low flow rate modifier addition in packed capillary column supercritical fluid chromatography

Low Flow Rate Modifier Addition in Packed Capillary Column Supercritical Fluid Chromatography Elena Ibaiiez, Wenbao Li, Abdul Malikl', and Milton L. Lee* Department of Chemistry, Brigham Young University, Provo, UT 84602-4672, USA

Key Words: Supercritical fluid chromatography Modifier addition Packed capillary columns

Summary

A new method to accurately deliver small amounts (0.5 to 20 mol%) of modifier into COz was used to study the effects of three different modifiers (methanol, water, and formic acid) in packed capillary column SFC. The method allows the use of different modifiers, with minimal instrument modification. The effects of the different modifiers at different concentrations on retention and peak shape are shown by analyzing a polarity test mixture and a sample of free fatty acids.

1 Introduction

Modifier addition in supercritical fluid chromatography (SFC) refers to the addition of a secondary solvent into the primary supercritical fluid to change [usually enhance) the solvating power of the mobile phase while maintaining the favorable critical properties of the primary fluid. Carbon dioxide is widely used as a primary mobile phase in SFC because of its mild critical parameters, together with its low toxicity, nonflammability, chemical inertness and compatibility with different detectors. However, because of its nonpolar nature, neat C02 is limited in its ability to dissolve, elute, and separate polar and high molecular weight compounds. A number of investigators have reported the addition of polar compounds to the primary supercritical fluid mobile phase [ 1-91 with the intention of dynamically masking the active sites in the stationary phase and, sometimes, enhancing the solubility of polar analytes in the mobile phase. Janssen and co-workers 1 10 I described three different ways in which modifiers can influence retention in packed column SFC: (a) increase mobile phase polarity, (b) increase mobile phase density, and (c) deactivate active sites on the surface of the packing material.

Among the different modifiers used in SFC, methanol is the most widely utilized. This modifier has shown its ability to effectively compete with the sample for the active sites in the column, contributing to changes in retention by simultaneously altering the interaction of the analyte in both the mobile and stationary phases.

When it is desirable to use flame ionization detection (FID) in SFC, only water, formic acid and formamide produce acceptably low background noise and enable the use of this universal detector [2 I . Water and formic acid have been suggested by some investigators as very useful modifiers in packed column SFC [4,11] because they significantly improve the resolution of some polar compounds such as free fatty acids. These compounds, when present in natural samples, are not easily detected by conven-

"Current address: Department ofchemistry, University of South Florida, Tampa, FL 33620. USA

I. High Resol. Chramatogr.

tional methods such as LC because of their low concentration. Both water and formic acid are only slightly soluble in supercritical C02 at low temperature (less than 0.3%), but their effects are appreciable even at this low level.

Packed capillary columns have shown their potential in SFC because they advantageously combine the positive characteristics of packed columns in terms of analysis speed and sample capacity, with low flow rates. low consumption of mobilc phase, and overall high efficiencies that are characteristics of open tubular columns [12]. Several papers on the use of packed capillary columns in SFC with neat CO2 have been published [ 13- 161, but their use in a mixed mobile phase system has not been extensively evaluated.

Binary mobile phases can be obtained either by off-line premix- ing of the components (in premixed cylinders or high pressure containers) or generated on-line during the chromatographic run by using a two-pump system. The first approach is undesirable because of the gradual change in modifier concentration that takes place in the premixed cylinder with time [ 171, or the limi- tation associated with working only at the premixed modifier concentration [ 181.

On-line generation of mixed mobile phaaes in SFC can be accomplished using the technology developed for microbore high perforniance liquid chromatography (HPLC). This involves high flow rates, and is useful only for conventional packed column SFC. The low microliter per minute flow rates required for open tubular and packed capillary columns are, however, difficult to realize in practice.

This paper describes the use of a home-made, single pump modifier addition system for capillary column SFC based on a pneumatic amplifier with an air-actuated high pressure primdpurge valve [19]. Using this system, the behavior of methanol, water, and formic acid as modifiers in C02 were studied for packed capillary columns.

2 Experimental

2.1 Instrumentation

The supercritical fluid chromatograph consisted of a Dionex Model 600 SFC pump (Sunnyvale, CA, USA) in combination with a Dionex Model 600 SFC oven. Modifier addition was achieved using a pneumatic amplifier constructed to deliver a 16% higher pressure on the high pressure side of the piston [19].

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Low Flow Rate Modifier Addition in Packed Capillary Column SFC

The modifier was introduced into the mobile phase by means of an air actuated, high pressure prime/purge valve (Valco, Houston, TX, USA). The valve was opened by means of a Model 180-4E2 12VDC solenoid valve (Koganei, Japan; Dionex 600 Series SFC part number 012683). The operation of the solenoid was control- led by a Model DVSP-2 digital valve sequence programmer (Valco). The amplifier containing the modifier was connected to the high-pressure valve by means of 50 pm i.d. fused silica tubing (Polymicro Technologies, Phoenix, AZ, USA). The connection from the valve to the chromatographic oven was also accomplished using 50 pm i.d. tubing. Calibration of the modifier concentration was accomplished using a standard procedure which was described in detail in a previous paper [19].

2.2 Reagents

SFC Grade CO2 (Scott Specialty Gases, Plumsteadville, PA, USA) was used as the primary mobile phase. HPLC grade methanol and water (Fisher, Fair Lawn, NJ, USA) and formic acid (99%) (Janssen Chimica, Gardena, CA, USA) were used as modifiers.

2.3 Columns.

A 32 cm x 200 pm i d . capillary column packed with 5 pm ODS (Phenomenex, Rancho Palos Verdes, CA, USA), and a 45 cm x 200pmi.d.fusedsilicacolumnpackedwith5 pmC18 deactivated phase (Phenomenex) were prepared according to a previously reported procedure [20].

2.4 Operating Conditions

The columns were connected to the injection valve via a flow splitter. Detection was accomplished using an FID or UV detector depending on whether water, formic acid, or methanol was used. Integral restrictors were prepared to give a gaseous flow of 1-2 mL min-' at the column outlet. The conditions of analysis are detailed in the figure captions.

2.5 Samples

Different mixtures of free fatty acids (C7-C18), 1-naphthol, 1 -phenyl- 1,2-ethanediol, methylbenzoate, menthol, 2,6-dimethylaniline, benLoic acid, anthracene, and fluoranthene (Sigma, St. Louis, MO, USA) were prepared and used as test samples .

3 Results and Discussion

3.1 Modifier Addition System Configurations

The modifier addition system used in this work is shown sche- matically in Figure 1. An extensive description of its operation has been reported previously [ 191, including the design of the Eystem and testing of the different parameters that can influence its operation. Each modifier studied in this report required some modification to the system. If water was used as a modifier, it was necessary to work at temperatures higher than 110 "C in order to increase the solubility of the water in the CO2 to more than 0.25%, which is the maximum value obtained when ambient temperature was used. Also, a pre-mixing chamber (1 5 cm x 0.5 mm i.d. stainless steel tube, packed with 30-40 wm silica parti- cles) was used to allow greater equilibration of the CO2/water

Puryelprime rb He fl Solenoid valve valve Digital controller j

T r-6-@-FC0 + Modifier

' I Modifier Jl

rump I

Figure 1. Schematic diagram of the single pump modifier addition system for capillary column SFC.

mixture. The injection valve was heated to the same temperature as the oven (120 "C) in order to avoid phase separation in the lower temperature zone 1211. Using these conditions, a 1.6% water mixture could be delivered without problems using the described modifier addition system (Figure 2). This same configuration was used when formic acid was added as a modifier.

CO, + Modifier

Injection valve

Split

Column

Oven

Heating system

JV delecior

detection

1

Figure 2. Instrumental configurations utilized when using (A) methanol and (B) water or formic acid as SFC modifiers in COz.

560 VOL. 18. SEPTEMBER 1995 J. High Resol. Chromatogr.


Some work has been previously reported using water as a modifier [S,21-23]. In these publications, a precolumn was always used to saturate the stream of C02 with water. Reports of equip- ment needed for saturating carbon dioxide with water have in- cluded the use of a precolumn (1 SO x 4.6 mm i.d.) packed with silica gel (100-200 mesh) inserted between the pump 'and the injector. The silica gel column was first saturated with about40% (dw) of water. As C02 passed through the column, water was desorbed from the silica gel, saturating the carbon dioxide.

Using the method described in this paper, only minimal modification of the system allowed the use of water and formic acid as modifiers at concentrations up to 2%. The simplest configuration of the system (Figure 2, top) was used when easily miscible modifiers that give response in the FID were utilized.

3.2 Efects oj'Methano1 on Retention and Peak Shapes

Methanol is one of the most widely used solvents as a modifier in SFC. The addition of methanol to CO2 decreases the adsorp- tivity of active sites, while increasing at the same time the polarity of the mobile phase. In Figure 3, a sequence of chromatograms is shown for a mixture of PAHs, an alcohol, and a diol, with increasing percentages of methanol in the mobile phase. From this figure it can be seen that increasing the amount of methanol decreases the retention for the more polar compounds, and also improves their peak shapes. When neat C02 is used, the most nonpolar compounds can be eluted, including 1 -naphthol which co-elutes with anthracene and produces a significantly tailing peak. The addition of only 2.6% methanol allows the elution of both polar compounds: 1 -phenyl-l,2-ethanediol that didnot elute when neat C02 was used, and 1 -naphthol that now can be sepa- rated from anthracene with good peak shape. Higher percentages of methanol provided an improvement in peak shapes while reducing the retention times for the alcohols by about 35%

i 3 4

B

\ I 7

10 min 0 8 min

3 4

C 3 4

7 I 0 8 rnin 0 8 min

Figure 3. Chromatograms of a polarity test mixture obtained by changing the percentage of methanol in the mobile phase. (A) neat C02, (€4) 2.6% methanol, (C) 5 .28 methanol, and (D) 1 1 3% methanol. Conditions: 90°C, 250 atm, UV detection at 254 nm. Peak identifications: (1) l-phenyl-1,2-ethanediol, (2) 1- naphthol, ( 3 ) anthracene, (4) fluoranthene.

(1 min) when comparing to the results obtained when 1 1.6% and 2.6% methanol were used. It seems that the methanol changes most significantly the retention characteristics of hydrogen- bonding solutes (alcohols) through deactivation of active sites on the stationary phase. Finally, for the PAHs, the effect of methanol became only appreciable in terms of changes in retention tiniewhenC02modified with the higherpercentage (1 1.3%) of methanol was used.

3.3 Effects of Water on Retention

The effect of water as modifier is shown in Figure 4 in which a separation of another mixture containing compounds of different polarities is shown. Several hydroxy compounds which interact strongly with polar modifiers were chosen along with a couple of PAHs that exhibit non-specific interactions with the mobile phase. As can be seen by comparing the chromatograms obtained using neat C02 and C02 modified with 1.6% water, the compounds that have strong interactions, such as alcohols (menthol and 1 -naphthol), acids (benzoic acid), or amines (2,6-dimethylaniline), were much more affected by the polar modifier. When neat COz was used, the alcohols and the amine were eluted with significant peak tailing, and benzoic acid was not eluted at all. Using a small percentage of water (1.6%), it was possible to elute all of the compounds present in the mixture with very good peak shapes, except for benzoic acid which still exhibited strong peak tailing. As can be seen in Figure 4, a decrease in retention was obtained for all of the compounds, with benzoic acid being the most affected by the presence of modifier. The percentage decreases in retention times for the compounds in the mixture were as follows 2,6-dimethylaniline (59%), methylbenzoate (55%), menthol (41%), 1-naphthol (30%), anthracene (17%), and fluordnthene (1 6%). The behavior observed can be attributed to the filling of the pores of the shtionary phase with water, as has already been suggested [24], as well as to the deactivation of active sites by the water [I, 251.

6 7

J J m 160 zoo 240 280atm 120 160 zoo 240 stm

lb 2'0 do 40 min b i a 20 30 min b 1

Figure 4. Chromatograms of a polarity test mixture obtained with (A) neat CO2 and (B) CO? + 1.6% water. Conditions: 120°C. pressure program from 120 atm to 300 atm at 4 alm min-', FID at 375°C. Peak identifications: ( 1 ) methylben- mate, (2) menthol, (3) 2,6-dimethylaniline, (4) benzoic acid, (5 ) 1-naphthol, (6) anthracene, (7) fluoranthene.

J. High Resol. Chromatogr. VOL. 18. SEPTEMBER 1995 561


" I

Neat I

0.5% water I

1.6% watei

Figure 5. Plots of retention factors (V) v\. percentage of water added to the COz mobile phase for various test solutes. (+) Methyl benmate, (+) menthol, (B) 2,6-dimethylaniline, (X)benmc acid, (*) I-naphthol, (A) anthracene, (0) fluoranthene.

Figure 5 shows the effect of water on the retention of compounds in the wide polarity mixture. It is interesting to point out that even a small amount of water (0.5%) has a great influence on the retention of all of the compounds tested in the mixture. In this test, identical chromatographic conditions were employed. As can be inferred from the data presented in Figure 5, the compounds with nonspecific interactions, such as the PAHs, show a decrease in retention, indicating that there is also a change in the solvating power of the mobile phase due to the addition of this small amount of water.

3.4 Efects of Formic Arid on Retention and Peak Shapes

The effect of using formic acid as modifier in SFC is shown in Figure 6 in which a separation of a free fatty acid mixture is preqented. As can be seen, in the absence of modifier, the acids are eluted with longer retention times and very poor peak shapes and selectivity. In the presence of 1% formic acid, the peak shapes and selectivity are improved; furthermore, the retention times are significantly reduced. By quantifying the percentage decreases in retention times when neat C02 and CO2 modified with 1% formic acid were used, the following observation was made: the percentagechange in retention time increases when the molecular weight of the free fatty acid decreases. The results show a retention reduction of 60% for heptanoic acid compared to 40% for oleic acid. This can be explained by the increase in alkyl chain that reduces the interaction of the acid functionality with the stationary and mobile phases.

The large effect of formic acid on the asymmetry factors for the free fatty acids can be clearly seen in Figure 7. Formic acid seems to be a very effective modifier in supercritical C02 because of its ability to compete with the polar solutes for proton accepting sites on the stationary phase. These sites are principally respon- sible for the undesirable chromatographic behavior manifested in asymmetric peaks and high retention when polar analytes are analyzed. For all of the acids ranging from C7 to C18, the asymmetry factors obtained with neat C02 are above 2, with most

I35 175 W5 255 atm 135 175 215 atm

b I 10 i0 3'0 min 0 rb min

Figure 6. Chromatograms of fatty acids obtained using (A) neat CO2 and (B) COz + 1% formic acid. Conditions as in Figure 4 except an initial pressure of 135 atm. Peak identifications: ( I ) heptanoic acid, (2) octanoic acid, (3) nonanoic acid, (4) lauric acid, ( 5 ) myristic acid, (6) oleic acid.

0 neat co2 .0.5% Formic acid

I % Formic acid

Figure 7. Asymmetry fdctor\ obtained for free fatty acids using neat COz and COz modified with formic acid.

greater than 6. However, supercritical carbon dioxide containing only 0.5% formic acid is able to improve the peak shapes for these compounds, reducing the asymmetry factors to around 1.

It is interesting lo note that depending on the packing materials used to prepare the columns, either poor or no elution of the free fatty acids occurred when neat C02 was used. This can be due to the different activities of the materials, and also to different deactivation procedures that are more or less effective in covering the active sites on the materials.

When higher percentages of formic acid were used (more than 2%), an increase in retention was observed. This effect seems to be related to the high dielectric constant of formic acid (E = 56.1 at 25°C). This effect has also been reported by Lesellier and co-workers [26 1, using solvents such as acetonitrile, methanol and nitromethane. They concluded that the increase in retention

562 VOL. 18, SEPTEMBER 1995 J. High Resol. Chromatogr.


is due to interactions between the solvent molecules when the dielectric constant is high. These interactions favor the cohesion and energetic stability of the mobile phase, but at the same time diminish the miscibility between the mobile phase and the hy- drophobic stationary phase or the solutes. The higher the constant, the higher the interfacial tension between the two phases. Finally, as the energy of transfer of the solute from the stationary phase toward the mobile phase increases, the retention also increases.

In general, the system reported in this work has proven to give very reproducible results, allowing in all cases the achievement of standard deviations of retention times of less than 4% for a minimum of 4 replicates, when either neat C02 or C02 with modifiers was used. However, we did observe restrictor plugging problems as reported by Engel and Olesik [27] when using formic acid as modifier. After continuous operation for an extended period oftime, it was found that the retention of solutes increased, the mobile phase flow rate decreased, and sometimes, the chromatography stopped. Careful checking under magnification revealed some black, solid material packed in the end of the restrictor. Further examination of this black material using scan- ning electron microscopy and energey dispersive X-ray spectros- copy revealed enriched concentrations of iron and silica. These results suggest that not only is polymerization of formic acid a problem during the analysis [27], but also the reaction of formic acid with stainless steel connections and packing materials is possible. In the case of water and methanol as modifiers, no such serious restrictor plugging problems were observed.

Acknowledgments

Elena Ibafiez thanks the Ministerio de Educacion y Cicncia of Spain for a grant in support of this work.

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low flow rate modifier addition in packed capillary column supercritical fluid chromatography

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