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Column Liquid Chromatography: Equipment andInstrumentation
William R. LaCourse
Department of Chemistry and Biochemistry, University of Maryland, Baltimore County, Baltimore, Maryland 21250 
Review Contents
Instrumentation 2813Hyphenated Instrumentation 2814High-Throughput Advances 2814Capillary Chromatography/ Electrochromatography2815Injectors/ Autosamplers 2815Temperature Effects In LC 2815Sample Preparation/ Derivatization 2816Microchip Technology in LC 2816Automation 2817Other Articles of Interest 2817Columns 2817Reviews 2817Columns Materials, Effects, and Packing 2817Supports 2817Stationary Phases 2818Ion-Exchange Phases 2819Monolithic Columns 2819Molecular Imprinted Polymer (MIP) Phases 2819Tunable Stationary Phases 2819Chiral Phases 2820Characterization and Assessment 2820Other Articles of Interest 2820Elemental Detectors 2821Atomic Absorption/ Emission 2821Inductively Coupled Plasma (ICP)-MassSpectrometry2821Microwave-Induced Plasma 2821Optical Detectors 2822UV/ Visible 2822IR/ Raman 2822Optical Activity 2822Evaporative Light Scattering 2822Refractive Index 2823Luminescent Detectors 2823Fluorescence/ Phosphorescence 2823Chemiluminescence/ Bioluminescence 2823Electrochemical Detectors 2823Reviews 2823Instrumentation 2823Potentiometry 2824Novel Material/ Modified Electrodes 2824Array Electrodes 2824Pulsed and Oscillometric Techniques 2824Indirect Electrochemical Detection Systems 2824Other Systems 2825Mass Spectrometry Detectors 2825Reviews 2825Time-of-Flight/ MALDI 2825Fourier Transform Ion Cyclotron ResonanceMass Spectrometry (FTICR-MS or FT-MS)2825Electrospray/ Thermospray 2826Atmospheric Pressure Ionization (API) 2826Particle Beam 2826Other Articles of Interest 2826Other Detection Systems 2826Gas Chromatography Detectors for LC 2826Nuclear Magnetic Resonance 2826Radioactivity Detectors 2827Surface Plasmon Resonance 2827Biosensors in LC 2827Immunochemical-Based Detection 2827Viscometry 2828Computation 2828Simulation 2828Software 2828Literature Cited 2828
This review covers fundamental developments in column liquidchromatography (LC) equipment and instrumentation for theperiod of January 2000 through December 2001. As with pastissues, separate reviews on theory, size exclusion chromatography(SEC), supercritical fluid chromatography (SFC), and capillaryelectrophoresis (CE) are not included in this review unless thereference has technology pertinent to LC. With the growing trendin miniaturization, this reviewer has chosen to include significantarticles in the area of capillary liquid chromatography (CLC),capillary electrochromatography (CEC), and microchip technolo-gies that have a focus on LC. High-performance liquid chroma-tography (HPLC) and LC are considered synonymous. Althoughthis review is not intended to cover applications, applications thatcontain developments and improvements to LC equipment andinstrumentation or were used to demonstrate equipment perfor-mance were also included.The databases for the majority of this review were CAPLUS,CHEMCATS, and CHEMLIST. This paper was not intended tobe a comprehensive review of all published papers during thistime period, but those papers that were considered to besignificant developments or improvements. This review has alsobeen largely restricted to the English language literature. Althoughthe vast majority ofarticles were from peer-reviewed publications,included are relevant articles from widely read periodicals suchas LC-GC and American Laboratory.
INSTRUMENTATION
Fundamental developments in column LC equipment andinstrumentation for the period of October 1997 through October1999 were reviewed by LaCourse (
 A1
). This paper is a continu-ation of that effort. Presently, major trends are toward miniaturiza-tion, speed, and throughput, important qualities when handlingcomplex, analysis-intensive samples such as those derived fromthe needs of research in proteomics, metabolomics, and combi-
Anal. Chem.
2002,
74,
2813
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Analytical Chemistry, Vol. 74, No. 12, June 15, 2002 
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natorial chemistry. There has been literally an explosion ofarticleson microchromatographic techniques (e.g., CLC, CEC, andmicrochip techniques), which also drives new detector design anddevelopments. Ultrafast separations, on-line analyte identification,high-throughput technology, and an orientation to biologicalapplications are all a reflection of the advances in genomics andbiotechnology.
Hyphenated Instrumentation.
This section is not inclusiveof all hyphenated techniques, in that numerous papers in thefollowing sections, especially detectors, also highlight hyphenatedtechniques. One area that underscores the goals of hyphenatedinstrumentation is in the full identification of eluting species inLC. To accomplish analyte identification, the approach is often tocombine mass spectrometry (MS) and nuclear magnetic reso-nance (NMR) on-line. Bailey et al. (
 A2
) used an HPLC NMR-MSsystem for identification of three metabolites (i.e.,
N 
-glucoside,
 N 
-malonylglucoside, and
O
-malonylglucoside) of 5-nitropyridone(2-hydroxy-5-nitropyridine), in maize plants grown hydroponically.Another approach used by Schrader et al. (
 A3
) was to coupleHPLC-infrared (IR) spectroscopy-NMR-MS to identify reactionproducts of pinene ozonolysis, which relates to the “blue haze”phenomenon above forests. Huber and Holzl (
 A4
) reviewed indetail the coupling of CEC with MS. The article also coversinstrumental aspects, separation systems, and applications.Multidimensional multimodal instrumentation greatly improvespeak capacity in complex mixtures. An on-line system for couplinga normal-phase LC column to a reversed-phase LC column isdescribed by Moret and co-workers (
 A5
). The interface consistedof an on-line solvent evaporator working on the principles of concurrent eluent evaporation and vapor overflow and two ad-ditional 10-port valves. Optimization of the on-line procedure andrecoveries, repeatability, and linearity characteristics were testedin a simplified system simulating detection of heavy polycyclicaromatic hydrocarbons (PAHs) in edible oils. Isaaq et al. (
 A6 
)presented a simple approach to the application of HPLC/ CE forthe separation of complex mixtures. A two-dimensional HPLC/ CE instrumental setup was assembled from commercially availableequipment. Fractions of the effluent from the HPLC system werecollected into microtiter plates with the aid of a microfractioncollector, which allows the collection of samples by time, drops,or external signal (peaks). The fractions are then dried undervacuum at room temperature in a special unit, reconstituted, andanalyzed by CE. Any size or type of HPLC or CE column can beused with no limitation on the amount of sample injected into theHPLC, and any CE detection procedure, such as laser-inducedfluorescence (LIF), MS, or UV, or other, can be used. Preliminaryresults show the utility ofthis system for the analysis ofa mixtureof two protein digests, cytochrome
c
and myoglobin.
High-Throughput Advances.
Morand et al. (
 A7 
) discussedgenerally recent advances in high-throughput MS. Hiller et al. (
 A8
)describes the application of a prototype dual sprayer electrosprayionization (ESI) source for the quantitative analysis of biologicalsamples. Quantitative performance for 180 compounds in amicrosomal stability assay was adequate when compared with aconventional single sprayer measurement. Sage and Giles (
 A9
)reported on the design and implementation of a multiplexed(MUX) electrospray interface capable of sampling four individualliquid streams in rapid succession. Data from single compoundsand mixed analytes under fast-gradient LC conditions using thefour-channel MUXtechnology source were also described. Usingfast-gradient LC on small-bore LC columns, samples can beanalyzed with inject-to-inject cycle times of 3 min. Anotherapproach is to process several inlet streams at one time todecrease cycle time, such as a new parallel LC/ tandem massspectrometry (LC/ MS/ MS) system, in which the mass detectorwas shared between two staggered parallel chromatographic runs(
 A10
). Shen et al. (
 A11
) reported on the design and applicationof a high-efficiency multiple-capillary LC system for high-throughput proteome analysis. The multiple-capillary LC systemusing commercial LC pumps was operated at a pressure of10 000psi to deliver mobile phases through a novel passive feedback valve arrangement that permitted mobile-phase flow path switch-ing and efficient sample introduction. Ahigh magnetic field (11.4T) Fourier transform ion cyclotron resonance (FTICR)-MS wascoupled on-line with this high-efficiency multiple-capillary LCsystem using an ESI interface. For yeast cytosolic tryptic digests,
>
100 000 polypeptides were detected and
∼
1000 proteins couldbe characterized from a single capillary LC-FTICR analysis usingthe high mass measurement accuracy (
∼
1 ppm) of FTICR andlikely more if LC retention time information was also exploitedfor peptide identification. Bayliss et al. (
 A12
) present parallelultrahigh flow rate LC using four columns in parallel and a four-way multiple sprayer interface to the mass spectrometer. Thistechnique enables the quantification of drugs from four plasmasamples simultaneously, at nanogram per milliliter concentrations,from small aliquots ofplasma without sample preparation and withthroughputs of 
>
120 samples/ h. High-throughput analysis in MScan also be accomplished via the use of an automatic functionswitching with direct infusion microspray quadrupole time-of-flight(TOF)-MS for peptide and protein analysis (
 A13
).The Karger group (
 A14
) described an approach, based on asubatmospheric ESI interface, which allows sample introductionfrom a commercially available microtiter plate without the needfor a separation fluid delivery system. The microtiter plate wasplaced vertically on a three-dimensional translation stage in frontof the sampling ESI interface. A flow-through wash device waspositioned between the microtiter plate and the ESI interface. Thisdesign allowed alternate filling of the capillary with (a) samplefrom the wells and (b) wash solution from the wash device. Sampleturnaround times of 10 s/ sample, with a 120-nL sample consump-tion/ analysis, and a duty cycle (percentage of total analysis timespent acquiring data) of 40%were achieved. Hoke et al. (
 A15
)increased throughput using packed-column supercritical fluid(pcSFC) coupled to MS/ MS. In contrast to traditional reversed-phase LC, the addition ofa volatile component to the mobile phase,such as CO
2
, produces a lower mobile-phase viscosity. This allowsthe use ofhigher flow rates, which can translate into faster analysistimes. In addition, the resulting mobile phase is considerably morevolatile than the aqueous-based mobile phases that are typicallyused with LC/ MS, allowing the entire effluent to be directed intothe MS interface. Using pcSFC and MS/ MS, dextromethorphanwas quantified in 96-well plates at a rate of 
∼
10 min/ plate withan average intraday accuracy of 9% or better. Daily relativestandard deviations (RSDs) were less than 10%for the 2.21 and14.8 ng/ mL quality control (QC) samples, while the RSDs wereless than 15%at the 0.554 ng/ mL QC level. Several configurations
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Analytical Chemistry, Vol. 74, No. 12, June 15, 2002 
 
using 6- and 10-port switching valves were studied for high-flow,on-line extraction of rat plasma coupled to an ESI-MS by Malletand co-workers (
 A16 
). After sample loading and sample cleanup,the analytes were eluted from the extraction column with a 1.0-min gradient at 0.4 mL/ min. The samples were analyzed eitherdirectly after elution from the extraction column or after additionalseparation using a short HPLC column, which resulted in a totaltime per sample of 3 min.
Capillary Chromatography/ Electrochromatography.
Oneof the reasons for the immense interest in capillary electrochro-matography is its promise to combine chromatographic selectivitywith the high efficiency and the miniaturization potential of CE.This interest is reflected in the numerous review articles of thistechnique. Instrumentation for CEC was reviewed by Steiner andScherer (
 A17 
) and Rozing et al. (
 A18
). General requirements,solvent delivery, and detection are covered in some detail. Otherreviews have focused on modes of separation (
 A19
), stationaryphases (
 A20
), and migration of charged species and electro-osmotic flow in CEC (
 A21
). Open-tubular CEC (
 A22
) and itscoupling with on-line MS (
 A23
) have also been reviewed. BothCEC and capillary LC are discussed in Tsuda’s (
 A24
) review.Powell and Tempst (
 A25
) described a microflow-based instrument,consisting of multiple rotary valves, capillary tubing, and miniatur-ized reaction vessels, for the purpose of performing automatedchemical and biochemical reactions on a very small scale (i.e.,submicroliter volumes). The novelty is that close to 100%of thereaction end products are available in a minimal volume (
<
5
µ
L)inside a pressurized microvial for subsequent analysis. This makesthe system compatible with capillary LC and, in principle, withcontinuous-flow nanoelectrospray MS. Instrument performancewas convincingly demonstrated by partially sequencing 100 fmolof an intact protein using classical Edman chemistry in combina-tion with CLC.Ye et al. (
 A26 
) reported on a novel mode of affinity chroma-tography (AC) based on an open-tubular (OT) capillary column.The OTAC column is prepared by immobilizing Cibacron blueF3GAonto the inner surface of a 50-
 µ
m-i.d. capillary column. TheAC experiment is performed on a CE instrument by using itspressure system as the driving force. Bovine serum albumin andlysozyme (Lys) are successfully separated with stepwise gradientelution. The RSD for the elution time of the retained Lys is 0.08%,and good repeatability of its peak area and peak height with anRSD value lower than 2.12%for 10 consecutive runs was observed.The loading capacity and detection limit for the retained Lys areabout 36 and 8.6 ng, respectively.
Injectors/Autosamplers.
Aseries ofarticles by Dolan (
 A27 
-
 A29
) focused on autosamplers in LC systems. The areas coveredare the common designs, advantages and disadvantages, problemsand solutions, and sample carryover. Kehl and Meyer (
 A30
)described an approach to the determination of LC injectionreproducibility based upon argentometric titration. The authorsfound the following repeatabilities (RSDs): titration, 0.07%; 10-
 µ
Lpull loop injection (partial filling of the loop), 0.6%; and 20-
 µ
L fullloop injection, 0.1%. Heron et al. (
 A31
) looked at the influence of the injection volume and the sample solvent on column efficiencyin packed nano LC using columns of 150-
 µ
m i.d.. When com-pounds are dissolved in a weak solvent (such as acetonitrile/ H
2
O,30:70) and whatever the injection volume (i.e., 60 or 200 nL), again in efficiency can be observed due to the known on-columnfocusing phenomenon. Layne et al. (
 A32
) studied the effects of sample solvent composition and injection volumes on the chro-matographic performance of ODS-bonded silica columns underfast-gradient running conditions. Chromatographic performancewas compromised as a function of both sample injection volumeand sample solvent strength, with earlier eluting analytes affectedmuch more than later eluting ones. In general, when injectingsamples dissolved in a strong solvent, performance was improvedby diluting the strong injection solvent and injecting a proportion-ally larger volume. Volume loading capacity can be increased byusing a longer column or by using a column of equivalent length,but with a larger inner diameter. It was conjectured that samplesolvent strength, not viscosity, is responsible for the noted effects.
Temperature Effects In LC.
The present state oftemperatureprogramming in LC, including CEC, is reviewed by Greibrokk and Andersen (
 A33
). As a part of the ongoing trend of miniatur-ization in chromatography, the active use of temperature as avariable in LC is expected to increase significantly. This paperincludes an overview of the effects of temperature on columnefficiency, retention, and selectivity, particularly in relation tonarrow-bore columns, instrumental features, and some selectedapplications. The Greibrokk group (
 A34
) also investigated tem-perature programming for gradient elution in packed capillary LCcoupled to electrochemical detection (EC).Xiao and Oefner (
 A35
) reviewed denaturing HPLC (dHPLC),which compares two or more chromosomes as a mixture of denatured and reannealed PCR amplicons, revealing the presenceof a mutation by the differential retention of homo- and hetero-duplexes. This technique relies on temperature programming, andthe authors discuss temperature-related resolution sensitivity andits optimum prediction by computational means. This work isextended further in an article by Premstaller et al. (
 A36 
) dealingwith temperature-modulated arrays in HPLC for mutation detec-tion. Analyses times are on the order of a few minutes andturnaround time is extremely short as there is no need for thereplenishment of the separation matrix between runs. Issaq andco-workers (
 A37 
) investigated the effect of experimental temper-ature on the separation of DNA fragments (i.e., 21
-
587 bp) byboth HPLC and CE. The optimum temperature was found to bebetween 40 and 50
°
C for HPLC, while 25
°
C was the optimumtemperature for the CE separation.The advantages of packed capillary columns in LC as well asin supercritical fluids were reviewed by Greibrokk et al. (
 A38
).In liquids, the active use of temperature as a variable resulted inreduced analyses time, increased column efficiency, increasedsignal-to-noise ratio, and allowed temperature programming forcontrolling retention and replacing solvent gradients in HPLC. Theadvantages of high-temperature separations lend themselves toultrafast LC, and the Carr group (
 A39
) described a novel LCsystem, which enabled high-temperature ultrafast liquid chroma-tography (HTUFLC). Careful consideration was given to heattransfer, band broadening, and pressure drop. Using pure wateras the mobile phase, five phenols were separated in
<
30 s.Thompson et al. (
 A40
) compared a narrow-bore column (2.1-mm i.d.) to a conventional-bore column (4.6-mm i.d.) at elevatedtemperatures under conditions where thermal mismatch broaden-ing is serious and showed that narrow-bore columns offer signifi-
Analytical Chemistry, Vol. 74, No. 12, June 15, 2002 
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Arun Raj

please help me to download it

reply10 / 27 / 2010
adetundetoyin

Thanks this is a very useful document. Thank you

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