Effect of Temperature in microHPLC Separation of

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Effect of Temperature in m;cro-HPLC Separation of PAHs under Isocratic Conditions Using Octadecyl and Alkylamide Phases Renata Gadzala-Kopciuch, Boguslaw Buszewski"

Department ofEnvironmental Chemistry, Faculty ofChemistry, Nicolaus Copernicus University, 7 Gagarin St. PL - 87 100 Torun, Poland

The main difficulty in micro-HPLC separation is the manipulation with the composition of the mobile phase (degassing of solvents and a slow establishment of equilibrium) but the problem of elution can he solved by temperature optirnalization. The effect of temperature in micro-HPLC separation of the 16 polycyclic aromatic hydrocarbons (PAHs) (mixture SRM 1647 of the US EPA) has been studied using conventional C LS and new developed AP phase (an amide group localized in the hydrophobic ligands). All the investigations have been performed under isocratic conditions (binary hydroorganic mobile phase: acetonitrile/water). The results have shown that, in the case of AP phase, application of temperature gradient (from 298 to 303 K). enabled the attainment of complete 16 PAHs separation (especially of the first 4 solutes). R, '= .!. 4

INTRODUCTION

Temperature is one of the most important influences on chromatographic parameters, especially in gas chromatography (GC), where temperature is the main factor deciding about the conditions of separation. L,2 In liquid chromatography, particularly high performance liquid chromatography (HPLC), the effects which accompany the separation mechanisms are more complicated. U Temperature, beside the type and composition of the mobile phase, influences not only retention but also selectivity and resoluuon.?" The "manipulation" of this parameter, during HPLC elution, can cause changes not only of viscosity of a mobile phase and/or orientation and organization of the stationary phase, but also in the structure of determined analytes.t" It is revealed in differences of the mass transfer, and in consequence, in the determination of thermodynamic parameters such as enthalpy (dHo) and entropy (dSo) that describe the partition coefficient: (1)

where: k' - capacity factor, dHo and dSo correspond to enthalpy and entropy, respectively, tor solutes which interact with the mobile and stationary phases during the determination of the mass transfer, R - gas constant, T - temperature [K], cp - phase ratio. This factor, being an element of the resolution (R.). determines two further important parameters, i.e, selectivity (0;) and a number of theoretical plates (N):

(ex-l J(~) (N)~ 0; II + k z

(2)

Gant et al." examined the effect of temperature on resolution and on selectivity, retention factor, and plate number, all of which determine the magnitude of resolution. They found that these data can be used together with the temperature dependence of solvent viscosity to optimize the analysis rate with the required resolution. The change of temperature may unfavourably be revealed also during the formation of the mobile phase gradient while two and three-component hydroorganic systems are being applied." Thus, different types of results of the perturbations and the disturbances ora baseline (e.g. solvent peaks, demixture, etc. 15. 17) may often be observed on the chromatogram. So, applying isocratic elution is decidedly simpler and cheaper. It holds well especially for routine determinations, where a temperature effect of a stationary phase 8 • IO may be used during the separation of a substance containing rt electrons, which although "flat", has an elaborate and well defined stereogeometry, e.g. polycyclic aromatic hydrocarbons (PAHs).13·18.20 Topography and surface properties of stationary phase in RP HPLC elution also significantly influence the separation properties of analytes with enlarged structures (e.g. PAHs). Commonly used is octadecyl phase for separation of bonded ligand with change of mobile phase composition.15,20,21 Several authors have investigated this effect, however in many cases the elution was realized during gradient conditions. 18,2L·24 In the present contribution, the influence of tempera-

This paper is dedicated to Prof. Mo-Hsiung Yang on his 60 birthday,

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J. Chin. Chern. Soc., Val. 45, No.2, 1998

area, Vp - pore volume, D - pore diameter) of bare Separon SOX were determined by a low temperature nitrogen adsorption-desorption method using a Model 1800 sorptomatic instruments (Carlo Erba, Milano, Italy). The degree of bonded ligands coverage on the SG support surface (O:RP)25.27 was calculated from carbon content (Pe) determined with a CHN analyzer, Model 240 (Perkin-Elmer, Norwalk, CT, USA). The total concentration of surface silanol groups (O:SiOH) was measured by the method based on the GC determination of methane formed during the reaction of dimethylzinc .... tetrahydrofurane complex with hydroxyl groups. The measuring instrumentation and conditions are described in detail elsewhere. 25.2lCiI)aftllI'''''_ :2_I£",n_pIl.ln.ytene ,-o,.:.hry""" a. K'UpNbc-ll' 11, Dtilzo(ta'hlor.llr'llNon"

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Temperature (K]

Fig. 4. Dependence In k' vs. temperature for selected PAHs obtained on the narrow-bore columns with the polymeric SG-CIS (A) and SG-AP (B) phases under hydroorganic (ACNfH20) conditions.

Fig. 5. Comparison of micro·HPLC separation of PAHs mixture (EPA method 610) on narrow-bore columns (250 x 1.0 mm i.d.) with polymeric SG-CIS (A) and SG-AP (B) phases. HPLC conditions: mobile phase: 75/25 acetonitrile/water; gradient temperature [T 298 K (3 min), 298 - 303 K (3 S min), 303 K (8 - 40 minj]: flow rate 150/011: detector: UV-Vis. A= 254 nm.

=

Effect of Temperature in micro- HPLC Separation

1. Chin. Chern. Soc., Vol. 45. No.2, 1998

255

Table 3. Selected PAHs Used for the Temperature Studies Compound

Naphthalene

LID

1.24

Acenaphthylcne

Phenanthrene

1.46

Mw

128

152

00

178

009

1.57

178

OO©

Fluoranthene

1.22

202

~

1.27

202

* Values obtained at temperature 298 K

SG·Clg

SG-AP

SG·Clg

SG-AP

1.10

1.19

0.93

1.55

1.30

1.25

1.49

1.47

1.22

1.23

1.12

1.53

00

Anthracene

Pyrene

Rs*

ex= k2/kJ'"

Structure

~

the separation conditions should cause the chemical ordering of a bonded film. In the case of SG- AP phase this effect is especially revealed, since due to the formed slots the penetration of molecules is possible. It seems to be confirmed by the chromatograms presented in Fig. 5a and b obtained on the SG-C 18 and SG- AP phases. In the case of SG-C 18 phase, the application of the temperature gradient within the range of 298 - 303 K after 3 - 8 minutes of elution (Fig. 5a) caused shortening of the test mixture analysis time as much as 45% compared to the analysis performed at constant temperature (Fig. 3). However, the complete resolution of 16 PAIls was not obtained. Two solutes lacked resolution (peaks No.3 and 4). Under identical conditions, applying the phase SG-AP. a complete resolution of the mixture components was obtained (R, > 1.25) (Fig. 5b). Similarly, the time of the analysis underwent further shortening by 35% (up to 24 min) in relation to th~ separation shown in Fig. Sa, undoubtedly, the result of differences in the interactions of PAHs due to the dltferentiated topography of those phases and their properties, especially under the influence of temperature.

ACKNOWLEDGEMENT This work was supported by Polish Committee of Scientific Research (KBN, Warsaw, Poland, Grant No.3 T09A 162 10).

Received September 30, 1997. Key Words Micro - high performance liquid chromatography; Polycyclic aromatic hydrocarbons; Temperature effect.

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