperatures and mobile phase compositions. Column: 22.5 cm L X 320 p m i.d., 15-25 prn CTA. Flowrate: 1.6 p L / min. A: 2YC, 100% ethanol, B: 60"C, 100% ethanol, C: 60"C, 90 / 10 ethanol / methanol.

J. Microcolumn Separations, Vol. 7, No. 3, 1995 263

11

R1 = H, R2 = p-methylbenzyloxy.

Figure 9. methyl-benzyloxy)-3,3-pentadienoate.

Radical racemization of methyl-9(p-

influence on the swelling state of CTA [21,22]. In general, efficiency increases while retention factor and selectivity decrease due to lower accessibility of the adsorption sites, since methanol and water are stronger competitors for adsorption compared to ethanol for the solutes. The fact that racemization is reduced after methanol or water, indicates that the solutes are adsorbed at specific sites on the CTA, which become less accessible after adding the modifiers. The thermostability of allenic compounds under inert conditions is also much higher than 50°C, as shown by the gas chro- matographic analysis of these compounds at temperatures above 100°C [26,27]. These findings support Rizzi’s theory about “quick” and “slow” type adsorption sites [20-221.

CONCLUSION The particle diameter of cellulose triac-

etate is of much less importance as for silica particles. The analysis time for the enantiose- lective separations on CTA can be drastically reduced for many compounds by increasing the column temperature. Care has to be taken to prevent that racemization occurs at higher temperatures.

ACKNOWLEDGMENTS The authors thank Merck (Darmstadt,

FRG) for the gift of a 10 p m cellulose triacetate research sample. D. Steenackers thanks the Instituut voor Wetenschappelijk Onderzoek in Nijverheid en Landbouw for a study grant.

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Received: September 22, 1993 Accepted: May 31, 1995

micro lc on cellulose triacetate (cta): influence of particle diameter and column temperature

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