8=MCPA, 9=Mecoprop, 10=Dichlorprop, 11=2,4,5-T, 12=2,4-DB, 13=MCPB, 14=2,4,5-TP,. 15=Dinoseb, 16=Dinoterb, 17=Pentachlorophenol. Reprinted from ...
Capillary Liquid Chromatography Capillary and Micro‐High Performance Liquid Chromatography Norman W. Smith1 Cristina Legido-Quigley2, Nicola D. Marlin2 and Virginie Melin2 1
Microseparations Group, Department of Pharmacy, King’s College London, Franklin-Wilkins Building, 150 Stamford Street, London SE1 9NN 2
Centre for Analytical Sciences, Department of Chemistry, Imperial College London, Exhibition Rd, London SW7 2AY
KEYWORDS Capillary HPLC, micro-HPLC, mass spectrometry, nano LC, sensitivity gain, flow splitter, monolithic stationary phase, micro-gradient, proteomics.
1 INTRODUCTION Liquid Chromatography is the term used to describe the separation of a solution following differential migration in a liquid flowing through a column packed with solid particles [1]. Miniaturized liquid separation techniques began in the late 1960’s. In 1967, Horváth et al. developed stainless steel columns ranging from 0.5 to 1.0 mm internal diameter (i.d.) packed with pellicular particles for the separation of nucleotides [2, 3]. In the mid-1970’s, Ishii et al. prepared 0.5 mm i.d. x 1.5 m long columns using pellicular particles and introduced slurry-packed teflon microcolumns [4, 5]. In the same decade, Scott et al. prepared 10 mm i.d. x 10 m long columns for the separation of alkylbenzenes [6, 7]. In the late 1970’s and early 1980’s, improvements were made to the technique and in this way the criteria were set for the next three decades of research [8, 9].
1
In conventional HPLC, columns are usually of 10–25 cm length and 2–4 mm internal diameter. Recently, capillary HPLC columns have become available on the market. The terminology, abbreviations and definitions have not yet been standardized. In the literature we can find microbore, micro-LC, semi-micro-LC, capillary LC and nano LC. In general, microbore or narrow-bore columns are considered as those with an internal diameter of less than 2 mm. From 1mm down, micro-LC is more appropriate. Capillary columns are described as those of less than 0.5 mm i.d. and nano LC columns usually have an i.d. of less than 0.1 mm. Table 1. Dimensions in chromatography
Application Micro LC Capillary LC Nano LC
Column I.D.
Flow rate
1 mm – 500 µm
40 µl/min – 20 µl/min
500 µm – 100 µm
20 µl/min – 300 nl/min
100 µm –75 µm –50 µm
300 nl/min – 180 nl/min 80 nl/min
The advantages of using micro-flows instead of normal flows are significant. An important advantage is the use of less mobile phase as illustrated in Table 1. This addresses both the cost of purchase and the cost of disposal of these solvents. It also diminishes the environmental impact of the toxic solvents. Arguably the most important advantage of using micro-flow through capillary columns is the higher sensitivity that can achieved. When scaling down the internal diameter of a column, the analysis becomes more sensitive. The increased mass sensitivity is attributed to the reduction in column internal diameter, which results in reduced dilution of the chromatographic band during analysis. This is very important when determining compounds present at low concentrations in limited sample volumes. Yet again, another important direct consequence of the small flow rates delivered is the excellent conditions for coupling micro-HPLC to a Mass Spectrometer.
2
1.1 Theory 1.1.1 The Rate Theory of Chromatography and Important Parameters The rate theory of chromatography, also known as the van Deemter model, examines the factors affecting band broadening, which is the amount of dispersion of a sample as it migrates through a column. The efficiency of the column can be approximated by the following expression known as the classical Knox equation [10]: (1)
The equation tries to account for all the kinetic processes that occur when a separation is undertaken. A, B and C are coefficients which relate to the three different processes. A being eddy diffusion, B/u the longitudinal axial diffusion, Cu the mass transfer term and u the mean linear velocity. Van Deemter et al. [11], Giddings [12], Huber [13], and Horváth et al. [14] have used alternative models to account for the relationship between the solute concentration in the stationary phase, its mobile phase concentration and the various parameters characterising the chromatographic system used [15]. In general, when all the contributions of the parameters are accounted for, the separation will be performed at the flow depicted by the smallest H value i.e. the greatest efficiency. Efficiency is a measure of how good the chromatographic column is performing. Another term that accounts for efficiency is the value N, which is called the number of theoretical plates. The term “plates” originates from the theory of distillation columns, where equilibrium between two phases occurs in each plate. For liquid chromatography, a column can be thought of as a series of “plates” where
3
equilibration of the analyte occurs between two phases. The greater the number of plates, the more efficient the separation. A useful way of describing efficiency is by taking into account the length of the column L, in this way calculating the height equivalent to a theoretical plate (H) or (HETP). (2)
Hence, an efficient separation will have a small H value. When columns of various lengths are compared it is more useful to use plates per meter N' as a measure of column efficiency: (3)
Where l is the length of the column in meters. N is widely used to compare columns, but this can only be done when the particle size and test analytes are the same. The reduced plate height (h) can also be calculated, which takes into account the diameter of the particles (dp):
(4) Next, we will consider the important factors when using these smaller diameters. The Down-Scale Factor was studied by Ryan in 1996 [16] and it applies to all components and parameters of the system, such as flow rates, injection, detection volumes and connecting capillaries [17]. This factor is very important for the successful transfer of a conventional method to a microscale equivalent. The equation below shows that this factor is equal to the partition of the squares of both diameters:
f=d2conv/d2micro
(5 )
Where dconv and dmicro are the diameters of the conventional and micro-scale columns.
4
1.1.2 Required Flow Rates When the column internal diameter is reduced, the mobile phase flow rate is also reduced to maintain the same efficiency. The column vacant volume can be expressed as:
Vo=πdc2ЄL/4
(6)
And the volumetric flow rate can defined as:
u = 4F/πdc2 Є
(7)
In order to maintain the same linear velocity for columns of different internal diameters, the volumetric flow rate should be decreased in proportion to the square of the ratio of the column internal diameters. 1.1.3 Maximum Injection Volume and Maximum Sample Mass As the column diameter decreases, the loading capacity of the column also decreases, thus the maximum sample injection volume has to be reduced accordingly. The following equation describes the maximum injection volume that can be injected into a column:
Vmax=θKπЄ dc2L(k+1)/√N
(8)
Where θ is the fractional loss of the column plate number caused by the injection, K is a constant defining the injection profile, L is the length of the column, k is the retention factor and N is the theoretical plate number [17]. The following equation relates the maximum sample mass to the maximum sample concentration that can be eluted from the column:
5
Mmax=CmπЄ dc2Lk/2√N
(9)
In practice, as stated by Chervet [17], for a 75 µm i.d. x 15 cm packed column, the maximum injected sample mass (loading capacity) for an unretained compound (k=0) should not exceed 50 ng. 1.1.4 Detection Volume From Equation 2, it can be deduced that the scaling down factor for the detection volume can also be applied, but a good compromise between sensitivity, noise and peak dispersion would consist in using detection volumes similar to injection volumes. 1.1.5 In-Column Pressure Drop The linear velocity can also be expressed based upon other column conditions as:
u=∆Pdp2/ΦηL
(10)
where ∆P is the pressure drop across the column, dp is the particle diameter, Φ is the flow resistance parameter and η is the viscosity of the mobile phase. Rearranging this equation gives:
∆P=ΦηLu/dp2
(11)
This demonstrates that the column backpressure is independent of the column internal diameter.
Therefore, microcolumns and analytical columns of the same length,
packed with the same particles and operated at the same linear velocity using the same mobile phase will generate similar backpressures. However, microcolumns are more susceptible to clogging, therefore care must be taken with filtering solutions and samples. The use of low dead-volume in-line filters are recommended.
6
1.1.6 Sensitivity Gain This describes how the sensitivity increases from a column of a defined internal diameter to a column of a smaller internal diameter. What is the maximum sensitivity gain possible in liquid chromatography by decreasing the internal diameter of columns? In 1995, Ryan proposed a model to adapt normal flow chromatography to micro-flow chromatography [16]. The theory states that when going from a column of diameter X to a smaller diameter Y, the gain in sensitivity would be equivalent to the partition of the squares of the internal diameters [X2/Y2]. Thus, in practical terms, when adapting a method from a 2.1 mm i.d. column to a 0.5 mm i.d. column, there would be a sensitivity gain of 2.12/ 0.52= 17 i.e. 17 times more response would be observed. From a 2.1 mm column to a 0.3 mm column the sensitivity increase could be as big as 49 times. Figure 1 illustrates the sensitivity gain going from a 1.0 mm i.d. column to a 0.3 mm i.d. column, after injecting the same amount of sample.
mA U
140 0 120 0 100 0 80 0 60 0 40 0 20 0 0
0.3mm
200ng Biphenyl Column: Zorbax SBC18
0.5mm 1mm
4.6mm
mi 2. 5 7. 1 12. 1 17. n 5 5 0 5 5 5 Figure 1. The response on four columns of different diameters after injecting the
0
same amount of sample; the sensitivity increases greatly with the smaller diameters.
7
2 INSTRUMENTATION Today micro-LC is a valuable analytical tool for sample-constrained applications such as proteomics and bioanalysis. The microliter flow rates are ideally suited for direct, splitless coupling with electrospray ionisation mass spectrometry (ESI-MS). Many benefits can be demonstrated from well-established HPLC theory, which allows for direct method transfer to micro-LC. Micro-HPLC has some key differences to conventional liquid chromatography. Microbore columns demand a very low flow rate, which needs to be delivered in a continuous flow. In generating sufficient flow through the columns, much higher backpressures are experienced, compared to standard HPLC. Also, the amount of analyte injected onto the column must be much lower, and all internal dead volumes must be minimized and reduced, otherwise band broadening effects will cause a significant problem. Conventional HPLC instruments cannot meet these specific requirements, and therefore are simply not suitable for micro-LC. It is possible to downscale standard HPLC equipment by employing flow splitters, capillary flow cells, micro-connectors and fittings, however these instruments can be unreliable. The problems can be grouped into three categories:
•
Detector signal to noise
•
Gradient formation
•
Flow splitting
The immediate result of attempts to scale down separations, is a lack of detector sensitivity. Analytical detector flow cells introduce excessive band spreading and low peak response, drastically reducing peak capacity and making peak integration all but impossible. Using on-line detection methods, by simply for example, passing the light across the capillary at the end of the column minimizes band spreading, but the short pathlengths can yield very low absorbance.
8
The other problem arises from the formation of accurate gradients under such low flow conditions. For gradients to be formed, one solvent must be accurately metered at only fractions of the total flow. One solution to this problem, ensuring microliter/minute flow rates, is to employ a flow splitter. Here the flow is split into two separate paths depending on the backpressure of the restrictor used, allowing flow to the column and to waste. While the ratio of these two pressures remains constant, a consistent flow rate can be achieved, however this ratio may change over time, due to the backpressure altering, resulting in the flow to the column changing. Another problem arising from splitting the flow as opposed to a system designed specifically to deliver micro-flows, are the greater costs due to the high solvent consumption. Figure 2 presents the two different configurations for flow splitting in micro-LC.
Column A
Pump
Detector Injector
Splitter
Waste or MS
Column B
Detector
Pump Splitter
Injector
Waste or MS
Figure 2. Flow splitting device. A shows the injector before the splitter, which means that both the flow and the sample are split before going on to the column. B shows the injector after the splitter, so just the flow is being split, and a full sample injection is going on to the column.
Micro-HPLC instruments are now commercially available and are designed to meet the exact requirements needed for micro-flows and offer the highest performance with ease of use. There are several micro/capillary LC instruments available on the market, and these include the Agilent 1100 Capillary LC™ system, LC packings Ultimate™ System, and Waters CapLC® System.
9
The Agilent 1100 Capillary LC works by splitting the relatively high flow rates to the column. The instrument utilizes a novel flow-monitoring device, called an electronic flow control (EFC), which monitors the flow rate and adjusts it accordingly. The device works by measuring the actual flow rate, rather than using calculations. This instrument is capable of providing flow rates as low as 1µl/min, running accurate gradients, and has a diode-array detector with a 500 nl flow cell. Waters CapLC® System The solvent delivery system in this instrument uses a syringe-style, positivedisplacement, continuous delivery design. Each syringe is motor driven and software controlled so that crossover-related flow phenomena are eliminated. Gradients can also be performed, and are mixed under high pressure to minimize the gradient delay. There is a dual wavelength detector, with patented low-volume flow cells (250 nl and 40 nl), which combine a long path-length and high light throughput (CapLC cell). This focuses and guides light from a deuterium lamp source along the length of the flow cell. Isocratic flow rates can be as low as 250 nl/min, while gradient flow rates can be generated from 1µl/min, while injection volumes can be as low as 20 nl. Ultimate™ fully integrated capillary HPLC system from LC Packings/Dionex This instrument uses a helium sparging device with four separately controlled solvent lines, ensuring optimal solvent degassing and improving check valve reliability at low flow rates, diminishing baseline disturbances at low UV wavelengths. The pumping system uses a high-precision reciprocating pump with proprietary microflow processing based on flow splitting. There are various calibrators that can be inserted to alter the extent of the flow splitting. This can generate flow rates from 200 µl/min to 50 nl/min. The system uses a micro-autosampler system to perform automated sample injection, but can also use a six-port low-dispersion injection valve for manual sample injection (1 µl minimum).
10
The instrument has a specially designed scanning UV-Visible absorbance detector, which can simultaneously monitor up to four different wavelengths. There are various interchangeable flow cells with volumes from 180 nl to 3 nl. They are used in conjunction with capillaries of 150 µm i.d. and 20 µm i.d. respectively. Micro pumps Pumps are now available that can deliver micro and nanoflows down to 100 nl/min, without the need for flow splitters. These are generally reciprocating or syringe pumps. One such pump is the MicroTech Ultra-Plus, which can deliver ultra-low flows using a micro-reciprocating piston, which is precise and digitally controlled. This can deliver flow rates to a minimum increment of 0.1 µl/min, and gradient flow rates as low as 0.01 µl/min with a split, and 5 µl/min without a split. Another pump, the Evolution 200 can also deliver low flow rates with high precision, with isocratic flows down to 0.1 µl/min, and gradient flows from 1 µl/min. This system uses binary high-pressure gradient formation using two linear drive piston pumps. With the use of very low diameter columns, dead volume becomes a key problem, and so manufacturers have gone to great lengths to try and minimize these effects. There are various column formats that have been employed for micro-LC, including fused silica capillary columns, glass-lined stainless steel, and glass-lined peek such as peeksil. Columns for capillary LC are often prepared in fused-silica capillaries. These capillaries have a polyimide outer coating to increase the robustness of the column. Generally the columns are packed with conventional HPLC chromatographic phases, with particle sizes of 3 and 5 µm diameter. Frits have to be manufactured in order to retain the packing material, and can be manufactured from sintering silica or directly sintering the packing material. The column can be packed using various methods, but the most common method is to use a slurry of the stationary phase. Once the initial end frit has been manufactured, the capillary can then be attached to a packing reservoir, which contains a slurry of the stationary phase. The packing reservoir in-turn is attached to a high-pressure packing pump that delivers the packing solvent. The packing material is generally
11
ultrasonicated during packing, to ensure a well-packed chromatographic bed, and then the second retaining frit is sintered directly from the packing material. More recently, columns have been developed where the stationary phase is formed of a porous polymer network inside the capillary. These are called monolithic phases, and have emerged as an alternative to traditional packed-bed columns for use in micro-HPLC. They hold many advantages over traditional packed bed columns, being easy to manufacture since the monolith is formed in-situ, often via a one-step reaction process, and its properties such as porosity, surface area and functionality can be tailored. Another major advantage is that they eliminate the need for retaining frits. These columns can be manufactured from a variety of polymer materials, but the most common include sol-gel, methacrylate-based, acrylamide-based and styrene-based polymeric structures. There are a few commercially available monolithic columns on the market today, one of the first being the Chromolith™ from Merck, which uses a silica-based polymeric network for the high-speed separation of compounds of low to medium molecular weight at high flow rates with only low backpressures. More recently, LC Packings (a Dionex company) have released the Monolith™, which is a polystyrenedivinylbenzene based monolith for the fast separation of proteins and peptides by micro-LC.
12
3 APPLICATIONS OF MICRO-HPLC Micro-HPLC has been used for the analysis of a wide variety of analytes, ranging from polyaromatic hydrocarbons (PAHs) and agrochemicals, to biomolecules. In these applications, the microcolumn (i.d. less than 1 mm) consisted of either a traditional packed bed or a monolith, i.e. a polymer network.
3.1 Proteomics Chen at al. [18, 19] reported the enantioseparation of dansyl amino acids on 100 µm i.d. ligand exchange-chiral monolithic microcolumns. Using continuous beds modified with chiral selectors such as L-phenylalaninamide, L-alaninamide, Lprolinamide, he achieved efficiencies of approximately 1000 plates/m. Czerwenka et al. [20] successfully performed the enantiomeric separations of a series of alanine peptides derivatised with different N-protection groups on packed beds containing a chiral stationary phase modified with the chiral selector tertbutylcarbamoylquinine. Huber’s research group [21] reported the high-resolution separation of peptides and proteins on poly(styrene-divinylbenzene) (PS/DVB) monoliths coupled with electrospray ionisation mass spectrometric detection (ESI-MS). More particularly, they were capable of separating and detecting very small amounts (lower femtomole range) of tryptic peptides of human transferrin (Figure 3), bovine catalase (Figure 4), standard proteins (Figure 5) as well as membrane proteins from the photosystem II of higher plants.
13
Figure 3 High-resolution capillary RP-HPLC separation of tryptic peptides of human transferrin in a monolithic column. Column, monolithic PS/DVB, 60 x 0.20 mm i.d.; mobile phase, (A) 2.0% acetonitrile, 0.050% TFA in water, (B) 80% acetonitrile, 0.050% TFA in water; linear gradient, 0-40% B in 30 min, 40-100% B in 10 min; flow rate, 1.7 µL/min; temperature, 50°C; detection, UV, 214 nm; sample, tryptic digest of 1.0 pmol of human transferrin. Reprinted with permission from [21]. Copyright 2003 American Chemical Society.
Figure 4. Separation and mass analysis of tryptic peptides of bovine catalase. Column, monolithic PS/DVB, 60 x 0.20 mm i.d.; mobile phase, (A) 0.050% TFA in water, (B) 80% acetonitrile, 0.050% TFA in water; linear gradient, 2-60% B in 15 min; flow rate, 1.8 µL/min; temperature, 50°C; scan, m/z 400-2000; electrospray voltage, 4.0 kV; sample, 5 pmol of tryptic digest of bovine catalase. Reprinted with permission from [21]. Copyright 2003 American Chemical Society.
14
Figure 5. High-resolution capillary RP-HPLC separation of 16 proteins in a monolithic capillary column. Column, monolithic PS/DVB, 60 x 0.20-mm i.d.; mobile phase, (A) 15% acetonitrile, 0.20% TFA in water, (B) 60% acetonitrile, 0.20% TFA in water; linear gradient, 28-93% B in 15 min; flow rate, 3.2 µL/min; temperature, 80°C; detection, UV, 214 nm; sample, mixture of 16 proteins, 200-350 fmol of each protein. Reprinted with permission from [21]. Copyright 2003 American Chemical Society.
Similarly, Huang et al. [22] reported the separation of standard proteins, tryptic digests of cytochrome c and myoglobin on both macroporous underivatised and octadecylated poly(styrene-divinylbenzene) monoliths. Hjerten’s research group [23] described the very fast separations (less than 100 s) of standard proteins and peptides on acrylamide-based monolithic capillary columns derivatised with C18 ligands. Schenk et al. [24] reported the quantification of cytokines in cell extracts using microHPLC packed microcolumns coupled with immunochemical detection. Various types of chromatographies were associated with the immunochemical detection system in order to perform the separation of cytokines. These included cation-exchange, size exclusion and reversed-phase chromatography, with the latter being the most successful. 15
3.2 Nucleic acids Huber’s research group [25-27] reported the rapid and highly efficient (efficiencies of up to 190 000 plates/m) separations of single-stranded oligodeoxynucleotides and double-stranded DNA fragments by ion-pair reversed-phase micro-HPLC (IP-RPmicro-HPLC) on poly(styrene-divinylbenzene) monoliths (Figure 6) and conventional capillary
columns
packed
with
micropellicular
octadecylated
poly(styrene-
divinylbenzene) particles. The coupling with electrospray ionisation mass spectrometry allowed the high resolution and identification of very small quantities of samples (femtomole amounts).
Figure
6.
High
resolution
capillary
IP-RP-HPLC
separation
of
phosphorylated
oligodeoxynucleotide ladders in a monolithic capillary column. Column, continuous PS– DVB, 60 x 0.20 mm i.d.; mobile phase, (A) 100 mM TEAA, pH 6.97, (B) 100 mM TEAA, pH 6.97, 20% acetonitrile; linear gradient, (a) 15–45% B in 3.5 min, 45–55% B in 2.5 min, 55–65% B in 4.0 min; flow-rate, 2.5 µl / min; temperature, 50°C; detection, UV, 254 nm; sample, p(dA)12-18, p(dT)12-30, 40-98 fmol of each oligodeoxynucleotide. Reprinted with permission from [25]. Copyright 2003 American Chemical Society.
16
Simek et al. [28] described the analysis and quantification of the purine bases hypoxanthine, xanthine and guanine in excreta of ticks using a traditional reversedphase C18 packed microcolumn.
3.3 Agrochemicals Cappiello’s research group [29-32] applied micro-HPLC to the screening of water samples for pesticide contamination. With electron ionisation mass spectrometric detection and the use of two microcolumns packed with a C18 silica-based stationary phase, they performed the pre-concentration of water samples and successfully detected trace levels of pollutants in similar samples. They also showed that ioninteraction micro-HPLC on a C18 stationary phase with hexylamine as the ion-pair reagent and coupling to particle beam mass spectrometry could be successfully used for the analysis of herbicides ranging from acidic species such as phenoxy-acids, to weak basic species such as phenol ureic herbicides, in a single analysis. An example of the separation of 17 acidic pesticides is featured in Figure 7.
Figure 7. Chromatographic separation of 17 acidic pesticides obtained injecting 50 µl of aqueous sample in different injection mode: (a) excluding and (b) including the loop in the
17
mobile phase path after completion of the injection process. Chromatographic conditions: (water+0.05%TFA)-(methanol+0.025%TFA), 100:0 to 20:80 in 40 min; flow-rate, 2 µl /min; sample concentration, 1 ng/µl. UV detection of pesticides was performed at 225 nm. UV detection of mobile phase concentration (solid line) was performed at 190 nm. 1=Picloram, 2=4-Nitrophenol, 3=Chloramben, 4=2,4-Dinitrophenol, 5=Dicamba, 6=Bentazone, 7=2,4-D, 8=MCPA, 9=Mecoprop, 10=Dichlorprop, 11=2,4,5-T, 12=2,4-DB, 13=MCPB, 14=2,4,5-TP, 15=Dinoseb, 16=Dinoterb, 17=Pentachlorophenol. Reprinted from [30], Copyright 2003, with permission from Elsevier.
Collins et al. [33] described the ultrafast (less than 1 min) separations of triazine herbicides and benzodiazepines on sol-gel bonded continuous beds filled with large pore octadecylsilica particles coupled with time-of-flight mass spectrometric detection. Efficiencies of up to 50 000 plates/m were achieved with the benzodiazepines.
3.4 Other aromatic compounds: PAHs (Polyaromatic hydrocarbons) and alkylbenzenes Maruska et al. [34] described the separations of polar aromatic solutes such as pyridine, 4-pyridylmethanol, 4-methoxyphenol, 2-naphthol, catechol, hydroquinone, resorcinol, and 2,7-dihydroxynaphthalene by normal-phase micro-HPLC on acrylic polymer-based continuous beds as illustrated by Figure 8. Efficiencies of up to 150 000 plates/m were achieved on columns II and III, which only differ by their monomer composition.
18
Figure 8. Normal-phase capillary chromatography of polar aromatic compounds: pyridine (1), 4-pyridylmethanol (2), 4-methoxy-phenol (3), 2-naphthol (4), catechol (5), hydroquinone (6), resorcinol (7), 2,7-dihydroxynaphthalene (8). UV detection at 220 nm. (a) Column II, mobile phase: hexane–ethanol–methanol, pressure: 62 bar; (b) Column II, mobile phase: pure methanol, pressure: 56 bar; (c) Column III, mobile phase: pure methanol, pressure: 5 bar. Column II: 100 µm i.d., 125 mm effective length, 175 mm total length, manufactured from a polymerisation mixture containing 300 mg N-isopropylacrylamide, 50 mg methacrylamide, 125 mg piperazine diacrylamide and 10 mg ammonium persulfate dissolved in 1 ml of 50 mM sodium phosphate, pH 7. Column III: 100 µm i.d., 126 mm effective length, 174 mm total length, manufactured from a polymerisation mixture containing 180 mg 2-hydroxyethyl methacrylate, 5 mg vinylsulfonic acid, 150 mg piperazine diacrylamide and 50 mg ammonium persulfate dissolved in 1 ml of 50 mM sodium phosphate, pH 7. Reprinted from [34], Copyright 2003, with permission from Elsevier.
Ericson et al. [35] described the separation of a series of PAHs (naphthalene, 2methylnaphthalene, fluorine, phenanthrene and anthracene) on a 25 µm i.d. continuous bed with C18 ligands and immobilized dextran sulfate. Efficiencies of up to 105 000 plates/m were obtained.
19
Tanaka’s research group [36-38] reported the analysis of a series of alkylbenzenes (C6H5-(CH2)nH, n=0-6) and polyaromatic hydrocarbons (naphthalene, fluorine, phenanthrene,
anthracene,
pyrene,
triphenylene
and
benzo[a]pyrene)
on
tetramethoxysilane-based macroporous silica gel monoliths and achieved efficiencies up to 80 000 plates/m. Tang et al. [39] successfully performed the separation of aromatic standards and aromatic amines on monolithic columns containing sol-gel bonded octadecylsilica particles.
4 CONCLUSION At present, micro-HPLC is not widely used despite its obvious advantages. For years, the lack of suitable instrumentation/hardware inhibited the use of micro-HPLC. Modification of conventional equipment helped kick-start the technique but the lack of suitable hardware (injectors/capillary columns) still prevented its widespread use. These issues have been addressed in the laboratories of micro-LC practitioners in academia and industry and by a few commercial suppliers. However, there still remains a perception that micro-LC is difficult to perform and therefore the exciting advantages of the technique go unrealised by many in the separation sciences. With the advance in instrumentation and the successful manufacture of robust capillary columns, it is believed that the technique is going to expand and may within a few years become the dominant technique of choice for analysis.
5 FURTHER READING 1. 2. 3. 4.
HPLC and CE: Principles and Practice, Andrea Weston and Phyllis Brown, Academic Press; 1st Edition. Introduction to Micro-scale High-performance Liquid Chromatography, Diado Ishii, VCH Publishing. HPLC Columns: Theory, Technology and Practice, Uwe. D. Neue, WileyVCH Publishing. Introduction to Modern Liquid Chromatography, Lloyd Snyder and Joseph Kirkland, Wiley-Interscience; 2nd Edition.
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6 REFERENCES [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28]
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