Packed Bed Columns

Chapter 4

Packed Bed Columns Luis A. C O L O N * , Todd D. M A L O N E Y and A d a m M. F E R M I E R t

Department of Chemistry, State University of New York at Buffalo, Natural Sciences Complex, Buffalo, NY 14260-3000, USA ?Present address." The R. W. Johnson Pharmaceutical Research Institute, Science and New Technology~Analytical Development, OMP Bld. B-236, 1000 Route 202, Raritan, NJ 08869, USA

CONTENTS

4.1 4.2

4.3

4.4 4.5 4.6 4.7 4.8

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Column fabrication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.1 The column . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.2 Chromatographic material . . . . . . . . . . . . . . . . . . . . . 4.2.2.1 Ion-exchangers and mixed-mode phases . . . . . . . . 4.2.2.2 Submicron particulate materials . . . . . . . . . . . . 4.2.2.3 Highly porous particles . . . . . . . . . . . . . . . . . 4.2.3 Retaining frits . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.3.1 Silica-base frits . . . . . . . . . . . . . . . . . . . . . 4.2.3.2 Fritless packed beds . . . . . . . . . . . . . . . . . . . 4.2.4 Fabricating columns . . . . . . . . . . . . . . . . . . . . . . . . Packing methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3.1 Pressure packing . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3.2 Packing with supercritical CO2 . . . . . . . . . . . . . . . . . . 4.3.3 Electrokinetic and pseudo-electrokinetic packing . . . . . . . . . 4.3.4 Packing by centripetal forces . . . . . . . . . . . . . . . . . . . 4.3.5 Packing by gravity . . . . . . . . . . . . . . . . . . . . . . . . . Comparison o f packing procedures . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgement ............................. Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. .

112 112 113

116 129 132 137

139

. . . . . .

140 144 145 150 150 152 152 154 155 156 158 158 159 159

112

Chapter 4

4.1 INTRODUCTION Capillary electrochromatography (CEC) can be performed in open tubes or packed structures. In the open tubular format, the stationary phase is fixed at the inner surface of a capillary column; columns with inner diameter of less than 20 gm are recommended for the best performance [ 1]. Packed structures, on the other hand, consist of a capillary tube filled with chromatographic media. These packed structures can be classified into three different groups: 1) columns packed with particles [2-30], 2) columns containing separation material that has been polymerized in situ, creating a "rod-like" monolithic structure also known as continuous beds [31-39], and 3) columns with entrapped particulate material, which are a combination of the first two groups [40-46]. Columns of the first group are the ones used the most in CEC and will be discussed in this chapter; the other two groups are discussed in Chapters 5, 6 and 7. The chromatographic packing material most commonly used in CEC is HPLC reversed-phase type on spherical particles (1.5-10 gm diameters); although new alternatives are being explored to fabricate materials with more applicability to CEC. As the CEC column technology develops, however, the preference of using columns packed with particles may change, particularly with the emerging approach of monolithic columns. Several protocols can be used to fabricate packed bed structures for use in CEC. In this chapter, we will discuss the packing techniques and column fabrication protocols that have been used for packing particulate material. We concentrate, therefore, on the different approaches used to deliver chromatographic particles into the capillary column. We present an overview of the different packing protocols available to the practitioner, as well as of the CEC column fabrication method, as performed in our laboratory. Our own experiences, practices, and views regarding packing procedures are also provided, when appropriate. 4.2 Column fabrication

Despite the several detailed procedures reported for the fabrication of packed columns for CEC [ 14,17,20,27,30,47-50], column fabrication may still be regarded as an art. A reliable and reproducible performance of a column depends on the column fabrication. Poorly packed columns can lead to low efficiency, poor resolution, and asymmetric peak shapes. The capillary tubes typically used to fabricate CEC columns are fused silica tubes with inner diameters of 100 gm or less, with 50 and 75 lam I.D. being the most popular. The small inner diameter allows for heat dissipation, which is generated by the applied electric field. Packing such columns is an elaborated process and a skill that requires experience.

Packed Bed Columns

113

4.2.1 The column

A packed column in CEC consists of two segments- a packed and an unpacked (or open) section, in most cases. A typical CEC column with a packed and an open segment is illustrated in Fig. 4.1. Most frequently, capillary tubes with less than 100 ~tm I.D. are packed with reverse phase HPLC materials of 1.5-10 ~tm diameter. The chromatographic material is kept in place by means of retaining frits (vide infra). The electroosmotic flow (EOF) velocity in each segment of the CEC column is different [51 ]. The overall EOF velocity depends on the fraction of the packed segment [51,52]. The resulting net EOF is thus a combination from both, the packed and the open segments. To facilitate detection through the column by spectroscopic means, the polyimide coating on the open segment, close to the outlet retaining frit, is removed; this provides the optical window for detection. This can be achieved by any of the methods already in use for capillary electrophoresis (CE) [53]; burning off the polyimide coating is the most common approach. Because of light scattering by particles, optical detection through the packed bed has been reported to decrease detectability [18,24,27]. The length of the column can alternatively be packed completely with the desired separation material (no open segment); however, if detection through the packed bed is not performed, connecting tubing to a detection system is required. Fig. 4.2 shows an example of a 75 ~tm I.D. fused silica column completely packed connected to a piece of a 50 ~tm I.D. capillary used for detection. The butt connector is made by inserting the two capillaries into a piece of PTFE shrinking tubing, which upon application of heat secures the two capillaries in place [54]. It has been reported that butt connection of capillaries has an insignificant contribution to band spreading [55]; however, care must be exercised since connecting of two different pieces of tubing

Packed Section

Open Section

..J

I ~176

""

"-'-'

.. . . . .

i

I Detection Window

Retaining Frits Fig. 4.1. Schematic of a typical packed-capillary column for CEC, illustrating the open and packed segments. References pp. 159-164

114

Chapter 4

Fig. 4.2. Photograph of a butt connection between a 75 ~tm I.D. packed fused silica capillary and a piece of a 50 ~m I.D. capillary tube. Reprinted from ref. [54] with permission. Copyright Wiley-VCH 1999.

2.0

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Fractional length of the capillary segment packed with bare silica

Fig. 4.3. EOF mobility as a function of the fractional length of the bare silica packed segment for a 100 p,m I.D. capillary containing a 20 cm ODS segment. Reprinted from ref. [56] with permission. Copyright Wiley-VCH 1999.

always has the potential of introducing band broadening to the system. Nonetheless, this option is often used to connect detection schemes that offer higher detectabilities, such as Z-cell for UV detection, mass spectrometry (MS) and NMR detection schemes (see Chapters 2 and 8). In such instances, the gain in detectability and/or structural information is far more important than the loss in efficiency. Totally packed capillary columns, having one segment packed with the stationary phase and a second segment with bare silica, have been fabricated to control the EOF [56]. In this case, the segment that is open in a typical CEC column is packed with bare silica to accelerate and provide a steadier EOF. Such a configuration has allowed an increased EOF that translates into shorter analysis times. Fig. 4.3 and 4 show the EOF mobility as a function of the fractional length of a column packed with bare silica and the effect on analysis time, respectively. As the porosity of the bare silica particles is increased, the EOF is also increased [56]. Columns have also been packed

Packed Bed Columns

115

'i 5

a, u = 0.8 mm/s Nay = I01,000 plates/m E tt)

b.

~g

u = 1.0 mm/s Nay = 108,000 platefdm

2

"~'5

C, u = 1.1

mm/s Nay = 130,000 plates/m

r

2'0

Min

Fig. 4.4. Electropherograms illustrating the effect of the length of the bare-silica segment on the separation of probe compounds, a) 0 cm, b) 6 cm, and c) 28 cm. Solutes: 1, benzene; 2, toluene; 3, ethylbenzene; 4, propylbenzene; 5, butylbenzene and 6, pentylbenzene. Reprinted from ref. [56] with permission. Copyright Wiley-VCH 1999.

with a blend of bare silica and reverse-phase silica supports [18,57]. This can provide enhanced EOF due to the amount of silanols groups introduced by the bare silica, decreasing analysis time. This approach also reduces retention because of the decrease amount of stationary phase, as the bare silica replaces the bonded one; hence, retention depends on the blend ratio. Initial work on CEC was performed on drawn-packed capillaries [ 11 ], a procedure originally introduced by Tsuda et al. [58]. In this approach, large bore columns (thick walled Pyrex tubing) were packed with underivatized packing material; then the columns were pulled at high temperatures to a desired diameter using a glass drawing machine. The stationary phase was attached to the underivatized support packing material after the columns were drawn. This column preparation procedure is not currently used because of the low success rate in fabricating the columns.

References pp. 159-164

116

Chapter 4

4.2.2 Chromatographic material Because of the column's dimensions in CEC, it is important to consider a narrow size distribution of the particles. The effect of particle size distribution on separation efficiency in CEC is expected to be similar of that in HPLC. Although of the same nominal particle size, different packing materials can yield different efficiencies in CEC. It has been pointed out that the different efficiencies reported for the separation of polycyclic aromatic hydrocarbons (PAH), for example, using different packing materials of the same sizes, can be attributed in part to the size distribution of each material [59]. The structure of the packed bed can be influenced by the size distribution. A homogeneous packing size leads to well-packed beds, approaching a closed packed structure. This can be seen in Fig. 4.5, where panels A and B show SEM of the packed bed for columns that were packed with silica particles of about 3 and 0.5 ~tm in diameter, respectively. It is apparent that the particle size in panel A is not as homogeneous as that of the particles in panel B. Notice how the particles with the tighter size distribution form a better-packed bed. One of the most important properties of a column packing material for CEC is the ability to support EOF. This is not only necessary for the separation of neutral compounds but also to separate charged species as the EOF is responsible for the bulk transport of the mobile phase and analytes [7,16]. In the absence of EOF, only species with the appropriate charge will reach the detector. Therefore, packing materials with very favorable characteristics for EOF generation are desired in CEC. Many silicabase HPLC packings from 1.5 to 40 ~tm in diameter have been utilized to pack columns for CEC; those with the C18 reverse- phase being used the most. Other materials include C8 [60-62], phenyl [61,62], and C30 [63,64]. The surface silanol groups of the silica impart a negative charge to the packing material, leading to the generation of the EOF upon application of the electric field. The high surface area of the silica-based packed beds provides for the EOF to be generated mostly at the particle surface, with negligible contributions from the fused silica capillary walls [7,16,65]. However, it is apparent that not all C 18 reversed phase HPLC materials are suitable for CEC. For example, Table 4.1 summarizes electrophoretic mobilities observed on typical reverse-phase HPLC chromatographic materials, and Fig. 4.6 illustrates the CEC separation properties of several C 18 packing materials under identical separation conditions. Faster analysis times are achieved with those materials capable of generating a strong EOF. CEC Hypersil and ODS-1 type are popular among the reverse-phase materials since they seem to support the fastest EOF. These materials are HPLC supports that have not been end-capped, and therefore, a relatively large amount of silanol groups are left on the surface, which can generate EOF. The EOF decreases as the alkyl substitution at the packing surface is increased be-

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References pp 159-164

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118

Chapter 4

TABLE 4.1 ELECTROOSMOTIC MOBILITIES OF VARIOUS CHROMATOGRAPHIC MATERIALS UTILIZED FOR CEC

Stationary phase material

Electroosmotic mobility (x 10-4 cm2/Vs)

BDS-ODS Hypersil a

0.99

CEC Hypersil C 18a

2.26

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0.14

LiChrospher RP-18 b

1.45

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1.56

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1.47

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<0.01 1.54 <0.01

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2.26

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0.68

aAdapted from [7]; separation conditions: (80:20) acetonitrile-Tris-HC1 50 mM, pH 8, 20~ EOF marker used: Thiourea bAdapted from [16]; separation conditions: (70:30) acetonitrile-3-cyclohexylamino-2-hydroxy-l-propanesulfonic acid 25 mM, pH 9.53, EOF marker used: Thiourea CAdapted from [63]; separation conditions: (95:5) acetone-1 mM borate buffer, EOF marker used: Acetone. cause of the concomitant decrease in silanols groups responsible for the EOF [65,66]. Packed beds formed with 3 lam particles containing C 18 phases have generated separation efficiencies above 300,000 plates/m [ 17,30]. Separation efficiencies larger than 500,000 theoretical plates/m have been achieved using 1.5 lam non-porous reversephase silica materials in several applications [67-71]. Fig. 4.7 shows an example of high separation efficiency in the separation of 14 explosive compounds using a packed bed containing 1.5 pm non-porous packing; over 500,000 plates/m were observed. SDS has been used in the mobile phase to prevent bubble formation (vide

Packed Bed Columns

119

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TNB

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References pp. 159-164

Chapter 4

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infra) and to stabilize the EOF through dynamic modification of the alkylated surface, which also has an effect on selectivity [69]. The effect of particle diameter on efficiency in CEC is better appreciated in a plot of plate height versus linear velocity of the mobile phase, known as a vanDeemter plot, as depicted in Fig. 4.8. It is clear that as the particle diameter is decreased, the separation efficiency is improved. Further, and contrary to pressure driven LC, the use of EOF in CEC also allows for the use of relatively high linear velocities of the mobile phase without a detrimental effect on efficiency. Silica-base stationary phases have also been employed for enantiomeric separations in CEC [6,72-81]. In the initial work on chiral CEC, commercially available HPLC materials were utilized, including cyclodextrins [6,74,81] and protein-type selectors [73,75,80] such as human serum albumin [75] and Otl-acid glycoprotein [73]. Fig. 4.9, for example, depicts the structure of a cyclodextrin-base stationary phase used in CEC and the separation of mephobarbital enantiomers by capillary LC and CEC in a capillary column packed with such a phase. The column operated in the CEC mode affords higher separation efficiency than in the capillary LC mode. Other enantiomeric selectors are also use in CEC, including the silica-linked or silica-coated macrocyclic antibiotics vancomycin [82,83] and teicoplanin [84], cyclodextrin-base polymer coated silicas [72,78], and weak anion-exchage type chiral phases [85]. Relatively high separation efficiency and excellent resolution for a variety of compounds have also been achieved using columns packed with naproxen-derived and Whelk-O chiral stationary phases linked to 3 ILtm silica particles [79]. Fig. 4.10 shows the

121

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122

Chapter 4

A :

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mAu 60 50 40 30 20 10

0

2

4

6

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Fig. 4.10. (A) The chiral stationary phases (S)-naproxen-derived and (3R,4S)-Whelk-O 1. (B) CEC enantiomeric separation of an antidepressant (N-[1-(4-bromo-phenyl)-ethyl]2,2-dimethyl-propionamide) on a column packed with (3R,4S)-Whelk-O 1 chiral phase immobilized on 3 t.tm silica. Adapted from ref. [79] with permission. Copyright Elsevier 1997.

materials used in CEC, including specific applications, is shown in Table 4.2. CEC applications have been discussed in details by Robson, et al. [86] and by Dermaux and Sandra [87].

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~m~

<

o

~

Oo o,

~

o

~ M

=

o

o M

o

o M

~

m

o

"n

",-"I

0

~

.0 ~ ~

•

~

o

o

.~ .~ ~

oa

e

...~ ~ "~, ~m -r.n~ ~m m

Chapter 4

Packed Bed Columns

129

4.2.2.1 Ion-exchangers and mixed-mode phases CEC chromatographic materials should have relatively high surface charge to afford a strong EOF. The EOF generated in commonly used silica-base packing materials depends on the pH of the mobile phase, requiring pH conditions suitable to deprotonate the silanol groups at the surface. At a pH of 8 or above, high EOF is obtained; however, at a pH of 3 or below the EOF is significantly reduced or non-existent. One approach to minimize the EOF dependence on the pH of the mobile phase is to use packing materials containing a fixed charge at the surface throughout a wide pH range. To this end, one can envision a variety of stationary phases attached onto the surface of the silica support by means of the well-known silane chemistry (see Fig. 4.11A). Such phases can be ion-exchangers or materials specifically designed for

A CH 3

CH 3

Where R is: C 8 , C18, C30, SCX, SAX, etc...

~

so 3-

B

so a-

C

.

.

so aFig. 4.11. (A) Generic representation of silane chemistry used to bond stationary phases onto silica supports. (B) Examples of packing materials that will enhance EOF. (C) Mixed-mode phase designed by E1 Rassi's group. Adapted from ref. [95] with permission. Copyright Wiley-VCH 1998.

Referencespp. 159-164

130

Chapter 4

CEC. Fig. 4.11B shows three possible materials suitable for CEC that provides high surface charge to generate EOF. Ion exchangers (i.e., SCX, SAX) are used in CEC as a way to increase surface charge, hence EOF. Columns packed with these materials have been used to separate charged organic and inorganic solutes [88-92]. Packing materials with cationic [65,90,91,93-106] and anionic [46,102,107,108] exchange moieties are one of the main subjects of study in packed beds for CEC. In principle, these materials should provide for a strong and stable EOF through a wide pH range. A material containing sulfonic groups, like those in SCX (e.g., C6-SCX, phenyl-SCX, C3-SCX) imparts a permanent negative charge at the surface, providing EOF towards the negative electrode. The typical quaternary amines of SAX phases, on the other hand, provide a positive charge at the surface of the packing leading to EOF in the direction of the positively charge electrode. Incorporation of an alkyl chain bonded to the silica in addition to the charged moiety (see Fig. 4.11B) has lead to the so-called mixed-mode phases for CEC, in which the separation mechanism can involve hydrophobic and ion exchange interactions, as well as differential electrophoretic migration [65,66,9397,99,101-103,109]. The EOF generated in these materials has been shown to be higher than in typical reverse-phase [65,95,97,101 ]. However, there is some dependence on the pH of the mobile phase, as shown in Fig. 4.12. Such dependence is attributed to the residual silanols present on the silica support. It has also been reported that rather lower EOF velocities than expected have been observed with the SCX phases [90]. More detailed studies on these types of materials are needed to

3.0 pH vs C 18.ODS 1 2.5 E 2.0

~. 1.5 o 1.0 ~" 0.5 O 0.0

i

4

i

i

i

5 6 7 pH of the mobile phase

i

8

9

Fig. 4.12. EOF velocity as a function of the mobile phase pH for a C18 bonded silica and a mixed-mode bonded silica (SCX/C18). Reprinted from ref. [ 101] with permission. Copyright Elsevier 2000.

Packed Bed Columns

to

(a)

i ~

131

,

,

4

|

8

,

12

t (rain)

3

1

(b) 2

2

6 345

0 0

1

2

3

i

|

4

5

t (min)

Fig. 4.13. Separation of acidic compounds in columns packed with (a) 5 p,m Spherisorb-ODS and (b) 5 pm Spherisorb-SAX materials. In (a) the mobile phase was composed of 60% acetonitrile in 2mM phosphate buffer (pH 2.2) and in (b) the mobile phase was composed of 50% acetonitrile in 20 mM phosphate buffer (pH 2.2). The compounds were: 1, 3,5-dinitrobenzoic acid; 2, p-nitrobenzoic acid; 3, p-bromobenzoic acid; 4, o-toluic acid; 5, benzoic acid; 6, o-bromobenzoic acid. Reprinted from ref. [102] with permission. Copyright Elsevier 2000. completely elucidate their behavior in CEC. Most of the mixed-mode stationary phases in CEC have incorporated two different moieties directly attached to a silica support, one with the SCX group and the other with an octadecyl group, for example (see Fig. 4.11B). One mixed-mode material specifically designed for CEC was introduced by E1Rassi's group [93-95,103], which is bonded to the silica support differently (see Fig. 4.11C). They synthesized a silicabased stationary phase that incorporates a (7-glycidoxypropyl)trimethoxysilane sublayer attached to the silica support and a sulfonated layer is covalently inserted between the sublayer and an octadecyl top layer. Although less popular, packed beds containing anion-exchanger groups have been shown to be useful in CEC. For example, using a commercially available SAX mixed mode phase, Lubman and coworkers [107] have been able to achieve excellent separations of peptides. The selectivity of the mixed-mode stationary phases, as well as the ion exchangers, in CEC have shown References pp. 159-164

Chapter 4

132

6

3

(a) pH 3.0

2

1

5

4

4

3 2

. . . . .

i .

2

.

.

.

.

.

.

.

i

4

,

6

t( rain ) (b) pH6.0

4 2

0 t

0

1

2

4 3 t( rain )

.

l

. .

5

J

6

Fig. 4.14. CEC separation of peptides using the mobile phase 60% acetonitrile in 30 mM phosphate buffer with (a) pH 3 and (b) pH 6. Peaks: 1, benzyl alcohol; 2, Gly-Gly; 3, Gly-Thr; 4, Gly-Gly-Gly; 5, Glu-Glu; 6, Gly-Gly-Gly-Gly-Gly; 7, Glu-Glu-Glu. Reprinted from ref. [108] with permission. Copyright Elsevier 2000. to be significantly different to that obtained in conventional reverse-phase silica materials. This is particularly the case for charged compounds where ion exchange-type of interactions have a significant contribution to the separation [65,94,97,102,103,107,108]. For example, selectivity can be tuned by changing the pH of the mobile phase, which influences ion exchanging. These effects are clearly illustrated in Figs. 4.13 and 14 for the separation of peptides and acidic compounds in a packed bed containing SCX phase.

4.2.2.2 Submicron particulate materials The pressure limitations found in HPLC as the particle diameter is decreased are not present in CEC, since the EOF velocity (u) does not depend on particle diameter as seen in Equation 4.1"

Packed Bed Columns

_

U--

~ o ~ r ~ E _ ~eoE q

133

(4.1)

where eo, er, ~, E, 11, and bteo are, respectively, the permitivity in vacuum, the relative permitivity of the medium, the zeta potential, the applied electric field strength, the viscosity of the medium, and the electroosmotic mobility. Therefore, the use of electroosmosis to drive the solvent through a packed bed should allow for the use of very small particles to generate high separation efficiencies. In principle, the limitation on particle diameter in CEC is imposed by overlapping of the electrical double layer in the flow channels. The reciprocal of the double layer thickness (~c) is known as the Debye-Htickel parameter given by Equation 4.2:

1( =

_1_= F N/ 21 8 R T ~o ~;,-

(4.2)

where 6, F,/, R, and T are, respectively, the thickness of the double layer, the Faraday constant, the ionic strength, the gas constant, and the temperature in K. The other variables are as in Equation 4.1. Equation 4.2 shows that the thickness of the electrical double layer is dependent on the dielectric constant of the mobile phase and the ionic strength of the electrolyte present. According to classical double layer theory, as applied to thin capillaries, the diameter of a channel must be at least 20 times the thickness of the electrical double layer (dc>205) in order to minimize parabolic flow [110]. Under such conditions, 80% of the volume transport due to electroosmosis should be retained [ 110]. Overlapping of the electrical double layer becomes significant in channels with diameters less than 206 and the plug-like profile is lost, decreasing EOF considerably. The double layer thickness has typical values of less than 10 nm, depending on the electrolyte concentration. The thickness of the double layer as a function of acetonitrile concentration at different ionic strengths, using univalent ions, is shown in Fig. 4.15. To at least maintain 80% of the flow transport for the double layer thickness in the typical range of 1-10 nm, the minimum flow channel dimensions must be 20-200 nm. In CEC, the EOF depends on the column packing structure and pore size of the packing material [51,111-116]. In a packed bed, there are many interconnected channels between particles, which leads to a porous packed structure. The porosity of the packed bed dictates the permeability through the column. The average channel size between particles in a CEC column can be estimated if the packed bed is assumed a

References pp. 159-164

Chapter 4

134 --- 40

[

E

~ /,o,,er

L_

(11 >,, J

0.1 mM 30

03 d3 .....

O

C3 2O

1 mM

JZ2

10 mM

100 mM

4,-.

O

o~ 10 09 (11 t',r O

'-" F--

0 I,

I

0

10

....

I

..

I

20

30

.

,~. .....

40

I

I

50

60

J_.

70

I

80

.l .......

90

I,

100

Percent of Acetonitrile Fig. 4.15. Thickness of the double layer as a function of acetonitrile concentration in a water-acetonitrile mixture, as per Equation 4.1. Constants for the mixed solvents were obtained from ref. [49,129].

collection of capillary tubes with an average diameter corresponding to the channels between particles. Equation 4.3 shows a relationship between the mean channel diameter and particle size (alp), accounting for the particle structure through the interparticle porosity [ 113]:

dc = 0.42 alp g 1-e,

(4.3)

where ~ is the interparticle porosity. A fairly well packed column is considered to have a random packing structure with an interparticle porosity of 0.4 [117]; therefore, the channel diameter is given by:

dc- 0.28 dp

(4.4)

A similar channel diameter (i.e., 0.25 dp) was originally suggested by Knox who

Packed Bed Columns

3.530

135

9

~

................ --m---- 3 pm

: ..................... !................. ~;~:: ..........

i ~o

2.5 --T--0.2 2.0

O

o

~.5

LL

1.0

0 LU 0.5 00

~m

!

.~ J

iiiiiii t

.......... i"<

o

::

iiiiii

........ .......... ! .......... i .......... ' ........... ,........... "....... ' 2OO

4OO Field

6OO

8OO

strength (V cm -1)

Fig. 4.16. EOF mobility as a function of applied voltage for different particle diameters. Reprinted with from ref. [66] with permission. Copyright Elsevier 2000.

obtained it using the flow resistance parameter of a packed column [ 118]. Using a minimum channel diameter of 206 as the limit to avoid a significant overlap of the electrical double layers, one can estimate the particle size required to preserve most of the volume transport with plug-like profile [11]. For 8 values of 1-10 nm, which corresponds to channel diameters of 20-200 nm, the minimum particle diameter would then be 0.071-0.71 lam, for a column having a randomly packed bed. Unger and coworkers have reported that EOF is independent of particle size down to 0.2 ~tm [66], as predicted by Equation 4.1 (see Fig. 4.16). According to Equation 4, the average interparticle channel diameter for 0.2 ~tm particles is about 0.06 ~m. The experimental conditions reported by Unger and coworkers indicate that 8 is about 3 nm, corresponding to a channel diameter of approximately 0.06 ~m, which satisfies the condition of dc>20 8. EOF linear velocities above 2 mm/s have been achieved in CEC using packed beds with submicron particles and organic/aqueous mobile phases [66,120]. So far, the separation efficiencies reported with the submicron packed beds have not offered a significant improvement over those obtained with particle diameters in the 1 ~m range [66,119-121]. Fig. 4.17 depicts the separation of a test mixture obtained in a packed bed with particles of about 0.5 ~tm in diameter. As reported by Luedtke, et al. [ 121 ], plate heights of about three times the particle diameter (H = 3dp) are achieved. This has been attributed to band dispersion due to temperature effects and instrumental limitations, such as the maximum electric field that can be applied with existing units and detection systems [121]. Plots of plate height versus linear

References pp. 159-164

Chapter 4

136

1) Thiourea

2

2) Naphthalene 3) Ethylnaphthalene 4) Amylbenzene

I

I

I

0.4

I

0.8

I

I

1.2

I

1.6

Time/minutes Fig. 4.17. Separation of a test mixture in a packed bed of 0.5 ~tm (C8) particles. Column: packed bed of 15 cm in a capillary of 35 cm total length; mobile phase: 80:20 acetonitrile-50 mM Tris buffer at pH 8; separation voltage of 30 kV; injection, 3 s at 300 V; UV detection (220 nm).

velocity indicate that a minimum in plate height has not been achieved and higher electric fields are needed to achieve higher velocities (see Fig. 4.18). Nevertheless, the small particles do provide for rapid separations with current systems. Further studies are still required to obtain a complete understanding of the effect of the submicron material in CEC.

137

Packed Bed Columns

3.5 (# c

.~

~, | ~ X

3.1

~ ~

~1'~" C

E v

=L

F- 2.7 "1-

T

(9

0.2

0.6

w -1-

1.0 1.4 Ttme/mln

1.8

LU 2.3 F< J &.

1.9

1.5 ,

0.1

I

,

0.6

I

1.1

~

I

1.6

,

I

2.1

,

I

~

2.6

I

3.1

LINEAR VELOCITY (mm/s) Fig. 4.18. Plate height versus linear velocity for 9-(1-pyrene)nonanol, last eluting peak in electropherogram in the insert, obtained in a column packed with particles of about 0.5 ~m diameter. Column: 12 cm packed, 35 cm total length; mobile phase 80:20 acetonitrile-50 mM Tris pH 8; voltage 27 kV.

4.2.2.3 Highly porous materials

The typical porous silica-base materials commercially available have pore sizes close to 10 nm. Based on our discussion on the channel diameter requirements to support EOF, it is unlikely that EOF through such pores can be generated under normal CEC conditions. However, it is evident that intraparticle EOF exists in materials with relatively

large pores,

exhibiting perfusion

through

the particles

[45,56,116,122,123]. Flow transport through a highly porous particle is schematically represented in Fig. 4.19. With appropriate channel diameters, EOF can be generated within the particle, transporting the solute through. In pressure driven systems, there is no flow through the particle pores and solutes can only have access to the pores by diffusion. Perfusive transport through wide-bore silica particles with nominal pore

Referencespp. 159-164

138

Chapter 4

Dapp~, ~:. ~?~"'"

Deft,~;~',~.~%~, ---;-9

~o

..~,:~ .......

[:::)m (a) Pressure-drivenFlow (HPLC) Diffusion only

'" "'~ "

'

(b) Voltage-drlven Flow (CEC) Dlffuslon + Convection

Fig. 4.19. Schematic representation of intraparticle flow. In pressure driven flow there is no flow through the particle (A); in electrically driven flow there is intraparticle transport. In (A) transport of solute into the pores is accomplished solely by diffusion, whereas in (B) the EOF enhances transport through the pores. Reprinted from ref. [125] with permission. Copyright Elsevier 1999.

sizes larger than 200 nm was initially reported by Remcho and coworker [ 123]. Later Stol et al. showed intraparticle EOF in particles with pore sizes between 50-400 nm [122,124]. The EOF transport through wide-pore silica has been reported even in particles entrapped in capillary columns via sol-gel processing [45]. Double layer interactions within the pores are minimized by controlling the concentration of the electrolyte in the mobile phase. EOF through the pores is observed at high concentration of electrolyte, while at low concentration of electrolyte, double layer interaction occurs and transport through the pores can be stopped [122,123]. The effect of electrolyte concentration on separation efficiency for columns packed with 7 ~m (C18) particles containing pores with a diameter of 400 nm (nominal value given by manufacturer) is illustrated in Fig. 4.20 [122]. The flow through the pores provides for an enhanced mass transfer, resulting in improved separation efficiencies. Separation efficiencies of 430,000 theoretical plates/rn have been reported for the columns packed with the 7 ~m (C18) particles having 400 nm pore diameter [122]. The efficiencies and linear velocities observed with the highly porous packing materials are comparable to those obtained with small particle sizes. Therefore, the use of the highly porous large packing materials have been proposed as an alternative to the very small particles to perform fast separations with high efficiency in CEC [122,123,125]. The wide-pore large particles also offer the advantage of easier packing than submicron material, since aggregation is minimal. However, highly porous particles have the disadvantage of being fragile; hence, care must be exercised during packing to avoid damage of the particles. The enhanced particle porosity will also affect sample capacity adversely and the increase in ionic strength to maintain a thin double layer can lead to heating of the column.

Packed Bed Columns

13 9

10

7.5

H (pm) 5

2.5

0

1

2

3

4

u (mm/s) Fig. 4.20. Effect of the electrolyte concentration in the mobile phase on separation efficiency in a packed bed containing 7 pm highly porous C18 particles (Nucleosil 4000-7). Reprinted from ref. [122] with permission. Copyright Elsevier 1999.

4.2.3 Retaining frits The flits retaining the chromatographic packing material inside the capillary column in CEC seem to be the "Achilles heel" of the packed column fabrication process. They are the major problem in column manufacturing and perhaps the most critical parameter influencing column performance in general [5,126,127]. Most typically, the flits are fabricated by sintering silica-base packing material by means of heating. Using this approach, the CEC column becomes fragile at the flit since during its fabrication the protective polyimide coating is removed. There also seems to be lack of reproducibility and reliability in the manufacture of the flits, particularly between laboratories. The characteristics of the packing material at the flit position change as heat is applied to produce the flits. This creates non-homogeneous packing at the flit, having different electrical resistivities when compared to the open and pack segments of the columns. The different electrical properties of the flit can contribute to non-uniformities in EOF, and lead to bubble formation at the boundary between the flit and the unpacked segment of the capillary [ 10,51,127]. Constructing flits with resistivities similar to either the packed or the open segment can minimize discontinuities in the column structure, hence decreasing non-uniformities in EOF. Bubble formation, which has also been attributed to heat generation as the electric field is applied, can be reduced by several means. The most common approach to avoid bubble formation is to pressurize the mobile phase at both inlet and outlet column reservoirs References pp. 159-164

140

Chapter 4

[ 10,14,17,27,128]. Other common practices include the use of well-degassed solvents, low concentrations of electrolytes, a relatively large amount of the organic component in themobile phase, working at reduced temperatures (e.g., 15~ when possible, and the use of low conductivity electrolytes (i.e., zwitterionic buffers). The addition of sodium dodecyl sulfate (SDS) into the mobile phase at low concentrations has also been used to minimize bubble formation [67]. The effects that frits and packing materials have on EOF seem inconsistent. For example, placement of two frits in a capillary column (without chromatographic packing material) has shown to be flow restrictive points, reducing EOF by 35% compare to a capillary without frits [56]. On the other hand, an increase in EOF has been reported for a column packed with ODS material when compared to an equivalent open tube [90,129]. The discrepancies can be attributed to differences in the materials and procedures used to fabricate the frits, since these appear to be the major differences reported [ 130]. The bed-retaining frits must posses high permeability to solvent flow, yet the flits must be mechanically strong to retain the packing material and resist the pressures used to pack and/or rinse the column. The heating conditions and method used to prepare the flits affects such characteristics. Pressure resistance studies have shown that, within certain constrains, correlation between pressure resistance of a frit and its influence on the EOF is likely to be insignificant [ 130]. 4.2.3.1 Silica-base frits

The fabrication of frits has been studied in detail by several researchers [27,55,56,129-131 ]. Behnke, et al. [27] studied the performance of columns fabricated using three different frit fabrication procedures. In one of the procedures, the frits were constructed by sintering (using heat) a plug of silica gel wetted with potassium silicate. The frits were mechanically stable; however, under CEC conditions the columns with these frits showed baseline and electrical current stability problems. Another procedure used the method by Cortes, et al. where the flits were formed by polymerization of a potassium silicate solution containing formamide [132]. The columns fabricated using these frits suffered from similar problems. The third method involved the sintering of a silica gel plug wetted with water. Packed beds retained by these frits showed a stable baseline and current. However, they lacked mechanical stability with relatively large column diameters (150 ~m I.D.); decreasing the column diameter to 50 ~tm I.D., increased the stability of the frit. In a different study, Chen and co-workers optimized the silicate polymerization method [ 131 ]. In their approach, the outlet frit is prepared by first filling the column with a sodium silicate solution. Then, the portion of the column at which the frit is desired is brought in contact to a heating element for a few seconds and the frit is

Packed Bed Columns

141

formed. The polyimide coating is not removed under the heating conditions, reducing column fragility at the flits. The excess of the silicate solution is removed by pressure. The flit is then silanized with a solution of 0.02 mol/1 trimethylchlorosilane in DMF, using imidazole as an acid acceptor. The column is packed and the inlet frit is constructed by a quick dip of the column entrance in another silicate solution and heating. Optimal sodium silicate solutions were found to be 10.8% and 5.4% (w/v) for outlet and inlet flits, respectively. The method uses short heating times: 5-6 seconds for the outlet flit and about 1 second for the inlet one, producing relatively short flits (-~75 ~tm). Columns using these flits have been run without pressurization and showed to be mechanically stable without electrical current stability or bubble formation problems over a wide range of acetonitrile-water mixtures, even under relatively high currents conditions (-27 ~tA) [ 131 ]. The most commonly used approach to fabricate silica-base flits, however, is to sinter the actual chromatographic material in place. Care must be taken, however, in order to minimize degradation of the alkylated silica that will not be part of the flit [24]. Heating time used for sintering depends on column I.D., particle size, and type of stationary phase material to be fritted [133]. During heating, a substantial number of silanol groups can be created, changing the surface charge of the material at the flit [ 130]. Excessive heating also damages the outer side of the capillary column, which can create possible adsorption sites. Fig. 4.21 illustrates the effect of using excessive heating to fabricate the flit. Panel A in Fig. 4.21 shows a magnification of the outer side of the column and panel B shows the entire end of the column. Fig. 4.22 shows an adequate sintered frit. It has been shown that decreasing the created silanols by resilanizing the flits can reduce the likelihood of bubble formation, creating a uniform structure that resembles the packed bed, and leads to an improved separation [55]. Fig. 4.23 depicts electroctrochromatograms for the separation of a test mixture on 3 ~tm Spherisorb ODS 1 packed beds (a) before and (b) after resilinazing the outlet frit with ODS. Silanization deactivates undesirable adsorption sites at the flit with which the analytes can interact [ 134]. The packing functionality can also affect the final properties of the flits. This has been shown on flits formed in open tubes using different silica with different functionalities: bare silica, strong cationic exchanger (SCX), strong anionic exchanger (SAX), and Hypersil ODS silica, without the contribution of any other packing material present [130]. The EOF velocity obtained with each flit is different, depending on residual groups of the different packing. Frits formed with bare silica and SCX showed EOF velocities higher than an open tube and flits of ODS material. The amount of residual groups on the surface of the silica support depends on the heating time applied during the formation of the flit, and can contribute considerably to the overall flow. In a column containing a small flit formed with SAX material a reversal

References pp. 159-164

142

m

<

Chapter 4

6

. ,....~

.,,.~

0

r.~

0

< . ,...~

. ,....q

0

0 ez~

~D r.~ r.~ X 9

("q

143

Packed Bed Columns

A

13

Fig. 4.22. Adequate frit without deformities on the capillary, particles are 1 pm in diameter.

of the EOF has been observed, when compared to that in an open tube, under otherwise similar experimental conditions. The temperature of the filament used to fabricate such a frit was 430~

References pp. 159-164

and heating time between 12 and 15 seconds. If the

Chapter 4

144

(a)

Before rccoating the fdt e

t ,oo-{

b a,

400

I] II

d

em

8l~

1000

1200

a Dimethyl phthalate b niU'obmzcne

1400

Ttme / M.mutes (b) After recoating the frit a

l l

I

x

5/)0

11 I [

I I

lO~X)

e

{~,_ D~.e~hylph~d~ a

II b

I

15oo

~obe~.e

It c. anisole

~1)o

2~/)o

Tune / 1,~u~es

Fig. 4.23. Electropherograms of a test mixture obtained in 3~m Waters Spherisorb ODS-1 packed bed (a) before and (b) after recoating of the outlet frit with ODS; mobile phase, 70:30 acetonitrile-water. Reprinted from ref. [55] with permission. Copyright Wiley-VCH 1999.

heating time is beyond 15 seconds, the EOF towards the positively charged electrode is reduced considerably, indicating a reduction of the residual positive charge responsible for the EOF reversal [130]. It is important to realize how critical is the heating during frit fabrication, as illustrated with Figs. 4.21; the process must be optimized, requiring experienced individuals. An alternative to sintering frits, which deserves mention here, is to form frits via UV photopolymerization of a glycidyl methacrylate and trimethylolpropane trimethacrylate solution (UV radiation, 365 nm for 1 hour) [135]. The photopolymerization process is similar to that used in the fabrication of monolithic columns (Chapters 5 and 6). Frits fabricated with this method have shown to be reproducible; since there is no sintering of packing material, weakening of the capillary column by removal of the polyimide coating and/or alteration of the stationary phase at the frit are avoided.

4.2.3.2 Fritless packed beds As an alternative to the formation of frits, the packing material can be retained by means of tapers fabricated on the fused silica column. In such a case, the packed bed is completely fritless. Two types of tapered capillary columns have been prepared for CEC: internally [136,137] and externally [133,138] tapered, which have been shown

Packed Bed Columns

145

A

B Fig. 4.24. Schematic representation of (A) external and (B) intemal tapers at the outlet of the CEC column. Adapted from ref. [ 136] with permission. Copyright Elsevier 1998.

to be useful in coupling CEC with mass spectrometry and NMR (Chapters 2 and 8). A schematic representation of the tapers is shown in Fig. 4.24. The internally tapered columns are fabricated by sealing the end of the capillary by means of a high temperature flame; the sealed end is carefully grinded to produce a small opening. A laser-based micropipette puller is utilized to make the externally tapered columns. In an attempt to have a fritless column, a capillary having an external taper was packed and the tapered end served as the entrance to the column; no outlet flit was constructed [133]. The problem with these columns is that they can only be used if the electrophoretic mobility of the packing material is larger than the EOF generated, so it can remain in place. Even in such cases, it can be problematic to keep the packing material inside the capillary. The external tapers are weak points on the column, prone to breakage; hence, externally tapered columns are inherently fragile. The internally tapered columns, on the other hand, are not fragile since only the inner diameter is reduced in dimension not the outer diameter and are easily connected to other tubing via shrink tubing. A manufacturing procedure for an internally tapered column is shown in Figs. 4.25 and 26. In Fig. 4.25, the internal taper is prepared at the outlet end of the packed bed, using a sintered frit at the entrance of the column. Fig. 4.26 describes the procedure for a fritless column. The internally tapered capillary columns can also facilitate the procedure of column packing by obviating the need for having a temporary retaining frit (vide infra). This approach holds great promise since it eliminates the problems associated with frits. Because of the connecting tubing, there is potential for band broadening, although it can be minimized.

4.2.4 Fabricating columns The most commonly used procedure to fabricate a packed capillary column for CEC is depicted in Figs. 4.27 A and B. Although several laboratories may have slight variations, the general procedure is as follows. A piece of fused silica capillary of a

References pp. 159-164

Chapter 4

146

Preparing the tapers A e.g. 60 cm length of fused silica capillary is sealed at the middle with a microflame torch.

fused silica capillary

The seal is cut to yield two tapered capillaries. Non-tapered end to be coupled are ground plane and smooth with P4000 (wet). Tapers are ground to form the desired orifice (i.d. 10-50 lam).

Coupling the segments dead volume free

internal tarter

The ground ends are carefully aligned and pushed together. The dual PTFE/FEP-connector is shrunk onto the junction. Slurry preparation 10 - 20 mg beads are ultrasonicated for 20 minutes in 70 - 150 lal acetone (or methanol). Packing the capillary The slurry is flushed in.

The stationary phase beads are allowed to settle under ultrasonication. Pressure drop to zero over a period of 20 min. The capillary is flushed with water.

e)

dual PTFE/FEP shrink-tube-connector

AP = 500 bar b.~ slurry

30 min. sonication AP = 500 bar acetone (resp.methanol)

30 min. sonication AP = 500 bar H20 r

Frit sintering (T=500~

AP = 500 bar

h~ p~

H20 f)

Burning the detection window

g)

Conditioning

inlet flit Column is flushed for 20 minutes with mobile phase (AP = 150 bar) followed by Electrokinetic conditioning:45 rain. at 10 kV with a 25 min. voltage - ramp 45 min. at 15 kV with a 5 rain. voltage - ramp g)

Storage Column is flushed for 30 rain with iso-propanol (AP = 150 bar) Capillary is stored with both ends immersed in iso-propanol filled vials

h)

~

[

UV - window

/

Internal taner

.............

25cm

i

0.5-2cm

i

lOcm

i

Fig. 4.25. Procedure to fabricate a single-flit CEC capillary column. Reprinted from ref. [137] with permission. Copyright Elsevier 2000.

desired length is selected, usually about 10 cm longer than the actual column length wanted. Usually, the capillary tube is rinsed before use; it is our practice to rinse the tube with a sodium hydroxide solution (-0.1 mol/1) and then water. A provisional frit is sintered at one end of the capillary tube. Typically, this is accomplished by tapping onto a pile of wet silica material, allowing a small section of the column end to pack;

Packed Bed Columns

147

fused silica capillary

a) Preparing the tapers

b)

c)

Slurry Preparation

internal taper

Packing the capillary

AP = 500 bar

The slurry is flushed in.

~,~

slurry

The stationary phase beads are allowed to settle.

60 min sonication AP = 500 bar

Pressure drop to zero over a period of 20 rain.

acetone (resp. methanol)

Dry out overnight. d)

dual PTFE/FEP shrink-tube-connector

Coupling the segments dead volume free The ground ends are carefully aligned and pushed together. The dual PTFE/FEP-connector is shrunk onto the junction.

d)

Burning the detection window

e)

Conditioning

Storage g)

T w o - peace Fritless Column internal taper

i

i)

I

25cm

2cm

UV - window

[

10cm

I

T h r e e - peace Fritless Column int~rnnl tarter

I

25cm

internal tarter

I

1-2cm

U V - window

I ~

I

,0om

I

Fig. 4.26. Procedure to fabricate a fritless CEC capillary column. Reprinted from ref. [137] with permission. Copyright Elsevier 2000.

then heat is applied to sinter the material. The amount of heat required to form the flits depends on the column diameter and particle diameter [133], as well as the heating element used. Different heating elements have been used and they vary from

References pp. 159-164

Chapter 4

148

A

B

,

C

D

>

===C,

.

.

.

.

.

.

_1 .

.

.

.

.

/" wet silica

heating element

frit

flush

Fig. 4.27A. Representation of the steps involved in the column fabrication processes: (A) introducing with wet silica material into the end of the capillary, (B) the silica material is ready to fabricate the temporary frit, (C) fabrication of the temporary frit with a heating element, and (D) excess of silica material is flushed out after temporary frit is made.

optical splicers [28,123] thermal wire strippers [28], microtorch [18], burners [56], to heating elements incorporated into more sophisticated assemblies [ 14]. One relatively inexpensive setup used in our laboratory, makes use of a soldering gun fitted with a Nichrome ribbon (1 mm thick) as the heating element. The ribbon is punctured making a small hole (-0.5 mm) through which the column can be inserted; this facilitates heating of a small spot at any desired point of the capillary. Once the temporary frit is in place, the column is flushed to remove the excess material used to fabricate the frits. The mechanical stability of the frit can be tested by applying pressure using a HPLC pump. The frit must resist the packing conditions; yet, it should be permeable enough to allow solvent flow. The temporary flit is eventually removed. An alternative to the temporary frit is to connect the end of the capillary column to a union containing a metallic frit; this will retain the packing material inside the column during the packing process. The capillary is then packed to a desired length (vide infra). Once packed, the capillary column is rinsed with water,

149

Packed Bed Columns

-

2nd

1st

<~

I

H20

temporary frit

B

I

Fig. 4.27B. Formation of retaining frits once the capillary column is packed, (A) packed column is pressurized with water and the retaining frits formed using a heating element and (B) a fabricated column with frits and detection window in place.

and while under pressure the retaining flits are fabricated. Typically, the outlet flit is formed at a predetermined distance from the temporary flit. The inlet flit is then fabricated at a desired length (typically 20-30 cm) from the outlet flit. The use of organic solvents to flush the capillary column during frit formation is not recommended since carbonaceous material, presumably from the solvent, can be formed and remained at the flit. After the frits have been fabricated, the temporary frit is cut off and the excess particles inside the capillary are flushed out using a pump. In most cases, the heat applied to form the outlet flit is sufficient to remove a portion of polyimide coating on the capillary that is extending towards the open segment of the column. This exposes part of the fused silica, serving as the optical window for spectroscopic detection methods. After the flits and the optical window have been fabricated, the column must be rinsed with well-degassed mobile phase prior to connecting it to the CEC system. Once the column is connected to the CEC separation unit, it is a good practice to equilibrate the column with the mobile phase by applying voltage across the column in a stepwise manner. This can aid to consolidate the packed bed that may have voids [139]. The effect of having voids in a packed bed on peak shape is shown in Fig. 4.28. One approach to condition the CEC column is to apply 5 kV with the mobile phase in the column, and then the voltage is increased in 5 kV steps until 25-30 kV is reached. The column is allowed to equilibrate at each step

References pp. 159-164

Chapter 4

150

n'l/~:

0 '

/

4

'

'

I

6

'

'

'

I

'

'

'

I

8

10

8

10

'

'

'

""1

rrin

1NI

0~

Fig. 4.28. Electropherograms showing the effect of having a void in the packed bed (a). The peak shape improves after the bed is consolidated under voltage. Column packed with ODS-1, 3 l.tm, 75 l,tm I.D., 25 cm long (33.5 cm total). Mobile phase was 60:40 acetonitrile-2 mmol/1 KH2PO4. Reprinted from ref. [139] with permission. Copyright Elsevier 2000.

until a stable electrical current is observed. 4.3 PACKING METHODS

Several methods have been used to pack chromatographic particulate into capillary columns for CEC. These packing methods have made use of pressure packing using slurries [ 14,17,20] or using supercritical CO2 [30,43], electrokinetic packing [47,48], pseudo-electrokinetic packing [140], centripetal forces [49,141], and packing by gravity [50]. CEC columns packed by either electrokinetic or slurry pressure packing are commercially available. 4.3.1 Pressure packing

The most commonly used method to pack capillary columns for CEC utilizes the slurry packing methods typically used in HPLC. The capillary tube with a temporary frit is connected to a packing reservoir, such as a short HPLC column or another viable unit, which is connected to a high-pressure pump for solvent delivery. The slurry at a concentration of 50-100 mg/ml is prepared in a suitable solvent. Table 4.3

Packed Bed Columns

151

TABLE 4.3 SELECTED LIST OF SOLVENTS USED TO FORM SLURRY OF VARIOUS PACKING MATERIALS TO PACKED COLUMNS FOR CEC

Packing material

Slurry solvent

Packing solvent

Alltech 1.5 gm silica

Water

Water

Alltech 1.5 ~tm ODS AB

Hexane, THF b

Acetone

Alltech 5 gm SCX/C 18

THF, AcN c

Acetone

Astec 5jam Cyclobond I

AcN, THF, MeOH d

Acetone

Hypersil 3 jam ODS

Hexane, 2-propanol, THF Acetone, hexane

Hypersil 3 jam MOS

AcN, hexane, THF

Acetone

Hypersil 3 jam Phenyl

Hexane, MeOH, THF

Acetone

Micra Scietific 1.5 jam NPS ODS I

Hexane, THF

AcN, acetone

Micra Scietific 1.5 jam NPS ODS II

Hexane, THF

AcN, acetone

Micra Scietific 1.5 jam NPS C 18

Hexane, THF

AcN, acetone

Micra Scietific 1.5 jam NPS TAS-1

THF

AcN, acetone

Micra Scietific 3 jam NPS

AcN, water

Acetone, water

Spherisorb 3 jam ODS-1

Hexane, THF

Acetone

Spherisorb 3 jam silica

AcN, water

Acetone

Spherisorb 5 jam SCX

THF

Acetone

aReproduced from from ref. [ 139] with permission. Copyright Elsevier 2000. bTHF - tetrahydrofuran CAcN- acetonitrile dMeOH - methanol

shows a list of solvents used to prepare slurry of packing materials [139]. Sonication of the slurry is recommended to aid dispersing the packing material before the slurry is placed in the packing reservoir. The packing material is delivered into the capillary column at pressures of about 35-70 MPa (5,000-10,000 p.s.i.). Sonication can also be used during packing [20]. The pump is tumed off once the capillary tube is packed. To ensure that disturbance is not introduced to the packed bed when disconnecting the column from the packing reservoir, the column is allowed to "bleed" for a period of time. This means that the column is not disconnected until the system reaches ambient pressure. Then, the column is rinsed with water and the retaining frits are fabricated. References pp. 159-164

152

Chapter 4

The column is rinsed with the mobile phase by pressure and further equilibrated while applying the electric field.

4.3.2 Packing with supercritical C02 Supercritical CO2 packing was originally used to pack capillary columns for HPLC and supercritical fluid chromatography (SFC) [142,143]. The method is also used to prepare columns for CEC [30,43]. In this approach, one end of the capillary is attached to a pressure reservoir, which can be a short 2 mm I.D. HPLC column containing dry packing material. The other end of the capillary is attached to a connecting union containing a metallic frit; a temporary frit can be used instead. At the other end of the connecting union, a restrictor such as a piece of small fused silica capillary with a diameter of about 10 ~tm is attached to maintain the pressures required for supercritical conditions. During packing, the capillary column is immersed in an ultrasonic bath. The temperature of the bath is maintained at 60-70~

well

above the critical temperature of CO2. The column is packed at a constant pressure above the CO2 critical pressure, typically at 20-30 MPa (about 3,000-4,500 p.s.i.). To avoid disturbances to the packed bed, the capillary column is depressurized over a 4-5 hour long period of time. The column is then rinsed with water and the frits formed as described above. The column is flushed with the mobile phase by pressure and then equilibrated with mobile phase while applying the electric field.

4.3.3 Electrokinetic and pseudo-electrokinetic packing An alternative to pressure packing is the use of an electrically driven flow [47,48] to deliver the packing material. It is claimed that the particles are driven into the capillary column by the EOF, while the column and packing reservoirs are vibrated. Particles are suspended in a methanol-water mixture containing an electrolyte and sonicated before packing. An electrokinetic packing system used in our laboratory is depicted in Fig. 4.29. The slurry containing the suspended particles is placed in the upper vial, maintained in a vertical upside down position. A rubber septum cap is used for the vial, through which the capillary is inserted to reach the slurry. An electrode is also placed in the vial, serving as the anode. The outlet end of the column containing the temporary frit is inserted through a septum cap on the second vial, which also contains an electrode serving as the cathode. To aid the packing process, each vial is mechanically agitated. The agitation can be performed by attaching each vial to a speaker connected to a music system or a frequency generator. The electric field is applied until reaching 30 kV and maintained constant until the column is packed. Once the column is packed, the voltage is turned off, the column removed and pressurized with water. Then, the retaining frits are fabricated and the column equilibrated

Packed Bed Columns

153

Islurry vial electrode

J

capillary column

y

particle flow

/'I"

e,ectrode/-/ solvent reservoir speaker Fig. 4.29. Schematic of electrokinetic packing apparatus. as stated above. One of the advantages of electrokinetic packing is that the use of special fittings and high-pressure pumps are not required. The method can also be carried out at low operational cost and allow for the simultaneous packing of multiple columns. A pseudo-electrokinetic packing procedure has also been utilized to pack CEC columns [ 140]. This approach incorporates the use of a high electric field and hydrodynamic flow to pack the capillary columns. The column, equipped with a temporary frit, is mounted in the CEC unit and flushed with background electrolyte (e.g., 75:25 methanol-water mixture containing 10 mmol/1 Tris buffer). The entrance of the capillary is placed in the slurry, which is made using the background electrolyte. A portion of the slurry is pumped into the column to partially fill the capillary tube. Voltage is applied and the material inside the capillary packs against the temporary frit; packing References pp. 159-164

Chapter 4

154

is completed in approximately 15 minutes. Once the column is packed, it is connected to a conventional HPLC pump, flushed with water, and the permanent frits fabricated. The column is then preconditioned with mobile phase prior to use.

4.3.4 Packing by centripetal forces Packed bed columns for CEC can also be obtained by using centripetal forces [49,141]. Packing of the particles is obtained by centripetal acceleration through the capillary column. The velocity of the particles is given by the sedimentation velocity (used) as follows [49]:

(4.5)

(Pdp -- 90) V 0) 2 r Used =

3 ~ rl dp

where 9dp and Po are the density of the particle and medium respectively, V is the volume of the particle, co is the rotational speed, r is the distance from the center of rotation, 1"1is viscosity of the solvent in which the particles are dispersed, and dp is the particle diameter. A schematic diagram of a system used to pack capillary columns by centripetal forces is presented in Fig. 4.30. Particles are slurried in an appropriate solvent (-10-50 mg/ml) and placed in the slurry reservoir; the velocity of the particles is higher in low viscosity solvents such as acetone. In the apparatus shown in Fig. 4.30, two columns containing temporary frits are connected to the reservoir. Two

cell

slurry reservoir Column i

l

stainless steel support arm

Motor

pu ley Fig. 4.30. Schematic of apparatus to pack by centripetal forces.

Packed Bed Columns

155

extending arms support the capillary columns. This means that the packed columns are inside the extending arms, which are made of 1 mm I.D. stainless steel tubing to avoid wrapping of the flexible fused silica tubes around the central reservoir during packing. Rotation of the apparatus forces the particles to move into and through the columns, sedimenting at the fritted-end. Columns are packed in about 5 to 15 minutes, depending on the solvent used to prepare the slurry. For example, 25 to 30 cm length of a 50 ~tm I.D. capillary column is packed in 5 min at 2,000 rpm using a 10 mg/ml slurry of 3 ~tm ODS packing material in acetone. Once packed, the column is submitted to the same procedure of rinsing and frit fabrication above-mentioned. Packing by centripetal forces also provides for the packing of multiple capillaries at once. A bed-drying step has been introduced into the packing procedure, once the column is packed by centripetal forces [ 141]; although this can be applied to any other packing procedure. The drying step is performed prior to frit fabrication and after frit formation the packed bed is resolvated. This step increases the column efficiency by about 15-20% and retention by 13%. However, despite the improvements, the drying step adds time to the column fabrication protocol because of both additional drying and rewetting of the capillaries. Perhaps it may not currently be worth to consider such step for a relatively small gain. However, if a more convenient dry-packing procedure is developed and the column preparation time can be similar to the wet procedures, it would be advantageous to use this approach.

4.3.5 Packing by gravity Particles can also be delivered into the capillary column by means of gravitational forces [50], which is also based on sedimentation velocity given by the following expression [ 144,145]:

Used =

(1-~) -k [13o(1-~,) + 19o(ec--1)] ~ g 18q

(4.6)

where Used is the sedimentation velocity, ~ is the volume fraction of the particles in the slurry solution, -k is a particle/solvent-dependent constant, 9p is the density of the skeleton of the particle material, 9o and 1] are the density and viscosity of the liquid, respectively, 8i is the particle porosity, ef is the fraction of the total hydrodynamic particle volume that is filled with suspension liquid, g is the gravitational constant, and dp is the particle diameter. In this method, gravity is used to transport the packing material into the column. The packing device is a 1-ml syringe containing about

References pp. 159-164

156

Chapter 4

100 pl of a 10 mg/ml slurry in acetone, which connects to the open end of the capillary to be packed via plastic tubing attached to the syringe needle. To avoid slurry solvent evaporation, the syringe plunger is attached and secured in place by means of a screw top. Capillary columns with a temporary frit are filled with acetone and connected to the packing apparatus. Sedimentation is allowed to proceed for a period of at least 10-12 h, replacing the slurry with a freshly sonicated one about every 4 hrs. Once packed, the columns are submitted to the rinses and frits fabrication procedures mentioned above. Columns with inner diameter of 50 ~tm and length of 20-28 cm have been packed with 3 ~tm particles [143]. Columns have also been packed with submicron packing material using this method [ 119]. This packing procedure is relatively simple, however, relatively long times of 12 to 48 hours, depending on particle size, are required to fill the capillary columns, before they are submitted to any pressurization step. 4.4 COMPARISON OF PACKING PROCEDURES The reported efficiencies for columns packed by the different methods vary considerably. Many factors can be attributed to this effect: experience of the analyst in making the frits, the use of different packing materials (although with the same nominal particle size), the use of different separations systems such as different sizes of the illumination spot on the optical window where detection is performed, that can be 200 ~m-2 mm long and localized after or before the end frit, and different separation conditions. These many variables make it difficult to assess which packing procedure offers the best-packed column by just examining results described in the literature. The effect of these variables must be minimized in order to achieve a true comparison among packing procedures. Ideally, one should compare columns fabricated using the same packing material under similar conditions, where the only difference is the packing method used. The same analyst should also construct the frits, perform packing and testing, using the same CEC apparatus. For a further comparison, experts in each packing technique can pack columns using the same packing materials and all columns would be then tested for performance by a single individual. This would indicate how much the experience on a particular packing method would influence the performance. In our laboratory, a study has been initiated taking into consideration all the above-mentioned factors. The column fabrication, packing, and testing have been performed by the same individual for columns packed by centripetal forces, electrokinetic packing, and pressure packing (slurry and supercritical CO2 packing). Fig. 4.31 shows electrochromatograms for a test mixture separated under similar conditions using capillaries packed by different protocols. Thus far, the data indicate that slurry

157

Packed Bed Columns

5

A

3 2

4 5

B

1

3 2 6

4

C

3

.......... v~ 5

D

3

.-____k I

I

I

1

0

2

4

6

Time/minutes Fig. 4.31. Separation of a test mixture on capillary columns packed by different methods: (A) pressure packing, (B) by centripetal forces, using supercritical fluid, and by electrokinetic packing. Columns were 50 pm I.D., 20 cm packed (30 cm total length); mobile phase 80:20 acetonitrile-4 mmol/1 aqueous borate. Separation voltage of 20 kV. Solutes: 1, thiourea; 2, benzyl alcohol; 3, biphenyl; 4, dimethylnaphthalene; 5, ethylnaphthalene; 6, amylbenzene.

References pp. 159-164

158

Chapter 4

pressure packing renders column with the lowest efficiency and that the highest retention is associated with columns packed by centripetal forces. Under the experimental conditions used, columns packed by supercritical CO2, centripetal forces, and electrokinetic packing showed similar separation efficiencies, about 216,000 plates/m, while pressure packed column showed about 150,000 plates/m, for the most retained compound. The columns packed by centripetal forces and by supercritical fluid showed retention factors slightly higher than the other two packing methods. This indicates that more retentive material is inside the column, which is the result of a tighter packed bed. From the four methods, electrokinetic packing is the simplest and easiest method to implement and use. A thorough investigation is in progress with all packing procedures at the time of this writings. 4.5 CONCLUSION Slurry pressure packing procedures have been the most commonly used to pack capillary columns for CEC; this departs from the well-established knowledge in packing columns for HPLC. The other methods discussed in this chapter, however, are also suitable to pack columns for CEC. In our experience, the other methods offer higher separation efficiencies. Further, packing of very small packing materials (< 1 lam) can be handled easier by the electrokinetic, centripetal, supercritical fluids and gravitypacking procedures, although with each technique the packing parameters must be optimized experimentally. Thus far, the preference of which method to use seems to depend on the familiarity of a given laboratory with a particular procedure. It is clear that column fabrication in CEC requires skill and experience, particularly constructing the retaining frits, a process that remains as one of the major problems in the column fabrication process, and improvement is still necessary. With the advances in CEC column technology, the problems associated with frit fabrication will be minimized. The monolithic and entrapped structures are clear alternatives, which will develop further. CEC practitioners may opt to obtain packed columns from commercial sources, as typically done with HPLC columns. This will avoid the implementation and optimization of a packing procedure in the laboratory, which can be laborious, time consuming, and require experience. 4.6 ACKNOWLEDGEMENT We acknowledge financial support from The National Science Foundation (CHE9614947).

Packed Bed Columns

159

4.7 ABBREVIATIONS

CEC CE EOF HPLC I.D. LC mL mM ~tm MS NMR ODS PTFE PAH p.s.i. SAX SCX SEM SDS SFC TRIS UV

capillary electrochromatography capillary electrophoresis electroosmotic flow high-performance liquid chromatography inner diameter liquid chromatography milliliter millimolar micrometer mass spectrometry nuclear magnetic resonance octadecylsilane polytetrafluoroethylene polyaromatic hydrocarbons pound per square inch strong anionic exchanger strong cationic exchanger scanning electron micrograph sodium dodecylsulfate supercritical fluid chromatography tris(hydroxymethyl)aminomethane ultraviolet

4.8 REFERENCES

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

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