Temperature Optimization for the Separation of PAHs on Micropacked LC ODS Columns Jian Chenl), Dirk Steenackers’), Andrei Medvedovici2), and Pat Sandra’’3)* ’) Department of Organic Chemistry, University of Gent, Krijgslaan 281 (S4), B-9000 Gent, Belgium 2, Department of Analytical Chemistry, University of Bucharest, Blvd. Republicii 13, 70031 Bucharest, Romania 3, Eindhoven University of Technology, Laboratory for Instrumental Analysis, P.O. Box 51 3, 5600 MB Eindhoven, The Netherlands

Key Words Micropacked liquid chromatography ODS stationary phases Temperature optimization Polycyclic aromatic hydrocarbons

Summary The effect of column temperature on the separation of the sixteen polycyclic aromatic hydrocarbons (PAHs) of mixture SRM 1647a of the US Environmental Protection Agency has been studied on different micropacked ODS columns. Isothermal temperature opti- mization was successfully used for complete separation of the PAHs on a polymeric ODS stationary phase, whereas temperature pro- grammed conditions were selected for separation on a monomeric ODS stationary phase.

1 Introduction

Column temperature is an important operating parameter in high performance hquid chromatography (HPLC) Selectimty and effi- ciency 1 e resolution, may be greatly improved by applylng sub- or super-ambient column temperatures Moreover, temperature pro- gramming in HPLC may be used with great advantage to reduce analysis time and/or to improve detectabihty for highly retained solutes offering an alternative to gradient elution

Temperature programming is not often used in HPLC This is mainly due on the one hand, to the success and ease of operation of solvent strength programmng and, on the other hand to the occunence of radial thermal gradients in conventional HPLC columns Mi- cropacked LC columns (< 0 5 mm i d ) show fast thermal equihbra- tion and radial thermal gradients created by viscous mobile phases at high velocities may disappear because of the narrow diameter of such columns

Micropacked LC in fused sihca tubing ranging from 100 to 320 pm i d and packed with 3 and 5 pm particles is routinely apphed in our laboratory In order to obtain reproducible retention data and selec- timties columns are normally thermostated at 25 “C With the same instrumental setup, chromatographic data can however also be obtained at higher or lower temperatures and in the temperature programmed mode

Temperature programmng has been apphed in conventional HPLC e g by Sheikh and Touchstone [ 11 for the separation of steroids, and in microbore ODS columns by Bowermaster and McNar [a] for the separation of alkylbenzenes

In the framework of temperature optimization in micropacked LC the sixteen PAHs of the EPA pnonty pollutant hst (SRM 1647a PAH mixture) were selected as solutes The resolution of several PAH pars on ODS stationary phases is affected not only by the charac- tenstics of the ODS matenal but to a considerable extent also by the temperature The influence of the chromatographic charactenstics of monomeric, polyrnenc and ohgomenc” ODS phases and the influence of temperature on PAH separation, using conventional columns has been thoroughly investigated by Sander and Wise 13-51

In this paper, the optimzation of isothermal temperature analysis is discussed for a fused sihca column packed with a polymenc ODS phase and of temperature programmation for a fused sihca column packed with a monomenc ODS phase

2 Experimental

2.1 Packing Materials

Two home made stationary phases were tested A monomeric ODS phase (phase A) was prepared starting from 3 pm RoSil particles (Biorad Eke, Belgium) by reaction with chlorodimethyloctadecylsi- lane (Aldnch, Belgium) and endcapping with trimethylchlorosilane (Aldrich, Belgium) The loading as determined by thermogravimet ric analysis (TGA) was 12 % A polymenc ODS phase (phase B) was prepared starting from 5 pm RoSil particles (Biorad, Eke, Belgium) with octadecyltrichlorosilane (Aldnch, Belgium ) and endcapping with trimethylchlorosilane TGA analysis 17 %

2.2 Columns and Packing Procedure

The columns were made of 0 32 mm i d fused sihca capillanes terminated with a FSOT polytetrafluoroethylene filter, held in place with an (epoxy)glued 0 1 mm i d capillary All columns were slurry packed using the same procedure A 10 % (w/v) slurry in CCh was somcated for 10 min and packed downwards at 600 bar utihzing a pneumatic high pressure pump (Haskel, Burbank,Cahfornia, USA), followed by acetonitrile as rinsing and pressurising solvent (2 hours) Columns were tested mth acenaphthene and further expen- ments were only started when a constant retention was obtained

The columns used in t h s work will be referred to as column A and column B

Column A 27 5 cm L x 320 pm i d , packed with the monomenc phase A Column B 50 0 cm L x 320 pm i d , packed with the polymeric phase B

2.3 Instrumentation and Materials

The instrumentation for the rmcro LC experiments consisted of a Varian 5000 LC pump with spht, a Valco C14W 60 nl inlector and a Vanan 2050 UV detector equipped with a home made 0 32 mm i d fused sihca cell, and worhng at 254 nm The column was placed in a water bath, the temperature of which was controlled using a Retsch RST I thermostat (Retsch Germany) For temperature pro- grammed analyses, the actual heating rate was measured using thermometer and stopwatch

Journal of High Resolution Chromatography VOL. 16, OCTOBER 1993 605

Separation of PAHs on Micropacked LC ODS Columns

The US Enwonmental Protection Agency SRM 1647a PAH mixture was obtamed from Supelco (Bellefonte, PA, USA) Acetonitnle, water, and carbon tetrachlonde were all of HPLC grade and pur- chased from Lab-Scan (Dubhn, Ireland)

3 Results and Discussion

Table 1 lists the PAH rmxture with the structures

The mixture contains several pars of isomers of which two pars, namely benz[a]anthracene/chrysene and indeno[ 1,2,3-cdjpy-

Table 1 List of the sixteen PAHs of the EPA SRM 1647a mixture.

__

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

Peakno Name -

Napthalene

Acenaphthylene

Fluorene

Acenaphthene

Phenanthrene

Anthracene

Fluoranthene

Pyrene

Benz[a]anthracene

Chrysene

Benzo(b1fluoranthene

Benzo[k]fluoranthene

Benzo[a]pyrene

Dibenz[a,h]anthracene

Inden01 1,2,3- cdlpyrene

Benzo[ghilperylene

Structure

rene/benzo[ghilperylene, are the most difficult to separate on ODS stationary phases Figure 1 shows the isocratic (86 14 acetomtnle- water) analysis at ambient temperature (24 "C) on column A and column B Complete separation cannot be obtained under these circumstances neither on the monomeric phase (coelution of benzlalanthracene and chrysene) nor on the polymeric phase (coelution of indeno[ 1,2,3- cdpyrene and benzo[ ghilperylene)

The different selectivity of columns A and B towards PAHs can be explained using the "slot model" as descnbed by Sander and Wise

Temperature: 2aOC Flow rate: 3.3 pl/min

I I I I I I - 0 10 20 30 40 50 min

Temperature: 24°C 2 FIOW rate: 2.1 Wmin

1 I I I I I I 0 20 40 60 80 100 120 min

Figure 1 Isothermal separation of PAHs on column A and 6.

[4] This model suggests that the separating abihty of ODS station- ary phases for PAH isomers is caused by a certain degree of "ordenng" within the bonded phase layer Order mthin this layer may be represented schematically as narrow "slots" into which the solute molecules can penetrate On the basis of this model and the trends seen experimentally on monomenc and polymenc ODS phases, the following predictions could be made

Long narrow solutes will penetrate a larger number of the slots than square solutes with the same molecular weight and thus are more retaned.

Nonplanar solutes have more difficulty in entering the slots than planar solutes and retention is decreased for nonplanar mole- cules

Polymenc ODS phases are better able to recognize shapes than monomeric ODS phases, and shape selectivlty for polymeric phases increases with increasing loading This suggests that polymenc phases are more "ordered" than monomeric phases

The lower the temperature, the higher will be the selectivity for both monomeric and polymenc phases

VOL. 16, OCTOBER 1993 Journal of High Resolution Chromatography

Separation of PAHs on Micropacked LC ODS Columns

The shape selectivlty of monomeric phase A at ambient tempera- ture is not high enough to separate chrysene [lo] from benz[a]an- thracene ([9] in Figure 1A) Benzolghilperylene [16] elutes after indeno[l,2,3-~d]pyrene 1151 The higher shape selectivity of poly- meric phase B causes the separation of chrysene [ lo] from benz[a]anthracene 191 but also the coelution of indeno[ 1,2,3-cd]py- rene [ 151 and benzolghilperylene 1161 m e n using gradient elution, indeno[l,2,3-cd]pyrene elutes even after benzo[ghilperylene on polyrnenc phases [3]

To improve the separating abihty of the monomeric phase A, the column temperature should be lowered Figure 2 shows the selec- tivities for the two critical pars of isomers between 0 and 40 "C The stationary phase selectively retards the elution of chrysene at tem peratures below 10 "C

4 4 4 . 4 4

4 4

0

4 0

- 4 0 0 0 0 0 0 0 0

0.95' ' ' ' ' . ' . ' ' ' . ' -10 0 1 0 20 3 0 40 SO

Temperature 'C

Figure 2 Selectivities for chrysene/benz[a]anthracene [0] and benzo[ghflperylene/in- deno[l,2,3-cdJpyrene [+I on the monomeric phase A in the temperature range 0-40 "C.

2 r I 5 1

0 10 20 30 40 50 60 min

Concerning the polymeric phase, operating at sub-ambient tern peratures would further increase the shape selectivity, causing indeno[ 1,2,3-cd]pyrene to elute after benzo[ghjperylene but would lead to very long analysis time as moreover capacity factors are much higher on the polymenc phase than on the monomenc phase In Figure 4, retention factors relative to benzolblfluoranthene are shown for all sixteen compounds in the temperature range 25- 80 "C

I I I

10 20 30 40 50 60 70 So 90 0.0 I Figure 4 Relative retention for the PAHs on the polymeric phase 6 in the temperature range 25-80 "C.

This figure clearly illustrates the loss of shape selectivity with increasing temperature At temperatures above 60 "C, the PAHs elute as groups of isomers Figure 5 shows selectivity factors for the two cntal pars of isomers From these data, together with the information in Figure 4, one can deduce that there is a small temperature range (3540 "C) in which all 16 PAHs are separated by isothermal analysis At higher temperatures, the pars benzjalan- thracenelchrysene [9,101 and benzo[a]pyrene/dibenzo[a,h]anthra- cene [13,14] start to coelute, while at ambient temperature (Figure 1B) indeno[1,2,3-cdpyrene [15] coelutes with benzo[ghi]perylene [16] The isothermal, isocratic analysis at 37 "C is shown in Figure 6

Figure 3 Temperature programmed analysis of PAHs on column A.

At super-ambient temperature, the selectivity for benzo[ghilperylene/inden0[1,2,3-~d]pyrene is higher than 1 05 and since capacity factors increase exponentially with decreasing tem- perature, a temperature programmed analysis is imposed to sepa rate all sixteen PAHs on the monomeric phase (Figure 3) After 30 mnutes isothermal at 0 "C, the temperature is programmed to 50 "C at 1 5 "/min

1.00 1 4

t 0.99' ' " * ' " " "

LO 2s 30 35 40 15 so Temperature ~C

Figure 5 Selectivity factors for chrysene benz[a]anthracene [V] and benzo[gh/]perylene/indeno[1,2,3-~~pyrene [+] on the polymeric phase 6 in the temperature range 25-50 "C.

Journal of High Resolution Chromatography VOL. 16, OCTOBER 1993 607

Separation of PAHs on Micropacked LC ODS Columns

Temperature: 37°C Flow rate: 2.1 wl/min

6

2 1

I I I I I I I I * 0 1 0 2 0 30 40 50 60 7 0

Figure 6 Optimized isothermal analysis of PAHs on the polymeric column 6.

Finally, as for gas chromatography [6] and supercritical fluid chro- matography [7] of PAHs, a good h e a r correlation is found between the logarithm of the capacity factors and the connectivity index. Whereas within a group of PAH isomers, the length to breadth ratio L/Bof the solutes is the best solute descriptor to descnbe the elution order of the isomers, the connectivity index is the descriptor that gves the highest correlation coefficients (compared to molecular weight, van der Waals volume, fused ring number etc.) when describing the elution of PAHs with increasing size. As an example Figure 7 shows this relationship for the PAH mixture on the polymeric phase B at three different temperatures.

Conclusions

All sixteen PAHs of the EPA SRM164a mxture could be separated wthout applyng gradient analysis on rmcrocolumns packed with monomenc and polymeric ODS phases using temperature optimi- zation The use of rmcrocolumns for temperature programmng avoids problems related with radial thermal gradients across the column

I 31°C

I / 2ooc

I " " " " I 3 5 x

Connectivity

Figure 7 Log k versus connectivity index x on column B.

Acknowledgments

D Steenackersthanks the Instituut voor Wetenschap en Onderzoek in Nilverheid en Landbouw for a study grant A Medvedovm thanks the European Commumty for financial support (TEMPUS JEP 0379- 91/2)

References

[l] S Sheiw? and i Touchstone J Chromatogr 455 (1988) 327

[Z] i Bowemasterand H McNair J Chromatogr 279 (1983) 431

131 L Sanderand S Wise Anal Chem 56 (1984) 504

[4] S Wise and L Sander, J HRC & CC 8 (1985) 248

[51 L Sanderand S Wise Anal Chem 61 (1989) 1749

[61 R Kaliszan and H Lamparczyk J Chromatogr Sci 16 (1978) 246

[7] d Rein C Cork and K Furton J Chromatogr 545 (1991) 149

Received May 28, 1993 Accepted August 2,1993

608 VOL. 16, OCTOBER 1993 Journal of High Resolution Chromatography