HPLCAPPLICATIONAND EDUCATIONNOTEBOOK - · PDF fileTable 1: LC ... Curr. Opin. Chem. Biol. 8; 418-423; 2004. ... Here we look closely at the problem and propose calculations that improve

APPLICATIONS• Applications for Optimal HPLC Separation

EDUCATION• Complimentary ChemStation and LC Online Courses• Glossary of Liquid-Phase Separation Terms

HPLC Application Guide and Glossary of Terms

HPLC APPLICATION ANDEDUCATION NOTEBOOK

Your Must Have Guide for Optimal HPLC Separations

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Achieving more with less, confidence in analytical results, and cost-effectiveness are keyobjectives in today’s lab. Agilent Technologies is committed to providing innovative measurement so-lutions that make a measurable difference in the lives of scientists and engineers everywhere, such aslife science research and quality control. With our leadership in liquid chromatographysolutions, Agilent continues its commitment to taking scientific discovery from dream to realityand then ensuring the highest quality possible in achieving those realities.

We are proud to provide laboratories with the most reliable, most flexible and cutting edge liq-uid chromatographs. As evidenced by over half a million modules already placed in labs worldwidesince the introduction of the 1100 and subsequent 1200 liquid chromatography portfolios, customershave recognized the value derived from Agilent’s commitment to the development of best-in-class in-struments, software, chemistries, and services, to help you realize your labs’ highest goals.Ranging from low nanoflow systems to extensive preparative LC solutions, the Agilent 1200 series of-fers unsurpassed performance in liquid chromatography. Innovations from within our research anddevelopment organizations have been the major contributors to achieving the high standards we setfor performance. Agilent’s HPLC-Chip technology was introduced in 2004, and coupled to mass spec-trometry has proven to reach sensitivity levels well beyond that which could be achieved with tradi-tional HPLC and has received accolades as the premier chip separation offering. Furthermore, themost significant advancement in liquid chromatography in the last eight years has been the utilizationof small particles for faster, higher resolution separations. In 2000, it was Agilent who led theindustry with our introduction of the Rapid Resolution High Throughput columns packed with sub2-micron particles. Our R&D labs continue to listen to your requests and strive for improved sepa-ration techniques — and we are truly excited about the possibilities of the future.

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Helmut Schulenburg-SchellWorldwide Liquid Chromatography Marketing ManagerAgilent Technologies Inc.

LC SOLUTIONS THAT REVOLVE AROUND YOUR NEEDS

OCTOBER 20084 HPLC RESOLUTION

HPLC APPLICATION AND EDUCATION NOTEBOOK

HPLC Applications That Revolve Around Your Needs

Page 5 RESOLUTION – Agilent 1200 Series Rapid Resolution LC systemand the Agilent 6210 TOF MS – Highest data content withhighest throughput

Page 10 SPEED – Improving the Effectivenss of Method Translation forFast and High Resolution Separations

`Page 14 SELECTIVITY – Unique Selectivity and High-Throughput

Applications of ZORBAX SB-Phenyl RRHT

HPLC Industry Applications

Page 18 Examples for Food, Environmental, Forensics, PharmaceuticalImpurity Profiling and Drug Discovery applications revealed.

Glossary of Liquid-Phase Separation Terms

Page 19 Updated HPLC Terminology and definitions.Authors : Ronald Majors and Peter Carr

LC Education Series

Page 42 Complimentary ‘Secrets of Agilent ChemStation' eSeminarSeries

Page 43 Complimentary 'Optimize your LC regardless of manufacturer'eSeminar Series

This notebook will both inspire and inform you by highlighting how Agilentliquid chromatography products and expertise effectively enhance yourlab’s progress. Whether selectivity, resolution, speed or sensitivity are mostimportant to you, Agilent solutions revolve around your lab’s needs. Theapplications selected in this compendium represent just a sampling of thesuccesses in analyses that are achieved using Agilent LC systems.

OCTOBER 2008 RESOLUTION HPLC 5

RESOLUTION – Agilent 1200 Series Rapid ResolutionLC system and the Agilent 6210 TOF MS –

Highest data content with highest throughputMichael Frank,

Agilent Technologies, Waldbronn, Germany

Fast and unambiguous determination of purity andidentity of compounds derived from screeninglibraries is a common task for many analytical labs inthe pharmaceutical industry. The method of choice todetermine the identity of compounds is mass spec-trometry, preferably with accurate mass. As yet, dataquality was usually compromised by gaining higherthroughput. This Application Note demonstrates howa daily throughput of far more than 1000 samples canbe achieved together with full spectral data acquisi-tion and accurate mass information with close toFT-MS mass accuracy.

In the quest to achieve highest throughput in LC/MS analyses,the quality of the data is often compromised. There are certainapproaches to increase the throughput of LC/MS systems. Oneapproach is to do flow injection analysis. This probably deliversthe highest possible throughput, however since no chromato-graphic separation occurs, the probability to lose compounds bythe ion suppression effect during the ionization process is high.Orthogonal detection methods like UV detection do not succeedat all in flow injection analysis as all compound signals are over-laid. Approaches to achieve at least minimal chromatographic sep-aration by using very short columns with 5 µm particles andballistic gradients are an improvement in view of data quality,however, not state-of-the-art. Some manufactures have establishedparallel working instrumentation with a shared mass spectrome-ter and shared UV detector. Obviously, this also compromisesdata quality as the full acquisition rate of each instrument has tobe shared on each LC channel1.With the introduction of an LC/MS system which facilitates

the use of columns with sub two micron particles it is now pos-sible to achieve short analyses times as well as high chromato-graphic resolution. Furthermore the system is able to acquire fullUV spectral data and mass spectral data with accurate masses.

ExperimentalThe Agilent 1200 Series Rapid Resolution LC system is set upfor alternating column regeneration (ACR)2 using 2.1-mm idcolumns. The pumps are in the low delay volume configurationwith an internal volume of only ca. 120 µL. All other modulesare optimized for lowest delay volumes by using the low delayvolume capillary kit (G1316-68744) and the alternating columnregeneration kit (G1316-68721). Consequently, from the injec-tion valve on only capillaries of 0.12 mm id are used. In the ther-mostatted column compartment the newly introduced low

dispersion heat exchangers consisting of 1.6 µL internal volumehave been used as well as the high pressure rated2-position/10-port valve.The instrument set-up is shown in figure 1:

• Two Agilent 1200 Series binary pumps SL with the new Agi-lent 1200 Series micro vacuum degasser placed between thetwo pumps eliminates the need for long tubing to the pumps.

• Agilent 1200 Series high performance autosampler SL.• An Agilent 1200 Series thermostatted column compartmentSL, equipped with a high pressure, 2-position/10-port valve,facilitating alternating column regeneration.

• An Agilent 1200 Series diode-array detector SL allowing a dataacquisition rate of 80 Hz and equipped with a 500 nano literflow cell with 0.12-mm id connecting capillaries.

• Agilent 6210 Time-of-Flight mass spectrometer allowing amaximum data acquisition rate of 40 Hz and equipped witha dual ESI source for parallel ionization of the analyte and areference mixture.

• Two ZORBAX SB C18, 2.1 mm id x 50 mm, 1.8 µmcolumns

• As mobile phase gradient grade water with 0.1 % trifluoroacetic acid and acetonitrile with 0.08 % trifluoro acetic acid-was used. No additional filtering of the solvents was made.Instrument control and data acquisition was done by the


Figure 1: Agilent 1200 Series Rapid Resolution LC system with Agi-lent 6210 TOF-MS with low delay volume for high speed applicationsusing 2.1-mm id columns with lengths ranging from 20 to 50 mm.

Figure 2: Feature of the TOF software to modify the MS parameterfrom run to run.

Agilent TOF-software A02.01 running on a Hewlett-Packardxw 4300 workstation with an Intel dual core Pentium™D840 CPU at 3.2 GHz.

Results and DiscussionBy applying elevated temperatures the viscosity of the solvent canbe reduced which allows higher flow rates and therefore shortergradient times. A maximum temperature of 80 °C was applied,which allowed a flow rate of 1.8 mL/min without hitting the pres-sure limit of the pump. This results in a linear velocity of ap-proximately 11 mm/s for the 2.1 mm x 50 mm column (1.8 µm).With the help of the regeneration pump and the 2-position/10-port valve in the column compartment, cycle times could be re-duced significantly because one column is flushed with highorganic content solvent and then re-equilibrated again with thestarting composition of the gradient while on the second columnthe separation of a sample occurs. After this sequence the 10-portvalve is switched and both columns are exchanged in the flowpath. Details of alternating column regeneration and the correctsetting of time points are described in another Application Note2.Despite the high flow rate (1.8 mL/min), the column effluent wasnot split prior to reaching the mass spectrometer. The standardESI source specifies a maximum flow rate of up to 1 mL/min,however even these higher flows are tolerated if the drying gastemperature and flow rate are set to maximum and little conden-sation occurs. Condensation of water is practically eliminatedwhen using ACR because equilibration is done on the columnwhich is not connected to the detector. Generally the use of anAgilent multi mode source with a specified flow rate up to 2mL/min even with pure water is recommended. The chromato-graphic conditions in table 1 were used to achieve gradient timesof 0.5 min. Under these conditions, the peak capacity for the MSdetection is in the range of >40 in 39 s. With the use of a 5-µmparticle size column of the same dimension the peak capacitywould only be half!The detector of the Agilent 6210TOFMS would be saturated

if the compound concentrations used here to give also significantUV signals would be injected into the MS without special set-

tings. Saturation of the MS detector would produce incorrect re-sults in mass determination. The solution is to intentionally de-sensitize the TOF MS. This can be done quite easily by applyingthe functionality of the TOF software to alter the MS parametersfrom one run to the other, simply by adding one or more “MS-parameter” columns to the worklist (figure 2). Select “addcolumns” from the worklist and then chose “MS-parameter” andthe desired parameter. As the reference mixture is also affected bythese settings, the concentration of the reference mixture was in-creased. Only the capillary voltage, the fragmentor voltage andthe skimmer voltage were varied. The optimal conditions deter-mined by this approach can be found in the method parametersin table 1.

In figure 3 the total ion chromatogram and the UV chro-matogram achieved with conditions above (80 Hz DAD, 30 HzTOF data acquisition rate) is shown for a five-component sample(58 ng/µL atenolol, 85 ng/µL primidon, 62 ng/µL metoprolol,125 ng/µL verapamil and 75 ng/µL beclomethasone-dipropi-onat). The peaks of the total ion chromatogram are inherentlybroader than the peaks of the UV chromatogram because of ad-ditional extra column volume from the flow cell and also from

Figure 3: Comparison of corresponding peaks in the UV (red trace)and the MS detection (black trace).


connecting the capillary between the UV detector and ESI inter-face. But as can be seen in figure 3, the additional peak broaden-ing of the MS peaks is only minor. The peak widths at half heightof the MS peaks obtained under the highest data acquisition rate(40 Hz) are shown in figure 4 with values from as little as 0.34 to0.42 s. The chromatograms shown in figure 5 were producedunder the same chromatographic conditions, but with differentdata acquisition rates of the time-of-flight MS. The peak formand resolution are improved by having high data acquisition ratesin the MS which shows clearly in figure 5. The effect is nicelydemonstrated on the little side peak next to the primidon peak –with 40-Hz data acquisition rate it is obvious that an additionalcompound shows up but with 5 Hz data acquisition rate thiscould not be differentiated from tailing of the primidon! The ad-vantage, especially if MS quantization is necessary, is clear.By applying the chromatographic conditions of table 1 and 80

Hz signal data acquisition of one wavelength and 30 Hz TOFcentroid data, a cycle time of 49 s was achieved. The achievablecycle time is not only dependent on the used run time (that is thegradient time plus additional flush and re-equilibration times, orin Agilent terminology the stop time plus post time) but also verymuch dependent on the instrument overhead time. This is usu-ally caused by communication between the data system and theindividual LC/MSmodules as well as the data system writing datato the hard disc and initiating certain processes. The overheadtime caused by the data system can be significant if the computer’sperformance is not sufficient to handle the data amount or ifother software programs or processes are consuming the poweravailable. To decrease the cycle time it might be worth decreasingthe amount of data acquired.Table 2 shows the cycle times and the possible daily through-

put depending on the DAD and MS settings. Since the MS data

Table 1: LC/MS method used for the data shown in figures 3-5. The method was also used to achieve the values in table 2.

are constantly written to the hard disc during data acquisition,whereas the UV data are buffered and added to the data file afterthe stop time of the method, the cycle time depends more on theUV data amount than on the MS data amount. The cycle timewas calculated from the time stamp each file gets assigned fromthe WindowsXP™ operating system after closing the file fol-lowing data acquisition.If using aTOFMS the attention is certainly focused on the ac-

curate mass. The question may arise if the possibility to obtainlow mass accuracy errors might suffer from these high speed con-

Figure 4: MS total ion chromatogram of highest speed LC-TOF-MSanalysis (40 Hz TOF data acquisition rate).

MMeetthhoodd::SSoollvveenntt:: A = water (0.1% TFA), B = ACN (0.08% TFA)TTeemmppeerraattuurree:: 80 °CFFllooww:: 1.8 mL/min GGrraaddiieenntt:: 0.00 min 5%B RReeggeenneerraattiioonn:: 0.00 min 5%B

0.50 min 90%B 0.01 min 95%B0.51 min 5%B 0.20 min 95%B0.65 min 5%B 0.21 min 5%B

0.65 min 5%BSSttooppttiimmee:: 0.65 min no limitPPoossttttiimmee:: off offDDAADD:: Wavelength: 210 nm (8), ref. off

Peak width: >0.0025 min (0.05s responsetime), 80 HzSpectra: noSlit: 8 nmBalance: pre-run

MMSS:: Scan range: 100-1000 m/zAcquisition rate: 5, 20, 30 and 40 cycles/sData type: profile dataCapillary voltage: 3000 VFragmentor: 180 VSkimmer: 40VGas temperature: 350 °CGas flow: 13 L/min

IInnjjeeccttiioonn vvoolluummee:: 1µLIInnjjeeccttoorr:: Overlapped injection, Automatic delay volume reduction,

Sample flush out factor = 10VVaallvvee ppoossiittiioonn:: Next position


ditions. Figure 6 shows the achieved mass accuracy errors of theanalysis of 140 members of a chemical library used in a screeningcampaign by a pharmaceutical company. The shown error-valueshave been extracted from an automated empirical formula con-firmation report and involved no manual interference. Sixteen ofthe compounds could not be ionized under positive ESI condi-tions and two compounds showed large mass errors of 11 and 15ppm, probably caused by co-eluting isobaric impurities. The cycletime was 90 s and was determined by a required injector programwhich allowed an on-line dilution of the samples directly prior tothe analysis. Chromatographic conditions applied a 5-100 %water-acetonitrile (0.1 % TFA) gradient in 0.7 min at a flow rateof 1.5 mL/min and 60 °C column temperature. UV data acqui-sition to determine purity was done in the wavelength range of210 to 500 nm with an acquisition rate of 80 Hz. The MS dataacquisition rate was at 8 Hz to reduce the file size. The scan rangewas 120 – 1200 Da, capillary voltage 4000 V and the fragmen-tor voltage at 215 V. No ACR was applied and the flow to the MSwas split in a 1:7.5 ratio. More compelling is the histogram of the mass errors of these

samples as shown in figure 7. More than 91 % of the ionizablecompounds (outliers included) have a mass accuracy error in therange of ±2.0 ppm. Excluding the outliers even 93 % of the an-alyzed samples lie in-between the ±2.0 ppm range. In the ±1.0ppm range which is FT-MS-like mass accuracy 71 % of the sam-ples can be found (72 % excluding the outliers).

Figure 5: Total ion chromatograms recorded with varying dataacquisition rates – dependence of the MS peak shape and resolutionon the data acquisition rate.

Figure 6: Mass accuracy errors of the analyses of a set of chemicallibrary members under fast-LC conditions.

Table 2: Dependence of the cycle time on the DAD and MS data acquisition settings, method stop-time was 0.65 min (39 s), pre-run balance was applied (ca. 2 s). The number in brackets for the DAD wavelength range stands for the scan width in nm.

DDAADD ((8800 HHzz)) TTOOFF ((110000 –– 11000000 DDaa)) CCyycclleettiimmee TThhrroouugghhppuutt

Type Wavelength Profile Data rate [Hz] [s] [Samples/day]spectral 190-900 (1) x 20 62 1394spectral 190-900 (1) 20 62 1394spectral 190-400 (2) x 20 59 1464spectral 190-400 (2) x 40 59 1464spectral 190-400 (2) x 30 58 1490signal 210/254 x 20 50 1728signal 210 30 49 1763


Figure 7: Histogram of the mass accuracy errors of the analysesof a set of chemical library members under fast LC conditions.The given populations of the ±1.0 ppm and ±2.0 ppm rangeinclude the outliers.

ConclusionThe Agilent 1200 Series Rapid Resolution LC system togetherwith the Agilent 6210 Time-of-Flight mass spectrometer allowsacquisition of a wealth of data to unambiguously determine thepurity and identity of compounds in samples as they are typicalfor the high throughput analytical departments of pharmaceuti-cal companies. In the time range of one minute high chromato-graphic resolution, full spectral diode-array data from 190-900nm wavelength in a band width of 1 nm at an 80 Hz acquisitionrate plus full MS spectral data from 100-1000 m/z with high ac-quisition rate and with an accurate mass with a mass error below±2.0 ppm for more than 91 % of the samples could be acquired.Using features like alternating column regeneration, over-

lapped injection, high temperatures, high flow rates together withhighest data acquisition rates and most importantly stable andeasy-to-use accurate mass, this system outperforms other highthroughput LC/MS techniques used as yet in throughput and/ordata quality. The linear velocities achieved were in the range of 11mm/s and cycle times were as fast as 49 s for a run time of 41 s.Due to the columns with particle sizes of 1.8 µm, the UV peakcapacities were still in the range of fifty and even the MS peakcapacities were in the range of forty for a gradient time of 39 s.

References1. Jeremy R. Kenseth, Shelly J. Coldiron, “High-throughput

characterization and quality control of small-molecule combinatorial libraries”, Curr. Opin. Chem. Biol. 8; 418-423; 2004.Jill Hochlowski, Xueheng Cheng, “Current Application of Mass Spectrometry to Combinatorial Chemistry”, Anal. Chem. 74, 2679-2690; 2002.

2. Udo Huber, “High throughput HPLC – Alternating column regeneration with the Agilent 1100 Series valve solutions” Agilent Application Note, Publication number 5988-7831EN; 2002.

Order your Agilent Rapid Resolution Liquid Chromatography Kit today • www.agilent.com/chem/futureLCnow | 800 227 9770

OCTOBER 200810 HPLC SPEED

SPEED – Improving the Effectivenss of MethodTranslation for Fast and High Resolution Separations

Michael Woodman,Agilent Technologies, Inc., 2850 Centerville Road, Wilmington, DE, USA

The increased availability of sub-2-micron (STM)columns and increased demand for methods friendlyto mass spectrometers has led to strong trend towardconversion of existing HPLC methods to smaller diameter and smaller particle size columns. While theconversion is a simple mathematical exercise requir-ing the scaling flow rates, gradient times and injec-tion volumes, many users observe less than perfectresults. Here we look closely at the problem and propose calculations that improve the speed and/orresolution in a more predictable and beneficial way.

Methods developed on older columns packed with large 5-or 10-µm particles are often good candidates for mod-

ernization by replacing these columns with smaller dimensioncolumns packed with smaller particle sizes. The potential benefitsinclude reduced analysis time and solvent consumption, improvedsensitivity and greater compatibility with mass spectrometer ion-ization sources.Simplistically, a column of 250-mm length and containing 5-

µm particles can be replaced by a 150-mm length column packedwith 3-µm particles. If the ratio of length to particle size is equal,the two columns are considered to have equal resolving power.Solvent consumption is reduced by L1/L2, here about 1.6-foldreduction in solvent usage per analysis. If an equal mass of ana-lyte can then be successfully injected, the sensitivity should alsoincrease by 1.6-fold due to reduced dilution of the peak as it trav-els through a smaller column of equal efficiency.LC/MS (Liquid Chromatography/Mass Spectrometry) ioniza-

tion sources, especially the electrospray ionization mode, havedemonstrated greater sensitivity at lower flow rates than typicallyused in normal LC/UV (UltraViolet UV/VIS optical detection)methods, so it may also be advantageous to reduce the internal di-ameter of a column to allow timely analysis at lower flow rates.The relationship of flow rate between different column diameters is shown in Equation 1.

The combined effect of reduced length and diameter con-tributes to a reduction in solvent consumption and, again as-suming the same analyte mass can be injected on the smallercolumn, a proportional increase in peak response. We normallyscale the injection mass to the size of the column, though, and aproportional injection volume would be calculated from the ratioof the void volumes of the two columns, multiplied by the injec-tion volume on the original column.

For isocratic separations, the above conditions will normallyresult in a successful conversion of the method with little or nochange in overall resolution. If one wishes to improve the out-come of the method conversion, though, there are several otherparameters that should be considered. The first of these parame-ters is the column efficiency relative to flow rate, or more cor-rectly efficiency to linear velocity, as commonly defined by vanDeemter [1] and others, and the second is the often overlookedeffect of extracolumn dispersion on the observed or empirical ef-ficiency of the column.Van Deemter observed and mathematically expressed the rela-

tionship of column efficiency to a variety of parameters, but weare most interested here in his observations that there is an opti-mum linear velocity for any given particle size, in a well-packedHPLC column, and that the optimum linear velocity increases asthe particle size decreases. Graphically, this is often representedin van Deemter plots as shown in Figure 1, a modified version ofthe original plot [2].In Figure 1 we observe that the linear velocity at which 5-µm

materials are most efficient, under the conditions used by the au-thors, is about 1 mm/sec. For 3.5-µm materials the optimum lin-ear velocity is about 1.7 mm/sec and has a less distinct optimumvalue, suggesting that 3.5-µm materials would give a more con-sistent column efficiency over a wider flow range. For the 1.8-µmmaterials, the minimum plate height, or maximum efficiency, isa broad range beginning at about 2 mm/sec and continuing pastthe range of the presented data. The practical application of thisinformation is that a reduction in particle size, as discussed ear-lier, can often be further optimized by increasing the linear ve-locity which results in a further reduction in analysis time. Thisincrease in elution speed will decrease absolute peak width andmay require the user to increase data acquisition rates and reduce

OCTOBER 2008 SPEED HPLC 11

signal filtering parameters to ensure that the chromatographicseparation is accurately recorded in the acquisition data file.The second important consideration is the often overlooked

effect of extracolumn dispersion on the observed or empirical ef-ficiency of the column. As column volume is reduced, peak elu-tion volumes are proportionately reduced. If smaller particle sizesare also employed there is a further reduction in the expected peakvolume. The liquid chromatograph, and particularly the areaswhere the analytes will traverse, is a collection of various con-necting capillaries and fittings which will cause a measurableamount of bandspreading. From the injector to the detector flowcell, the cumulative dispersion that occurs degrades the columnperformance and results in observed efficiencies that can be farbelow the values that would be estimated by purely theoreticalmeans. It is fairly typical to see a measured dispersion of 20 to100 µL in an HPLC system. This has a disproportionate effecton the smallest columns and smallest particle sizes, both of whichare expected to yield the smallest possible peak volumes. Caremust be taken by the user to minimize the extracolumn volumeand to reduce, where practical, the number of connecting fittingsand the volume of injection valves and detector flow cells. For gradient elution separations, where the mobile phase com-

position increases through the initial part of the analysis until theanalytes of interest have been eluted from the column, successfulmethod conversion to smaller columns requires that the gradientslope be preserved. While many publications have referred to gra-dient slope in terms of % change per minute, it is more useful toexpress it as % change per column volume. In this way, the changein column volume during method conversion can be used to ac-curately render the new gradient condition. If we think of eachline of a gradient table as a segment, we can express the gradientby the following equation:

Figure 1: van Deemter plot with various flow rates and particlesizes.

Note that the use of % change per column volume rather than% change per minute frees the user to control gradient slope byaltering gradient time and/or gradient flow rate. A large value forgradient slope yields very fast gradients with minimal resolution,while lower gradient slopes produce higher resolution at the ex-pense of increased solvent consumption and somewhat reducedsensitivity. Longer analysis time may also result unless the gradi-ent slope is reduced by increasing the flow rate, within accept-able operating pressure ranges, rather than by increasing thegradient time.Resolution increases with shallow gradients because the effec-

tive capacity factor, k*, is increased. Much like in isocratic sepa-rations, where the capacity term is called k', a higher value directlyincreases resolution. The effect is quite dramatic up to a k valueof about 5 to 10, after which little improvement is observed. Inthe subsequent examples, we will see the results associated withthe calculations discussed above.

Experimental Conditions

SystemAgilent 1200 Series Rapid Resolution LC consisting of:G1379B micro degasserG1312B binary pump SLG1367C autosampler SL, with thermostatic temperature controlG1316B Thermostatted column compartment SLG1315C UV/VIS diode array detector SL, flow cell as indicated inindividual chromatograms ChemStation 32-bit version B.02.01

ColumnsAgilent ZORBAX SB-C18, 4.6 mm, 250 mm, 5 µmAgilent ZORBAX SB-C18, 3.0 mm, 150 mm, 3.5 µm

Mobile phase conditionsOrganic solvent: AcetonitrileAqueous solvent: 25 mm phosphoric acid in Milli-Q water

Gradient ConditionsGradient slope: 7.8% or 2.3% per column volume, as

indicated. See individual chromatograms for flow rate and time

SampleStandard mixture of chlorinated phenoxy acid herbicides, 100 µg/mL in methanol

OCTOBER 200812 HPLC SPEED

ResultsThe separation was initially performed on a standard 4.6 × 250mm, 5-µm ZORBAX SB-C18 column thermostatted to 25 °C(Figure 2) using conditions referenced in US EPA Method 555.The method was then scaled in flow and time for exact transla-tion to a 3.0 × 150 mm, 3.5-µm column (Figure 3). Solvent con-sumption is reduced from 60 mL to 15.5 mL per analysis.The separation was then re-optimized for faster separation with

the identical slope, 7.8%, by increasing the flow rate from 0.43to 1.42 mL/min, and proportionately reducing the gradient time(Figure 4). Finally, increased resolution is demonstrated by keep-ing the original times used in Figure 3 with the increased flowrate (Figure 5). This yields a gradient with identical time but a re-duced slope of 2.3%. The increased resolution of peaks 4 and 5is readily apparent. The conditions in Figure 4, 7.8% slope at increased linear ve-

locity on 3.0 × 150 mm, 3.5-µm material, yield a separation withcomparable resolution to the original 4.6 × 250 mm method, butwith only a 12-minute total analysis time. This is excellent forhigh throughput screening and quantitation of a large number ofsamples. Figure 5, with the gradient slope reduced to 2.3%, re-sults in a high-resolution separation with a calculated R value of3.3 vs. the standard 3.0 × 150 mm separation value of 1.9, for thecritical pair seen in Figure 5 at 7.5 to 8 minutes.

Figure 2: Gradient separation of herbicides on 4.6 × 250 mm 5-µmZORBAX SB-C18.

Conditions

EPA Method 555 with ZORBAX SB-C18 columns and fastDAD detectorZORBAX SB-C18 4.6 mm x 250 mm, 5 µmColumn temp: 25 °CGradient: 10% to 90% ACN vs. 25 mM H3PO4Gradient slope: 7.8% ACN/column volume Analysis flow rate: 1 mL/min

Group A CompoundsTotal analysis time: 60 minDetection: UV 230 nm, 10-mm 13-µL flow cell,

filter 2 seconds (default)

Figure 3: Gradient separation of herbicides on 3.0 × 150 mm,3.5-µm ZORBAX SB-C18.

Conditions:

EPA Method 555 with ZORBAX SB-C18 columns and fastDAD detectorZORBAX SB-C18 3.0 mm x 150 mm, 3.5 µmColumn temp: 25 °CGradient: 25 mm H3PO4/ACN, 0% to 90%

ACN in 18 minutesGradient slope: 7.8% ACN/column volumeAnalysis flow rate: 0.43 mL/minDetection: UV 230 nm, 3-mm 2-µL flow cell,

filter 0.2 secondsTotal analysis time: 36 min.

In Table 1 the column has been replaced with a low dead vol-ume connecting union in a system fitted with 0.12-mm id capil-lary tubing at all points of sample contact. A 1-µL injection ofdilute actone is made to determine the bandspreading contribu-tion of the system, with various flow cells. Multiple flow cells weretested, and the average result reported, where possible. The elu-tion volume summarizes the total volume of all tubing in the sys-tem. While the absolute volume from the 2-µL to the 13-µL flowcells is 11 µL, we observe an increase of 15 to 16 µL because ofthe larger diameter inlet tubing integral to the larger volume flowcells.

ConclusionCareful analysis of the existing gradient conditions, coupled withan awareness of the need to accurately calculate new flow and gra-dient conditions can lead to an easy and reliable conversion ofexisting methods to new faster or higher resolution conditions. Inaddition, awareness of extracolumn dispersion, especially withsmall and high resolution columns, will ensure good column ef-ficiency which is critical to a successful translation of the method.

OCTOBER 2008 SPEED HPLC 13

Figure 4: High speed gradient separation of herbicides on 3.0 ×150 mm, 3.5-µm ZORBAX SB-C18.

Conditions

EPA Method 555 with ZORBAX SB-C18 columns and fastDAD detectorZORBAX SB-C18, 3.0 mm x 150 mm, 3.5 µm Column temp: 25 °C Gradient: 25 mM H3PO4/ACN, 10% to 90%

ACN in 5.4 min.Gradient slope: 7.8% ACN/column volumeAnalysis flow rate: 1.42 mL/minDetection: UV 230 nm, 3-mm 2-µL flow cell,


Figure 5: Reduced slope gradient separation of herbicides on 3.0 ×150 mm, 3.5-µm ZORBAX SB-C18.

Conditions

EPA Method 555 with ZORBAX SB-C18 columns and fastDAD detectorZORBAX SB-C18, 3.0 mm x 150 mm, 3.5 µmTemp: 25 °CGradient: 25 mM H3PO4/ACN, 10% to 90%

ACN in 18 min.Gradient slope: 2.3% ACN/column volume Analysis flow rate: 1.42 mL/minDetection: UV 230 nm, 3-mm 2-µL flow cell,


References1. J. J. van Deemter, F. J. Zuiderweg, A. Klinkenberg,

Chemical Engineering Science 1956, 5, 271–289

2. The Influence of Sub-Two Micron Particles on HPLC Performance, Agilent Technologies, application note 5989-9251EN, May 2003

Elution Half height 5 SigmaFlow cell volume (µL) width (µL) width (µL)

New SL 11 5 122 µL 3 mm

Micro 14 6 186 mm 1.7 µL(n = 2)

Semi-micro 13 6.5 18.56 mm 5 µL (n = 2)

Standard 26 11 2610 mm 13 µL

New SL 27 11 2510 mm 13 µL

Table 1: Volumetric Measurements of Various Flow Cells


OCTOBER 200814 HPLC SELECTIVITY

SELECTIVITY – Unique Selectivity and High-ThroughputApplications of ZORBAX SB-Phenyl RRHT

William J. Long and John W. Henderson Jr.,Agilent Technologies, Inc., 2850 Centerville Road, Wilmington, DE, USA

Examples of pharmaceutical products employingZORBAX SB-Phenyl Rapid Resolution High Through-put (RRHT) columns are shown. SB-Phenyl possessesuseful selectivity for compounds containing phenyl-type moieties. Using RRHT columns, several combi-nations of stationary and mobile phase were quicklyinvestigated to determine the best selectivity and res-olution for analgesics and steroids. In both cases,ZORBAX SB-Phenyl yielded excellent peak shape inacetonitrile, but in methanol even more selectivitywas obtained.

Currently, much HPLC method development is being drivenby the need for faster analysis times, resulting in higher pro-

ductivity. This trend to faster analysis is reflected in the configu-ration of HPLC columns currently used for method development.Whereas several years ago, a 250-mm long column packed with5-µm particles was standard, today’s methods often involve 3.5-µm particles packed in column lengths of 100 mm and shorter.In addition, contemporary HPLC column technology is focusedon even smaller particles (< 2 µm) delivering higher theoreticalplate counts and with the capability of running at very fast flowrates. The high-throughput separations thus obtained are highlydependent on column efficiency alone with the assumption thatif sufficient resolution can be obtained on a “standard column,”the use of smaller particles, higher flow rates, and optimized in-strumentation will make the separation faster.Increasing column efficiency is one way to increase resolution.

It can be accomplished by increasing column length or by de-creasing particle size. Resolution, however, is a function of thesquare root of N as shown:

Large changes in N result in small changes in chromatographicresolution. Another approach, which can be far more effective, isto work with selectivity, (a). Selectivity can be altered by chang-ing bonded phase (column) or changing the mobile phase.An accepted standard for equilibrating an HPLC column is 10

column volumes. This can be a rather time-consuming task onconventional columns (4.6 × 150 mm, 1 mL/min), but by usingshort columns (4.6 × 50 mm, 2 mL/min), this can be accom-plished in less than 5 minutes. By taking advantage of this fastequilibration, columns and mobile phases can be used for selec-tivity improvements in less time.Phenyl is an alternative to ODS phases and is particularly use-

ful for the analysis of aromatic-containing compounds. Theunique selectivity for the phenyl phase is derived from an inter-action of the pi electrons found in the phenyl groups. Phenylcolumns are typically used in applications involving pharmaceu-ticals such as analgesics and other aromatic compounds

ExperimentalAn Agilent 1200 HPLC system was used throughout these ex-periments. This system included an autosampler, binary pump,temperature-controlled column compartment, and a diode arraydetector at 254 nm. Samples were purchased from Sigma-Aldrich(St. Louis, MO USA) and include the analgesics in Figure 1: tol-metin, naproxen, diflusinal, ibuprofen, and diclofenac. Thesteroids in Figure 2, hydrocortisone, prednisone, betamethasone,dexamethasone, and corticosterone were also purchased fromSigma- Aldrich. The analgesics and steroids were prepared indi-vidually in methanol at approximately 1 mg/mL. The individualcomponents were then mixed at 1 part each tolmetin, naproxenand diclofenac with 0.5 parts diflusinal and 2 parts ibuprofen.Columns used in this work include StableBond SB-C18, SB-C8,SB-CN, and SB-Phenyl, all 4.6 × 50 mm with 1.8-µm particles.The part numbers for these rapid-resolution high-throughput (RRHT) columns are listed in Table 1.

Results and DiscussionBecause of the high resolution and short length of these RRHTcolumns, evaluations of all column and mobile phase combina-tions were accomplished in less than 1 day. Short high-resolutioncolumns can allow many column options to be examined withvery little time, typically equilibrating to most mobile phases infewer than 10 mL or about 5 minutes.Analgesics such as ibuprofen and others depicted in Figure 1 re-

duce pain, fever, and inflammation. They are categorized as non-steroidal anti-inflammatory drugs or NSAIDs. As can be seen in

Table 1: Columns Used in This Work

1 ZORBAX SB-Phenyl RRHT1.8 µm, 4.6 × 50 mm, Part Number 827975-912

2 ZORBAX SB-CN RRHT1.8 µm, 4.6 × 50 mm, Part Number 827975-005

3 ZORBAX SB-C8 RRHT1.8 µm, 4.6 × 50 mm, Part Number 827975-006

4 ZORBAX SB-C18 RRH1.8 µm, 4.6 × 50 mm, Part Number 827975-002

OCTOBER 2008 SELECTIVITY HPLC 15

Figure 1: Steriods used in this study.

Figure 2: Structures of analgesic compounds used in this study.

Figure 3, these compounds can be readily separated at pH 2.5 inacetonitrile. Since these are small-particle columns, in many caseshigher flow rates can be used to speed the separation without lossof efficiency. The optimum flow rate for maximum efficiency isdependent on a method’s specific operating conditions. The bestflow rate, therefore, should be determined experimentally. In thismobile phase, the optimal efficiency was achieved at 1.85mL/min. In Figure 4, several columns from the StableBond family are

compared. Using the optimized mobile phase conditions fromFigure 1, some interesting selectivity is revealed. StableBond C18shows the longest retention of these analgesics, followed by SB-C8, phenyl, and Cyano (CN). An interesting elution order changebetween C18 and C8 for the last two eluting peaks was observedfor ibuprofen and diclofenac. On the C18 column we observeibuprofen as the latest eluter, while on the C8, it can be seen topartially co-elute with diclofenac. Phenyl yields a similar elutionorder to C8, but in this case we have complete resolution of thelast two components. CN shows another selectivity change mov-ing the third component, diflusinal, to the last position. The peakshape for diflusinal is better, however, on the phenyl and the C8column than on the C18 or CN. Comparing methanol and acetonitrile mobile phases in Figure

5 show some interesting results. Since acetonitrile is a strongersolvent than methanol, a higher concentration of methanol is usedto achieve an iso-elutropic comparison. In the methanol com-parison, one quickly observes the co-elution of tolmetin andnaproxen, but also the longer retention of these phenyl-like struc-tures. Longer retention of analytes can be advantageous in caseswhere sample matrices such as serum are not strongly retained.The use of methanol may help selectively retain aromatic com-pounds on phenyl columns.Figure 6 shows a separation of five steroids in an acetoni-

trile/water mobile phase using several columns. The separation ofthese compounds on StableBond C8 and Phenyl columns yieldssimilar chromatograms; the peaks are all well shaped and well re-solved. However, the more highly aliphatic C18 is found to beslightly less retentive than the C8. The StableBond CN columnhas a very good separation of the first two peaks followed by a co-elution of the peaks corresponding to betamethasone and corti-costerone (peaks 3 and 5). Figure 7 shows a separation of the steroids in a methanol mo-

bile phase. In choosing a mobile phase where the steroids separateon the phenyl column, no complete separation is achieved on theother columns. Methanol again shows more retention and betterselectivity. A recently published work proposes that acetonitrileoverwhelms or blocks the pi-pi (π-π) interactions between the an-alytes and phenyl stationary phase[1]. This work supports thatproposal, using steroids and analgesics as examples.

ConclusionsWhile the initial column offerings of sub-two-micron columnswere primarily C18, selectivity advantages offered by alternativephases such as C8, phenyl, and cyano can yield improvements inresolution and speed. Short, 1.8-micron columns quickly equili-

OCTOBER 200816 HPLC SELECTIVITY

Figure 3: Determining optimum flow rate for RRHT columns foranalgesics method.

Figure 4: Analgesic separation on fast and selective family of StableBond RRHT columns

Figure 5: Comparison of acetonitrile and methanol solvent conditions.

Figure 6: Steroid separation on fast and selective SB RRHTfinish of columns.

OCTOBER 2008 SELECTIVITY HPLC 17

brate, allowing selectivity to be quickly evaluated using many dif-ferent columns or mobile phases. When choosing a stationaryphase, a separation mechanism that employs differences in thechemical structures of the target analytes should be used. Foranalyses in which the target analytes are structurally very similar,this is especially critical. For analgesics and steroids, this includesseparations based on π-π interactions between aromatic or un-saturated groups and a stationary phase containing phenyl group.In order to take best advantage of these interactions, the use ofmethanolic mobile phase should be considered when using aphenyl column.

References1. M. Yang, S. Fazio, D. Munch, and P. Drumm, “Impact of

methanol and acetonitrile on separations based on π-πinteractions with reversed phase phenyl column,” J. of Chromatography, 1097, 124-129. 2005.

Figure 7: SB-Phenyl with methanol shows substantial differ-ences in k and a.


18 OCTOBER 2008

Food and Flavor Analysis Rapid Screening and Analysis of Components in Nonalcoholic DrinksPublication Number – 5989-5178EN

Multiresidue Analysis of 301 Pesticides in Food Samplesby LC Triple Quadrupole Mass SpectrometryPublication Number – 5989-8614EN

Environmental AnalysisEPA Method 1694: Agilent’s 6410A LC/MS/MS Solutionfor Pharmaceuticals and Personal Care Products in Water,Soil, Sediment, and Biosolids by HPLC/MS/MSPublication Number – 5989-9665EN

Rapid Separation and Identification of Carbonyl Compounds by HPLC Publication Number – 5989-7483EN

Specialty Chemical Analysis Analysis of Phenolic Antioxidant and Erucamide Slip Additives in Polymer by Rapid-Resolution LCPublication Number – 5989-5850EN

A Total Solution for the Analysis of Melamine and Cyanuric Acid in Pet Food by GC/MS and Aqueous Normal-Phase LC/MS/MS Publication Number – 5989-7546EN

Forensic Analysis Determination of Benzodiazepines in Oral Fluid Using LC/MS/MS Publication Number – 5989-7201EN

Pharmaceutical Impurity ProfilingAnalysisImpurity Profiling with the Agilent 1200 Series LC SystemPart 1: Structure Elucidation of Impurities with LC/MS Publication Number – 5989-5617EN

Impurity Profiling with the Agilent 1200 Series LC System Part 3: Rapid Condition Scouting for Method DevelopmentPublication Number – 5989-5619EN

Impurity Profiling with the Agilent 1200 Series LC SystemPart 5: QA/QC Application Example Using a Fast LCMethod for Higher Sample Throughput Publication Number – 5989-5621EN

Pharmaceutical Drug Discovery AnalysisDeveloping a fast, generic method for rapid resolution LC with quadrupole MS detection – Combining the Agilent 6140 quadrupole MS and multimode source with the Agilent 1200 Series Rapid Resolution LC system:Fast, generic LC/MS method enables drug analysis in lessthan one minutePublication Number – 5989 -7592EN

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Improve Peak Shape and Productivity in HPLC analysis ofPharmaceutical compounds with Eclipse Plus C8 ColumnsPublication Number – 5989-5803EN

Access the complete applications online by visitingwww.agilent.com/chem/library and entering the publication number specified.

GLOSSARY 19

Glossary of Liquid-PhaseSeparation TermsRonald E. Majors and Peter W. Carr


OCTOBER 2008

In 2001, the second glossary of com-mon and not-so-common terms and

“buzz words” for reference to HPLCcolumns and column technology waspublished (1). It is time for an updatesince new terms have arisen or, in somecases, their original meanings have ex-panded or changed. We have also de-cided to expand the terms dealing withHPLC and LC to cover some ofthe common terms that we neglected toinclude in the earlier glossary. To makeroom for this expansion, we have de-cided to separate the terms referring tocapillary electrophoresis (CE) since thistechnique is rather specialized and notall liquid chromatographers are also per-forming the various forms of CE. Wewill also stick to the conventions of theInternational Union of Pure and Ap-plied Chemistry (IUPAC) in their“Nomenclature for Chromatography”that provides guidance and changes insome of the more commonly acceptedterms (2). Since there is still widespreadusage of nomenclature that is not inalignment with the IUPAC definitions,those terms specifically recommendedby them will be followed by a (IUPAC)in parens.The article is not intended to be an

in-depth listing or highly theoretical cov-erage. For example, we have elected notto cover the myriad of terms used in in-strumentation, detection, data handling,and validation associated with HPLCanalysis but have chosen to use termsthat may be encountered in everydaylaboratory work around columns,phases, method development, and gen-eral usage. The listing should be helpfulto those just starting in HPLC but it canalso serve as a refresher for longtimeusers in the field.The entire listing, including the removed

CE terms, can also be found on the LCGCwebsite at www.chromatographyonline.com.

A ��a: See separation factor.A solvent: Usually the weaker solvent

in a binary eluent or gradient elutionseparation. In reversed-phase liquidchromatography (LC), the A solvent typ-ically is water or a water-rich mixture.A term: The first term in the van

Deemter equation. See eddy dispersionterm and van Deemter equation.Absorption: The process of retention

in which the solute partitions into a liquid-like coating.Activity: The relative strength of the

surface of the packing in adsorptionchromatography. For silica gel, the moreavailable the silanol groups, the more active the surface. Activity can be con-trolled by adding water or other polarmodifier that hydrogen bonds to the active sites, thereby reducing the surfaceactivity.Additive: A substance added to the

mobile phase to improve the separationor detection characteristics; for example,a competing base to negate the effectsof silanols, a chelating agent to blockmetal sites, or a UV-absorbing com-pound to perform indirect photometricdetection.Adjusted retention time (tR’): A

measure of the retention time adjustedfor the holdup time; tR’ = tR – tM,where tR is the retention time and tM isthe holdup time (the time it takes for asmall, unretained compound that com-pletely permeates the pores to be elutedfrom the chromatographic column).Adjusted retention volume (VR’):

Adjusts the retention volume for theholdup volume; VR’ = VR – VM,where VR is the retention volume of thepeak of interest and VM is the holdupvolume (the volume corresponding tothe total volume of mobile phase in thecolumn). See also dead volume andholdup volume.

Adsorbent: Packing used in adsorp-tion chromatography. Silica gel and alu-mina are the most frequently usedadsorbents in high performance liquidchromatography (HPLC).Adsorption: The process of retention

in which the interactions between thesolute and the surface of an adsorbentdominate. The forces can be strongforces (hydrogen bonds) or weak (vander Waals forces). For silica gel, thesilanol group is the driving force foradsorption, and any solute functionalgroup that can interact with this groupcan be retained on silica. The termadsorption places emphasis on the sur-face versus penetration or embedding inthe stationary phase coated or bondedto a surface.Adsorption chromatography: One

of the basic LC modes that relies uponadsorption to the surface of an activesolid to effect the separation. Silica geland alumina are the most frequentlyused normal-phase adsorbents, and mol-ecules are retained by the interaction oftheir polar function groups with the sur-face functional groups; for example,silanols of silica. Carbon also is used asan adsorbent in a reversed-phase mode.Adsorption isotherm: A plot of the

equilibrium concentration of sample inthe mobile phase per unit volume versusthe concentration in the stationary phaseper unit weight in adsorption chro-matography. The shape of the adsorp-tion isotherm can determine thechromatographic behavior of the solute;for example, peak tailing, peak fronting,and column overload.Aerogel: A packing prepared when

the dispersing agent is removed from agel system without collapsing the gelstructure. Silica gels and glass beads usedfor size-exclusion chromatography (SEC)are examples of aerogels that can retaintheir structures even at the high pres-

20 GLOSSARY OCTOBER 2008

sures used in HPLC. See also xerogels.Affinity chromatography: A tech-

nique in which a biospecific adsorbent isprepared by coupling a specific ligand— such as an enzyme, antigen, or hor-mone — for the macromolecule of in-terest to a solid support (or carrier).This immobilized ligand will interactonly with molecules that can selectivelybind to it. Molecules that will not bindwill be eluted unretained. The retainedcompound later can be released in a pu-rified state. Affinity chromatography isnormally practiced as an on–off separa-tion technique.Agarose: High molecular weight poly-

saccharide used as a separation mediumin biochromatography. It is used in beadform, often in gel-filtration chromatog-raphy, with aqueous mobile phases.

Alkoxysilane: A reactant used for thepreparation of chemically bondedphases. It will react with silica gel as follows: R3SiOR + [SiOH ®[Si–OSiR3 1 ROH, where R is analkyl group.Alumina: A normal-phase adsorbent

used in adsorption chromatography.Aluminum oxide is a porous adsorbentthat is available with a slightly basic sur-face; neutral and acidic modificationsalso can be made. Basic alumina canhave advantages over silica, which isconsidered to have an acidic surface.Amino phase: A propylamino phase

used in normal bonded-phase chro-matography. It is somewhat reactive forsolute molecules such as aldehydes ormobile-phase additives that can reactwith amines. The amino phase hasfound some applications as a weak anionexchanger, and it also is used for the

separation of carbohydrates with awater–acetonitrile mobile phase. It is arelatively unstable phase.Amphoteric ion-exchange resin:

Ionexchange resins that have both posi-tive and negative ionic groups. Theseresins are most useful for ion retardationin which all ionic materials can be re-moved from solution because the an-ionic and cationic functionalities coexiston the same material.Analyte: The compound of interest to

be analyzed by injection into and elutionfrom an HPLC column.Analytical Column: An HPLC col-

umn used for qualitative and quantitia-tive analysis; a typical analytical columnwill be 4.6-mm I.d. X 50-250 cm inlength but columns with smaller diame-ters (down to 0.05-mm I.d.) can also beconsidered as analytical columns; can beconstructed of stainless steel , glass,glass-lined SS, PEEK and other metallicand non-metallic materials.Anion exchange: The ion-exchange

procedure used for the separation ofanions. Synthetic resins, bonded-phasesilicas, and other metal oxides can beanalyzed in this mode. A typical an-ionexchange functional group is thetetraalkylammonium, which makes astrong anion exchanger. An aminogroup on a bonded stationary phase isan example of a weak anion exchanger.Asymmetry: Factor describing the

shape of a chromatographic peak. Chro-matographic theory assumes a Gaussianshape and that peaks are symmetrical. Aquantitative measure is the peak asym-metry factor, which is the ratio of thedistance from the peak apex to the backside of the chromatography curve overthe distance from the peak apex to thefront side of the chromatography curveat 10% of the peak height. Other meas-ures of asymmetry are commonly used,especially the U.S. Pharmacopeia (USP)method. See Figure 1. See alsoFoley–Dorsey equation.Asymmetry factor: A factor that

denotes band shape. The asymmetryfactor is calculated from the chromato-graphic peak by dropping a perpendicu-lar at the peak apex and a horizontal lineat 10% of the peak height; at the inter-section, the distance to the tail of the

peak along the horizontal line (distanceB) divided by the distance along the hor-izontal line to the front of the peak (dis-tance A) produces a ratio called the peakasymmetry factor (see Figure 1). Theratio is 1 for a symmetrical peak, lessthan 1 for a fronting peak, and greaterthan 1 for a tailing peak. The higher thevalue, the less symmetrical the peak; val-ues greater than 2 are unacceptable.Atmosphere (atm): A measure of the

pressure drop across an HPLC column;1 atm = 14.7 lb/in.2 (psi). See also barand pascals.

B ��b: See phase ratio.Bo: See permeability.B solvent: Usually the stronger sol-

vent in a binary eluent or gradient sepa-ration; typically the organic modifier ormodifier-rich binary mixture with waterin reversed-phase LC.B term: The second term of the van

Deemter equation. See also longitudi-nal diffusion and molecular diffusionterm.Backflushing: A column-switching

technique in which a four-way valveplaced between the injector and the col-umn allows mobile-phase flow in eitherdirection. Backflushing is used to elutestrongly held compounds at the head ofa column. It can be used for analyzingthese compounds or merely removingthem from the column.Back Pressure: Same as head pres-

sure, column pressure.Back Pressure Regulator: A device

placed on-line after the detector tomaintain a positive pressure on the flowcell minimizing solvent outgassing prob-lems in the detector.Band: Refers to the chromatographic

peak as it moves down and is elutedfrom the column.Band broadening: The process of

increasing width and concomitant dilut-ing of the chromatographic band as itmoves down the column. The peak isinjected as a narrow slug and, ideally,each separated component would beeluted as a narrow slug of pure com-pound if not for the process of band

Figure 1: Example of a tailing peak: (Mod-ified with permission from reference 3.)

GLOSSARY 21

broadening. The measure of bandbroadening is bandwidth (tw) or, morecorrectly, the number of theoreticalplates (N) in the column. Sometimescalled band dispersion or band spread-ing. See Figure 2.Bandwidth (tw): The width of the

chromatographic band during elutionfrom the column. It usually is measuredat the baseline by drawing tangents tothe inflection points on the sides of theGaussian curve that represents the peak.Small bandwidths usually represent effi-cient separations; also called peak width.See Figure 2.Bar: A unit of pressure measurement

in HPLC equal to 1 atm, ;15 lb/in.2,or 0.1 MPa.Baseline: The baseline is the line

drawn by the recording device repre-senting the signal from the detectorwhen only mobile phase is passingthrough. It also represents the pointfrom which calculations are often madeon peaks to determine peak area or peakheight.Baseline Noise: Irregular variations

(short term) in the chromatographicbaseline due to electrical noise or tem-perature fluctuations, outgassing in theflow cell, or poorly mixed mobile phasesolvents.BET method: Developed by Bruner,

Emmett, and Teller (BET), a method formeasuring surface area that uses nitro-gen adsorption–condensation in poresat liquid nitrogen temperature. Pore vol-ume and pore size distribution also canbe obtained from BET method calcula-tions.Bidentate silane: A specific type of

bonded phase in which a short hydro-carbon bridge connects two siliconatoms in a silane that is bound to thesurface through two siloxane groups.Binary mobile phase: Mobile phase

comprising two solvents or buffers.Biocompatible: A term to indicate

that the column or instrument compo-nent will not irreversibly or stronglyadsorb or deactivate biomolecules suchas proteins. Frequently means metal-free

or ceramic surfaces and components.Bonded-phase chromatography:

The most popular mode in LC in whicha phase chemically bonded to a supportis used for separation. The most popularsupport for bonded-phase chromatogra-phy is microparticulate silica gel, and themost popular type of bonded phase isorganosilanesuch as octadecyl for re-versedphase chromatography. Approxi-mately 70% of all HPLC applicationsare performed using chemically bondedphases.Bonded-phase concentration: See

coverage.Boxcar chromatography: See

column switching.Breakthrough volume: The volume

at which a particular solute pumpedcontinuously through a column willbegin to be eluted. It is related to thecolumn volume and the retention factorof the solute. It is useful to determinethe total sample capacity of the columnfor a particular solute.Buffer: A solution that maintains con-

stant pH by resisting changes in pHfrom dilution or addition of smallamounts of acids and bases.Buffer capacity: A quantitative meas-

ure of the potential of a buffer solution(defined as the number of equivalentsof strong acid or base to cause a one pHunit change in 1 L of a buffer solution)or simply the ability of a buffer to with-stand injections of a buffered samplesolution without changing mobile-phasepH; capacity determined by pH, bufferpKa, and buffer concentration.Buffer Strength: See Ionic Strength.

C ��C term: The interphase mass transfer

term of the van Deemter equation. Seealso mass transfer and van Deemterequation.C8: See octylsilane.C18: See octadecylsilane.C4, C8, C18, etc.: Refer to the alkyl-

chain length of a reversed bondedphase.

CS: See Langmuir isotherm.Capacity: See sample capacity.Capacity factor (k9): Old term for a

chromatographic parameter that meas-ures the degree of retention. Nowdefined as the retention factor (k) bythe International Union of Pure andApplied Chemistry (IUPAC). See alsoretention factor for method of calculation.Capillary column: Refers to columns

with inner diameters less than 0.5 mm.Capillary electrochromatography

(CEC): A hybrid technique in which cap-illary columns are packed with chro-matographic sorbents and electroosmoticflow rather than pressure moves mobilephase through the column; techniquehas the surface-mediated selectivitypotential of HPLC and the high effi-ciency of capillary electrophoresis (CE).Capillary LC: Generally refers to

HPLC performed in a fused-silica orother type of capillary column; the innerdiameters typically are less than 0.5 mm;has also been called micro-LC.Capillary micellar electrochro-

matography: The CEC version of mi-cellar electrokinetic capillarychromatography (MEKC).Capillary tubing: Tubing to connect

various parts of a chromatograph anddirect flow to the proper places. Mostcapillary tubing used in HPLC is lessthan 0.020 in. in inner diameter. Thesmallest useful inner diameter is approx-imately 0.004 in.Capping: Same as endcapping.Carbon Load: For a bonded phase

silica, term usually used to describe thesurface coverage or the degree to whichthe available silanols on the columnpacking's surface have reacted and beenreplaced with the bonded phase; thehigher the carbon load, the lower num-ber of residual silanols. The carbon loadis normally expressed as a % carbon(e.g. 12% carbon). In reversed-phaseLC, the higher the carbon load, thegreater the analyte retention.Carrier: A term most often used in

affinity chromatography; refers to the


OCTOBER 2008


upport that binds the active ligand,usually by a covalent bond; can also referto the support in other chromatographymodes such as liquid–liquid chromatog-raphy.Carrier gas: The mobile phase in gas

chromatography (GC).Cartridge column: A column type

that has no endfittings and is held in acartridge holder. The column comprisesa tube and packing contained by frits ineach end of the tube. Cartridges are easyto change and are less expensive andmore convenient than conventionalcolumns with endfittings.Cation-exchange chromatography:

The form of ion-exchange chromatog-raphy that uses resins or packings withfunctional groups that can separatecations. An example of a strong cationfunctional group would be a sulfonicacid; a weak cation-exchange functionalgroup would be a carboxylic acid.CE: Capillary electrophoresis.CEC: See capillary electrochromatogra-

phy.

CGE: See capillary gel electrophoresis.CZE: See capillary zone electrophoresis.Chain length: The length of carbon

chain in the hydrocarbon portion of areversed-phase packing. It is expressedas the number of carbon atoms (C8,C18, etc.). It specifically excludes theshort chains — typically methyl, iso-propyl, and sec-butyl groups — that alsoare attached to the silane.Channeling: Occurs when voids cre-

ated in the packing material causemobile phase and accompanying solutesto move more rapidly than the averageflow velocity, which in turn allows bandbroadening to occur. The voids are cre-ated by poor packing or erosion of thepacked bed.Check Valve: A device inserted into a

moving liquid stream that allows flow ofthe stream in only one direction; mostoften used on the inlet and outlet sidesof an HPLC pump.Chemisorption: Sorption caused by a

chemical reaction with the packing.Most of these interactions are irre-

versible and usually occur on packingswith reactive functional groups such assilanol or bonded amino phases.Chemisorption is common with metaloxide phases that have strong Lewis acidsites.Chiral recognition: The ability of a

chiral stationary phase to interact differ-ently with two enantiomers leading totheir HPLC separation.Chiral stationary phases: Stationary

phases that are designed to separateenantiomeric mixtures. The phases canbe coated or bonded to solid supports,created in situ on the surface of thesolid support, or exist as surface cavitiesthat allow specific interactions with oneenantiomeric form.Chlorosilane: A chemical reagent used

to prepare siloxane bonded phases; reac-tivity changes from a monochlorosilane< dichlorosilane < trichlorosilane; thealkyl portion (octadecyl, octyl, etc.) willdictate the hydrophobicity of the result-ing bonded phase; alkoxysilanes can beused but are less reactive.Chromatogram: A plot of detector

signal output or sample concentrationversus time or elution volume during thechromatographic process.Chromatograph: As a noun: a device

used to implement a chromatographicseparation. As a verb (IUPAC): the actof separating by elution through a chro-matographic bed.Chromatographic Conditions: Those

chromatographic method experimentalparameters that describe how an analysiswas performed. Sufficient informationmust be presented so that the analysiscan be duplicated for verification pur-poses.Classification: The process of sizing

column packing particles; generally inHPLC, small particle-size distributionprovides better efficiency and a greaterpermeability because of the absence offines. Classification can be performed bysedimentation, elutriation, and centrifu-galair techniques.Column back pressure: See head

pressure.Column chromatography: Any form

of chromatography that uses a columnor tube to hold the stationary phase.

Figure 2: Widths of a Gaussian peak at various heights as a function of the standard devia-tion (?????) of the peak. (Modified with permission from reference 2.)

GLOSSARY 23

Open-column chromatography, HPLC,and open-tubular capillary chromatogra-phy all are forms of column chromatog-raphy. Most often refers to open-columnchromatography used for prepara-tivescale work.Column Dead Time: The time associ-

ated with the dead volume; determinedby the dead volume divided by the flowrate; in reversed phase LC, uracil is oftenused to measure dead volume and deadtimes.Column length (L): The length of

chromatography column in HPLC orcapillary in CE used to perform the liquid-phase separation.Column Packing: The solid material,

usually a porous solid with or without achemically interactive surface, placedinside of the column used to differen-tially retain analytes; referred to as thestationary phase; common packings areunbonded and bonded silica, resins,inorganic-organic hybrids, graphtizedcarbonColumn performance (N): Refers to

the efficiency of a column; the numberof theoretical plates for a given test compound.Column plate number (N): Denotes

the column efficiency; the larger theplate number, the more theoretical platesthe column possesses; a typical well-packed column with a 5-µm dp porouspacking in a 15 cm x 4.6 mm columnshould provide 10,000–12,000 plates.Column switching: Using multiple

columns connected by switching valvesfor better chromatographic separationsor sample cleanup. Fractions from a pri-mary column can be switched to two ormore secondary columns, which in turncan be further diverted to additionalcolumns or to detectors; sometimescalled multidimensional chromatogra-phy.Column volume (Vc): The volume of

the unpacked column; Vc = Ac L, whereAc and L are the cross-sectional area ofthe tube and the tube length, respec-tively.Competing base: Adding a small

basic compound such as triethylamine or

dimethyloctylamine at 10–50 mM con-centration to the mobile phase inreversed-phase chromatography toinhibit basic analytes from interactingwith residual silanols; works by the lawof mass action because concentration ofcompeting base is much greater thananalyte. See also additive.Comprehensive two-dimensional

chromatography: Two-dimensionalchromatography applied to every frac-tion. See also two-dimensional chro-matography.Controlled surface porosity support:

Same as porous-layer bead and pellicu-lar packing.Counterion: The ion in solution used

to displace the ion of interest from theionic site in an ion-exchange process. Inion pairing, it is the ion of oppositecharge added to the mobile phase toform a neutral ion pair in solution.Coupled columns: A form of columnswitching that uses a primary columnconnected to two secondary columns bya selector valve. Fractions from the firstcolumn can be selectively transferred tothe second and third columns for addi-tional separations. This term also is usedto describe two or more columns con-nected in series to provide an increasednumber of plates.Coverage: Refers to the amount of

bonded phase on a silica support inbonded-phase chromatography. Cover-age usually is described in micromolesper square meter or in terms of percent-age carbon (w/w).Critical micelle concentration: The

concentration of an ionic surfactantabove which a micelle is formed byaggregation; micelles added to a mobilephase improve the separation of non-ionic substances in HPLC and CE(MEKC) by a partitioning mechanism.Cross-linking: During the process of

copolymerization of resins to form athree-dimensional matrix, a difunctionalmonomer is added to form cross-link-ages between adjacent polymer chains.The degree of cross-linking is deter-mined by the amount of the monomeradded to the reaction. For example,

divinylbenzene is a typical cross-linkingagent for the production of polystyreneion-exchange resins. The swelling anddiffusion characteristics of a resin aregoverned by its degree of cross-linking.Cyano Phase: A chemically bonded

phase that terminates with the -CNfunctional group; it can be used in nor-mal phase as a moderate polarity sorbentand in reversed-phase as a short chainbonded phase.Cyclodextrins: Cyclic oligomers of

several D-(+)-glucopyranose units usedin chiral HPLC and CE separations;popular ones are named a-, b-, and g-cyclodextrins; they have a truncatedcone shape, a relatively hydrophobiccavity, and primary and secondary hy-droxyl groups at their ends; they sepa-rate on the basis of differential inclusionof enantiomers; modified cyclodextrinswith derivatized hydroxyl groups also areused for selectivity modification.

D ��Data Acquisition Rate: A term refer-

ring to the rate of sampling of a detec-tor output. To characterize achromatographic peak at least 20-30data points must be collected. The dataacquisition rate, usually measured inHertz, defines how many data points persecond are collected while the peak ismoving through the detector. For fastchromatography, the data acquisitionrate must be sufficiently rapid to charac-terize a narrow peak. Modern detectorshave data rates up to 80 Hz; also knownas data rate and sampling rate.Dead volume (VM): The column dead

volume comprises the entire space ac-cessible to a small molecule that canfully permeate all pores of a packingmaterial. It includes the interstitial vol-ume and the unoccupied pore volume. Itis denoted as VM. The system dead vol-ume includes the additional volume inthe tubing that connects the injector anddetector to the column. The systemdead volume usually is approximated byinjecting a small, essentially unretainedspecies. Uracil, acetone and thiourea are


OCTOBER 2008


most commonly used species inreversed-phase chromatography. See alsoadjusted retention volume, holdup vol-ume, and void volume.DEAE: See diethylaminoethyl.Degassing: The process of removing

dissolved gas from the mobile phasebefore or during use. Dissolved gas maycome out of solution in the detector celland cause baseline spikes and noise. Dis-solved air can affect detectors such aselectrochemical (by reaction) or fluores-cence (by quenching) detectors. Dis-solved gases also can cause pumps tolose their prime. Degassing is performedby heating the solvent, helium sparging,or using vacuum (in a vacuum flask) oronline evacuation from a tube made of agas-permeable substance such as polyte-trafluoroethylene (PTFE).Denaturing HPLC: Using reversed-

phase HPLC to investigate geneticmutations by the investigation of DNAbase pairs.Desalting: Technique in which low

molecular weight salts and other com-pounds can be removed from nonionicand high molecular weight compounds.An example is using a reversed-phasepacking to retain sample compounds byhydrophobic effects yet allowing salts topass through unretained. Using an SECcolumn to exclude large molecules andretain lower molecular weight salts isanother example.Dextran: Polydextran-based packing

material primarily used for low-pressurebiochromatography; an example wouldbe Sephadex (Amersham PharmaciaBiotech, Piscataway, New Jersey).Diethylaminoethyl (DEAE): A popu-

lar weak anion-exchange functionality(typically attached to cellulose orSepharose [Amersham PharmaciaBiotech]) used for separating biomole-cules.Diffusion coefficient (DM or DS): A

fundamental parameter of a molecule ingas, solution (DM), or the stationaryphase (DS). Expressed in square cen-timeters per second. DM is dependenton the molecular weight of the solute,temperature, solvent viscosity, and molarvolume of the solute. A typical value fora 100-Da molecule in reversed-phasechromatography at room temperature is

10–5 cm2/s.Diol phase: A hydrophilic phase that

is useful in normal and reversed phase.It is a diol structure (two –OH groupson adjacent carbon atoms in an aliphaticchain). In normal-phase work, it is lesspolar than silica. It has been used to sep-arate proteins and polypeptides inreversed-phase chromatography.Displacement chromatography: A

chromatographic process in which thesample is placed onto the column headand then is displaced by a compoundthat is more strongly sorbed than thecompounds of the original mixture.Sample molecules then are displaced byeach other and by the more stronglysorbed compound. The result is that theeluted sample solute zones may besharpened; displacement techniqueshave been used mainly in preparative-scale HPLC applications.Distribution constant (coefficient)

(Kc): The total equilibrium concentra-tion of a component in all forms or onthe stationary phase divided by the totalequilibrium concentration of the com-ponent in the mobile phase; also calledthe distribution coefficient or the parti-tion coefficient in partition chromatog-raphy. In partition chromatography, Kcis used when the concentration in thestationary phase is expressed per unitvolume of the phase(VR = VM + KcVS). In a solid station-ary phase, KVg is used and is expressedper mass (weight) of the dry solid phase.In adsorption chromatography with awell characterized adsorbent of knownsurface area, the concentration in thestationary phase is expressed per unitsurface area.DM: See diffusion coefficient.dp: See particle size.DS: See diffusion coefficient.Dwell time: The time equivalent to

dwell volume; determined by the prod-uct of flow rate and the dwell volume.Dwell volume: The volume between

the point of mixing of solvents (usuallyin the mixing chamber or at the propor-tioning valves in the liquid chromato-graph) and the head of an LC column.Important in gradient elution or in iso-cratic elution situations when changes in

solvent composition are made so thatthe column experiences the compositionchange in the shortest possible time.Low-pressure mixing systems generallyhave larger dwell volumes than highpres-sure mixing systems.Dynamic coating: The formation of

in-situ coatings on the packing in HPLCor on capillary walls in CE by adding asubstance to the mobile phase thatadsorbs onto (or absorbs into) the pack-ing or at the wall surface. The purposeof a dynamic coating is to generate anew stationary phase or to deactivate thepacking material or capillary wall to pre-vent unwanted interactions. One simpleexample is the adjustment of the mobilephase or running buffer to less than pH3 to protonate silanols and negate theireffect. Another example is coating thephase with a hydrophilic polymericmaterial to prevent adsorption ofproteins.

E ��E: See separation impedance.«: See interparticle porosity.Eddy dispersion (diffusion) term (l):

The A term in the van Deemter equa-tion. It is the contribution to plateheight from the heterogeneity in axialvelocities as a result of the particle sizeand geometry of the packing, as well aswall effects; A 5 2ldp, where l is anempirical column constant. Typical val-ues of l for well-packed columns are0.8–1.0. Some theories of chromatogra-phy indicate a velocity-dependent contri-bution to the height equivalent to atheoretical plate (HETP) from thisprocess. Also known as eddy diffusion,flow-heterogeneity induced broadening,and the multipath term. See also vanDeemter equation.

«e: See interstitial porosity.Effective plate height (Heff): The

column length divided by the effectiveplate number.Effective theoretical plates (Neff):

Also called the effective plate number byIUPAC. The true number of plates in acolumn, because it corrects theoreticalplates for dead volume. Neff =16[(tR9/wb)2], where tR9 is the adjusted

GLOSSARY 25

retention time and wb is the bandwidthof the peak (see Figure 2). It is a betterfigure of merit than simple plate num-ber for comparing devices of very dif-ferent geometries and phase ratios.Efficiency (N or H ): A measure typi-

cally determined by the number of theo-retical plates (N) calculated from theequation N 5 16(VR/wb)2 5 16(tR/wb)2, where wb is the peak widthmeasured at the base (see Figure 2). Ifthe peak width is measured at halfheight, the following equation is used: N5 5.545 (VR/wh)2. The plate height (H)or HETP is determined by H 5= L/N.The efficiency of asymmetric peaks isbetter determined from the peak cen-troid and variance by mathematicalanalysis of the peak shape. See alsoFoley–Dorsey equation.Effluent: The mobile phase leaving

the column; same as eluate.«i: See intraparticle porosity.Eluate: Combination of mobile phase

and solute exiting the column; alsocalled effluent.Eluent: The mobile phase used to per-

forma separation.Eluite: The species being eluted, the

analyte, or the sample.Eluotropic series: A series of sol-

vents (eluents) with an increasing degreeof solvent strength generally used in liq-uid– solid or adsorption chromatogra-phy. In normal-phase chromatography, anonpolar solvent such as pentane wouldbe at the low end of the scale, an inter-mediate solvent such as methylene chlo-ride would be in the middle of the scale,and a strongly polar solvent such asmethanol would be near the upper endof the scale. In reversed-phase chro-matography, the reverse order ofstrength would be observed; waterwould be weak and acetonitrile strong.Thus, when developing a method orrunning a gradient, an eluotropic seriesis useful for selecting solvents. See alsoSnyder «o.Elute: To chromatograph by elution

chromatography. The term elute is pre-ferred over develop, which was used inolder nomenclature.

Elution: The process of passing mo-bile phase through the column to trans-port solutes down a column.Elution chromatography: The most

commonly used chromatographicmethod in which a sample is applied tothe head of the column as a narrowzone and individual analytes are sepa-rated and eluted from the end of thecolumn. Compare with displacementchromatography and frontal analysis.Elution volume (VR): Refers to the

volume of mobile phase necessary toelute a solute from a column. It is thevolume from the point of injection tothe volume at maximum concentration(apex) for a symmetrical peak; VR =FtR, where F is the flow rate and tR isthe retention time of the peak of interest.Elutriation: A technique used to frac-

tionate packing particles by size basedon the difference in their Stokes termi-nal velocities. It most often is used forthe separation of ion-exchange resinsthat require a particularly narrow sizerange, such as amino acid resins. Thetechnique involves the upward flow ofwater into a large tube. The unsizedbeads are added to the moving water,and the particles seek their own level,depending upon their density and parti-cle size. They are removed at certain levels in the tube. High-purity sphericalsilica gels sometimes are sized by elutriation.Enantiomeric Compound: Chemical

compounds that display chiral activity;such compounds will require a separa-tion mechanism that can differentiatebetween the R- or S-enantiomer andspecialty columns are available for thispurpose.Endcapping: A technique used to

remove silica gel silanol groups that mayremain after reaction with a large sily-latin agent such as octadecyltrichlorosi-lane. The column is said to beendcapped when a small silylatingreagent (such as trimethylchlorosilaneor dichlorodimethylsilane) is used tobond residual silanol groups on a silica-gel–based packing surface. Most often

used with reversed-phase packings tominimize undesirable adsorption ofbasic, ionizable, and ionic compounds.Endcapping reactions also are used toremove terminal silanol groups frompolymeric phases.Endfitting: The fitting at the end of

the column that permits connection tothe injector or detector. Most HPLCendfittings have frits to contain thepacking and low dead volumes for mini-mum band spreading. They usually areconstructed of stainless steel, but poly-etherether ketone (PEEK) and otherpolymeric materials also are used.«T: See total porosity.Exchange capacity: See ion-exchange

capacity.Excluded volume: See interstitial

volume.Exclusion chromatography: See

ionexclusion chromatography andsteric exclusion chromatography.Exclusion limit: The upper limit of

molecular weight (or size) beyond whichmolecules will be eluted at the sameretention volume, called the exclusionvolume. Many SEC packings are knownby their exclusion limit. For example, a105 column of porous silica gel willexclude any compounds with a molecu-lar weight greater than 100,000, basedon a polystyrene calibration standard.Exclusion volume (V0, Vei): The

minimum retention volume of a mole-cule on an SEC packing in which allmolecules larger than the size of thelargest pore are totally excluded. Thesemolecules are incapable of penetratingthe pores and are eluted at the interstitial(interparticle) volume of the column.Exponentially modified Gaussian

peak: An asymmetric peak resultingfrom passing a Gaussian peak through adetector that is excessively slow or hasan excessive volume. Frequently used tomodel peak tailing arising from the col-umn per se. The basis for theFoley–Dorsey equations. See alsoFoley–Dorsey equation.Extracolumn effects: The total band

broadening effects of all parts of the


OCTOBER 2008


chromatographic system outside of thecolumn itself. Extracolumn effects mustbe minimized to maintain the efficiencyof a column. Sources of band broaden-ing can include the injector design,injection volume, connecting tubing,endfittings, frits, detector cell volume,and internal detector tubing. The vari-ances of all of these contributions areadditive.Extracolumn volume: The volume

between the effective injection point andthe effective detection point, excludingthe part of the column containing thestationary phase. It comprises the vol-umes of the injector, connecting linesand frits, and the detector. It determinesthe extracolumn effects.

F ��F: See flow rate.F: See flow resistance parameter.Fast LC: Use of HPLC of short

columns (1.5–7 cm) with conventionalinner diameters (2–6 mm) packed withsmall particles (3- or 5-µm dp). Separa-tion times in the range of minutes, oreven seconds, are common.Fast protein LC (FPLC): A termed

coined to cover the specific use ofHPLC for separating proteins. Gener-ally, glass columns, moderate pressure,and spherical microbeads are used forFPLC.Flash chromatography: A very fast

form of classic LC used by syntheticorganic chemists for rapid purification.Performed primarily in the normal-phase mode, sometimes with reversed-phase chromatography.Flow rate (F): The volumetric rate of

flow of a mobile phase through an LCcolumn. Typical flow rates are 1–2mL/min for a conventional 4.6-mm i.d.HPLC column.Flow resistance parameter (F): F 5

dp 2/Bo, where Bo is permeability. Seealso permeability.Fluoro phase: One of a family of

aliphatic and aromatic reversed-phasematerials in which a substantial fractionof the bonded phase is fluorinated.Sometimes called fluorous phases orperfluoro phases. Typically these phases

have different selectivities than hydro-carbon phases.Foley–Dorsey equation: A correction

of the plate count and retention timefor peak tailing from extracolumnsources of broadening. See reference 3.FPLC: See fast protein LC.Fractionation range: Refers to the

operating range of a gel or packing inSEC. This range is where a packing canseparate molecules based on their size.At one end of the range, molecules thatare too large to diffuse into the poresare excluded. At the other end of therange, molecules that can diffuse into allof the pores totally permeate the pack-ing and are eluted (unseparated) at thepermeation volume.Frit: The porous element at either end

of a column that contains the columnpacking. It is placed at the very ends ofthe column tube or, more commonly, inthe endfitting. Frits can be stainless steelor other inert metal or plastic such asporous PTFE or polypropylene. The fritporosity must be less than the smallestparticle in the HPLC column; otherwiseparticles will pass through the frit, andthe packing will be lost.Frontal analysis: A chromatographic

technique that involves continuous addi-tion of sample to the column with theresult that only the least sorbed com-pound, which moves at the fastest rate,is obtained in a pure state. The sec-ondleast-sorbed compound is elutedwith the first-eluted compound, thethirdleast-sorbed compound with thefirst and second compound and so onuntil the original sample is eluted at thecolumn exit. Frontal analysis is seldomused and is mainly a preparative tech-nique.Frontal chromatography: Same as

frontal analysis.Fronting: Peak shape in which the

front part of the peak (before the apex)in a chromatogram tapers in advance ofthe remainder of the peak; that is, thefront is less steep than the rear. Thepeak has an asymmetric distributionwith a leading edge. The asymmetry fac-tor for a fronting peak has a value ofless than one. Tailing is the opposite ef-fect. Fronting can result at high sampleloads because of positive curvature in

the isotherm and from using poorlypacked columns.

G ��g: The obstruction or tortuosity fac-

tor. Molecular diffusing term. See alsotortuosity.Gaussian curve: A standard error

curve, based on a mathematical func-tion, that is a symmetrical, bell-shapedband or peak. Most chromatographictheory assumes a Gaussian peak. Usingthe peak maximum position as a meas-ure of retention and the efficiency equa-tions mentioned above assume Gaussianpeak shape. See Figure 2.Gaussian peak: A peak whose shape

conforms closely to the equation: C =Cmax exp[-(t - tR)2/2s2].Gel: The solid packing used in gel

chromatography or gel-permeationchromatography (GPC). An actual gelconsists of two parts: the dispersedmedium (solid portion) and the dispers-ing medium (the solvent). Also definedas a colloidal dispersion of a solid andliquid in which the solid is the continu-ous phase.Gel-filtration chromatography

(GFC): Also called aqueous size-exclu-sion chromatography. Performed withaqueous mobile phases. Generally refersto molecular size separation performedon soft gels such as polydextrans, butanalysts also can use highly cross-linkedpolymers, silica gels, and other porousmedia. Most gel-filtration separationsinvolve biopolymers and water-solublepolymers such as polyacrylic acid.Gel-permeation chromatography(GPC): SEC performed with organic

mobile phases used for the separationand characterization of polymers. SECwith aqueous mobile phases is calledaqueous GPC, GFC, or aqueous SEC.GFC: See gel-filtration chromatogra-

phy.Gigapores: See perfusion chromatog-

raphy.GPC: See gel-permeation chromatog-

raphy.Gradient: A process to change solvent

strength as a function of time (normallysolvent strength increases) thereby elut-

GLOSSARY 27

ing progressively more highly retainedanalytes. Typically gradients can bebinary, ternary, and quaternary solventmixtures in which solvents are blendedto achieve the proper strength.Gradient Delay Volume: See dwell

volumeGradient elution: Technique for

decreasing separation time by increasingthe mobile-phase strength over timeduring the chromatographic separation.Also known as solvent programming.Gradients can be continuous or step-wise. Binary, ternary, and quaternary sol-vent gradients have been used routinelyin HPLC.Graphitized carbon: Graphitized car-

bon is a graphitic carbon with more orless perfect three-dimensional hexagonalcrystalline order prepared from non-graphitic carbon by graphitization heattreatment; this packing material has astrong affinity for polar compounds inaqueous samples and water miscibleorganic extracts. Commonly used in pes-ticide analysis of food samplesGraphitized carbon packing: A

reversed-phase packing material consist-ing of pure graphitic carbon. Possessesinteresting sorbent properties such aspreferential separation of geometric iso-mers such as o-, m- and p-aromatics andcis–trans isomers.Guard column: A small column

placed between the injector and the ana-lytical column. It protects the analyticalcolumn from contamination by sampleparticulates and strongly retainedspecies. The guard column usually ispacked with the same material as that inthe analytical column and is often of thesame inner diameter. It is much shorter,costs less, and usually is discarded whenit becomes contaminated. Integratedguard–analytical column systems oftenare preferred to minimize extracolumneffects caused by connecting tubing withseparate guard and analytical columns.

H ��h: Reduced plate height. Defined as

HETP/dp, where HETP is the height

equivalent to a theoretical plate and dp isthe particle diameter. See also reducedplate height.H: Same as HETP. See also efficiency.h: See viscosity.Head pressure (Dp): The difference

in pressure between the inlet and outletof a column measured in pounds persquare inch. Governed by the followingapproximate equation for a columnpacked with spherical particles of typicalinternal porosity (0.5): Dp =3000Lh/tMdp 2, where L is the columnlength in centimeters, h is the mobile-phase viscosity in centipoise, tM the col-umn holdup time in minutes, and dp isthe particle diameter in micrometers.Pressure can be expressed in pounds per square inch, bars, atmospheres, orpascals.Heart cutting: Refers to collection of

the center of the peak at which purityshould be maximum in preparative LC.The term also is used in column switching.Heff: See effective plate height.Helium sparging: See degassing.

Helium has a very low solubility in mostcommon liquids.HETP: Height equivalent to a theoreti-

cal plate. A carryover from distillationtheory; a measure of column efficiency;HETP = L/N, where L is columnlength and N is the number of theoreti-cal plates. HETP should be approxi-mately 2–3 dp for 5-µm particles with atypical well-packed HPLC column,HETP (or H) values usually are in therange of 0.01–0.03 mm. See also effi-ciency and h.High performance CE: A technique in

which small-diameter capillaries,buffered conducting solutions, and highvoltages (as much as 30,000 V) separateionic molecules based on their differen-tial electrophoretic mobilities. Nonionic(neutral) molecules can be separated byMEKC. High performance liquid chro-matography(HPLC): The modern, fully instrumen-

tal form of liquid-phase chromatogra-phy technique that uses small particles

and high pressures. Sometimes calledhigh-pressure LC.High Pressure Mixing: A configura-

tion of a gradient HPLC system wherethe solvents are mixed on the high pres-sure side of multiple pumps (usually 2,binary); such a system offers a lowergradient delay volume than low pressuremixing systems where the solvents aremixed by proportioning valves prior to asingle pump.Holdup volume (VM): The total vol-

ume of mobile phase in the columnregardless of where it exists; VM = Ve+ Vi, where Ve is the interstitial volumeand Vi is the intraparticle volume. Alsocalled the column void volume. IUPACindicates that use of the term dead vol-ume should be eliminated for this con-cept. The use of dead volume is limitedto regions not swept by the flowingmobile phase system. Holdup volume ismeasured by injecting an unretainedspecies that fits in all the pores. See alsointerstitial porosity and intraparticleporosity.HPLC: See high performance liquid

chromatography.Hybrid silica: Silica gel comprising

both organic and inorganic moietieswith hybrid properties of polymericpackings and silica packings. Synthesizedfrom silanes containing organic func-tionality. Different selectivity but betterhigh-pH stability than bare or uncoatedsilica gel.Hydrodynamic volume: The molecu-

lar volume defined by the effective di-ameter of a molecule in free solution atwhich the hydrodynamic sphere wouldbe a sphere defined by the molecule as itrevolves around its central axis in solu-tion. Term used in SEC to define molec-ular shape and to explain why moleculeswith the same molecular weight oftenhave different elution volumes. Meas-ured by determining the Stokes radius.Hydrophilic: Greek word for water

loving. Refers to stationary phases thatare fully compatible with water and towater-soluble molecules in general.Many columns used to separate proteins— such as ion-exchange, SEC, and


OCTOBER 2008


affinity columns — are hydrophilic innature and should not irreversibly sorbor denature protein in an aqueous envi-ronment.Hydrophilic interaction chromatog-

raphy: An alternative technique toreversed-phase HPLC (RPC) for theseparation of highly polar analytes thatmay be only slightly retained by RPC,HILIC requires a high percentage of anonpolar mobile phase and a polar sta-tionary phase, similar to the require-ments in normal phase chromatography(NPC). However, unlike NPC whichuses nonpolar solvents such as hexaneand methylene chloride and tries to ex-clude water from the mobile phase,HILIC requires some water in the mo-bile phase to maintain a stagnant en-riched water layer on the surface intowhich analytes may selectively partition.In addition, watermiscible organic sol-vents are used instead of the water-im-miscible solvents used in NPC. WithHILIC, sorbents such as bare silica,bonded diol, and polyhdroxyethylaspar-tamide are used. Polar analytes are wellretained and elute in order of increasinghydrophilicity, just the inverse of RPLC.Hydrophobic: Greek word for water

fearing. Refers to stationary phases thatare incompatible with water or to mole-cules that in general have little affinityfor water. Hydrophobic molecules havefew polar functional groups. Most havea high content of hydrocarbon (aliphaticand aromatic) functionality.Hydrophobic interaction chro-

matography: A technique in whichweakly polar (nonhydrocarboneous)packings are used to separate moleculesby the interactions of their hydrophobicmoieties and the hydrophobic sites ontheir packing surface. High concentra-tions of salt solutions are used in themobile phases, and separations are gen-erated by changing the salt concentra-tion. The technique is analogous tosalting-out molecules from solution.Gradients are run by decreasing thesalt concentration. The technique oftenis used to separate proteins that are sensitive to denaturization by the organicsolvents used in regular reversed-phasechromatography. Usually little or noorganic solvent is used in the mobile

phase in hydrophobic interaction chro-matography.Hydroxyapatite: A porous calcium

hydroxy phosphate solid that chemicallyresembles bone and tooth. Used as apacking material in biochromatographyfor nucleic acid constituents, mono-clonal antibodies, and proteins.Hyphenated techniques: Refers to

the family of techniques best known bytheir acronyms, including LC–massspectrometry (MS), LC–Fourier trans-form IR spectroscopy (FTIR), and LC–MS–MS. See also multidimensionalchromatography.

I ��IC: See ion chromatography.Immobilized metal-affinity chro-

matography: See metal-affinity chro-matography.Immunoaffinity Chromatography: A

specific form of separation where anantibody is bonded or immobilizedonto the surface of an HPLC supportmaterial. Based a molecular recognitionmechanism, analytes that are specificallytargetted by the antibody can be selec-tively retained via antibody-antigeninteractions from a complex mixture.After interferences are washed away,retained analytes can be released bychanging the mobile phase conditionssuch that the strong binding is dis-rupted.Imprinted phases: Polymer and silica

phases generated in the presence of atemplate or printing molecule. Thesephases have enhanced selectivity for thetemplating molecule.Included volume: Also known as

totally included volume. The volume atwhich a small molecule that explores theentire pore space of a column is eluted.See also size-exclusion chromatogra-phy.Indirect detection: Used for non-UV

absorbing or nonfluorescing analytes. AUV-absorbing or fluorescent compoundadded to the mobile phase maintains ahigh background signal; when a nonab-sorbing or nonfluorescing analyte iseluted, the background is diluted and anegative peak is observed for that ana-

lyte. When an analyte acts to increasethe concentration of the indicatingspecies, it produces a positive peak.When a negative signal is detected, thedetector signals are reversed to the out-put device.Infinite diameter column effect: At

a certain column length, a sample in-jected into center of a packed bedspreads by radial diffusion but neverreaches column wall, where wall effectscan cause band broadening. Phenome-non observed by John Knox, whoshowed that a sample peak collected inthe exact center of the column exit dis-played a higher efficiency than a samplepeak collected near the wall. The infinitediameter effect depends on columnlength, internal diameter, particle size,and mobile-phase properties. Very sel-dom applied in HPLC.Injection Solvent: Solvent used to

inject sample into an HPLC column;solvent should be of equal or lowerstrength than the mobile phase to pre-vent premature movement down thecolumn due to the presence of astronger solvent.Inlet: The initial part of the column

where the solvent and sample enter. Aninlet frit usually holds the packing inplace and, in some cases, protects thepacked bed.Inlet/Outlet Check Valves: The check

valve(s) on an LC pump that allow(s)mobile phase to flow in one directionbut not in the reverse direction. Theinlet check valve allow flow from thereservoir into the pump and the outletcheck valve allows mobile phase to flowto the column from the pump.Inlet Filter: Filtration devices attached

to the inlet lines of the pump thatremoves particulate matter from themobile phase before the solvent reachesthe pump; reservoir filters are an inletfilter that resides in the solvent bottle.In-line filter: A device that prevents

particulate matter from damaging thecolumn. Modern low-volume, in-line fil-ters can be placed between the injectorand the column without major contribu-tions to band broadening. A filter in thisposition prevents sample particles fromentering the packed bed or column inlet frit.

GLOSSARY 29

Interparticle porosity («e): The inter-particle volume of a packed column perunit column volume; «e 5= Ve/Vc,where Ve is the interstitial volume andVc is the total column volume. See alsointerstitial porosity.Interparticle volume (Vo): The vol-

ume of mobile phase located outside theparticles.Interstitial porosity («e): The frac-

tion of the volume in the column lo-cated in the interparticle (interstitial)space; «e = Ve/Vc.Interstitial velocity (ue): The actual

velocity of the eluent as it movesthrough the column flowing around theparticles; ue = F/Ac«e. The interstitialvelocity is the basis for computing thereduced velocity.Interstitial volume (Ve): The volume

between the particles. It does not in-clude the volume in the pores of theparticles. Also called the excluded vol-ume (see SEC) and interparticle volume.Measured by injecting a molecule thatdoes not permeate any pores and doesnot interact with the surface of the particles. In SEC, this volume is denoted Vo.Intraparticle porosity («i): The frac-

tion of the particle volume that is thepore volume; «i = Vpore/Vparticle.Intraparticle volume (Vi): The vol-

ume inside the pores of the particles.Also called the internal and includedvolume. Can be measured by the BET method or mercury-intrusionporosimetry.Ion chromatography (IC): An ionex-

change technique in which low concen-trations of organic and inorganicanions or cations are determined usingion exchangers of low ion-exchangecapacity with dilute buffers. Conductiv-ity detectors often are used. IC is prac-ticed in two forms: In suppressed IC, asecond column or a membrane separa-tor is used to remove the buffer counterion from the analyte and simultaneouslyreplace it with a hydrogen or hydroxideion that concomitantly converts thebuffer to an uncharged species therebysuppressing background and enhancing

sensitivity. In nonsuppressed IC, low-concentration, weakly conductingbuffers are carefully selected, the entireeffluent is passed through the detector,and ions are detected above the back-ground signal.Ion-exchange capacity: The number

of ionic sites on the packing that canparticipate in the exchange process. Theexchange capacity is expressed in mil12liequivalents per gram. A typicalstyrene–divinylbenzene strong anionex-change resin may have 3–5 mequiv/gcapacity. Exchangers for IC have verylow capacity. Capacity of weak anionand cation exchangers varies dramati-cally with pH.Ion-exchange chromatography: A

mode of chromatography in which ionicsubstances are separated on cationic oranionic sites of the packing. The sampleion, usually with a counterion, willexchange with ions already on the iono-genic group of the packing. Retention isbased on the affinity of different ionsfor the site and other solution parame-ters such as pH, ionic strength, andcounterion type. Ion chromatographybasically is an ion-exchange technique.Ion exclusion: The process in which

ionized solutes can be separated fromun-ionized or partially ionized solutesusing ion-exchange resins. Separationresults from Donnan potential in whichionic solutes exist at a higher concentra-tion in solution than in the stationaryphase, whereas nonionic solutes areevenly distributed between the mobilephase and resin. Therefore, ionic soluteswill move faster down the column thannonionic solutes. Ion exclusion occurs inreversed-phase chromatography whenanions are separated at pH values atwhich the silanol groups are ionized.Ionic Strength: Ionic strength is a

characteristic of an electrolyte solution.It is typically expressed as the averageelectrostatic interactions among an elec-trolyte’s ions. It is related to electrolyteconcentration but the main differencebetween ionic strength and electrolyteconcentration is that the former ishigher if some of the ions are more

highly charged. The higher the ionicstrength of a mobile phase the more themobile phase competes with the analytefor ionic or adsorptive sites.Ion-moderated partitioning chro-

matography: A technique used for separating carbohydrates using strongcation exchange packings that are in specific cationic form (for example, calcium, hydrogen, silver). The separa-tion mechanism is complexation ratherthan ion exchange.Ion-pair chromatography: Form of

chromatography in which ions in solu-tion can be paired or neutralized andseparated as an ion pair on a reversed-phase column. Ion-pairing agents usuallyare ionic compounds that contain ahydrocarbon chain, which imparts a cer-tain hydrophobicity so that the ion paircan be retained on a reversed-phase col-umn. Retention is proportional to thelength of the hydrophobic chain and theconcentration of the ion-pair additive.Ion pairing also can occur in normal-phase chromatography when onepart of the pair is dynamically loadedonto a sorbent, but this technique is notas popular as reversed-phase chromatog-raphy. Also known as ion-interactionchromatography or dynamic ionex-change chromatography, which stressesthat users sometimes do not know theprecise mechanistic details of how theadditive controls retention.Ion retardation: Refers to using

amphoteric ion-exchange resins, whichretard ionic molecules and allow non-ionic molecules or nonelectrolytes to beeluted preferentially.Ion suppression: Buffering in an

aqueous mobile phase at a particular pHto suppress solute ionization. For exam-ple, weak carboxylic acids can have theirionization suppressed by the adjustmentof the pH below their pKa value. Usefulfor improving peak shape of weak acidsand bases in reversed-phase chromatog-raphy.Irregular packing: Refers to the shape

of a column packing. Irregular packingsare available in microparticulate sizes.The packings are obtained from grind-


OCTOBER 2008


ing solid materials into small particlesand sizing them into narrow fractionsusing classification machinery. Sphericalpackings are used more often than irreg-ular packings in analytical HPLC, butthe less-expensive, irregular packings arestill widely used in preparative-scale LC.Irreversible adsorption: When a com-pound with a very strong affinity for anadsorbent is injected onto a column, itcan be adsorbed so strongly that it can-not be eluted from the column. A chem-ical reaction between the sample and thesurface of the adsorbent is an exampleof irreversible adsorption. See alsochemisorption.Isocratic: Using a time invariant–

eluent composition in LC.Isotherm: See adsorption isotherm.Isothermal chromatography: Using

conditions of constant temperature. Thevast preponderance of all LC is per-formed under isothermal conditions.

K ��k: See retention factor.k9: An old term that has been re-

placed by the IUPAC-approved term, retention factor (k).K: See partition coefficient.kA/B: See selectivity coefficient.Kc: See distribution constant (coefficient).Kieselguhr: A diatomaceous earth

used in column chromatography andalso as a sample cleanup media. Onlyweakly adsorptive, it can be used as asupport in liquid–liquid chromatogra-phy. Rarely used in HPLC.Kinetic Plot: Kinetic plots are meth-

ods to characterize the practical limits ofcolumn performance, where theoreticalplates (H) and separation impedance (E)are plotted as a function of the pressure-drop limited plate number (N). Thekinetic plot retains the informationshown in van Deemter plots but com-pletes it with the information on the bedpermeability. See Poppe Plot.Knox equation: A modification of the

van Deemter equation developed byJohn Knox in which the A term thatrepresents eddy dispersion multiplied byu1Y3, where u is the interstitial eluent

velocity. Usually written in terms of thedimensionless or reduced plate height(h) and reduced velocity (v) as h = Av1Y3

+ B/v + Cv. See also van Deemterequation.

L ��L: See column length.Laminar flow: The smooth timein-

variant flow that develops when a liquidis moving under conditions in whichviscous forces dominate inertial forces.Laminar flow is characterized by a lowReynolds number (see Reynoldsnumber). In a cylindrical tube, fluidstreams in the center flow faster thanthose at the tube wall, which results in aradially parabolic distribution in axialfluid velocity. This nonuniformity ofaxial velocities in the interstices in apacked bed also causes substantial peakbroadening in packed columns.Langmuir isotherm: A specific form

of an isotherm; CS = N0CM/(Kd +CM), where CS and CM are the equilib-rium stationary and mobile-phase con-centrations of the solute, N0 the totalnumber of surface sites available forsorption, and Kd the sorption bindingconstant.LC: See liquid chromatography.Ligand: In ligand-exchange chro-

matography, it refers to the analyte thatundergoes ligand exchange with the sta-tionary phase. In affinity chromatogra-phy, it refers to the biospecific material— enzyme, antigen, or hormone —coupled with the support (carrier) toform the affinity column. In bonded-phase chromatography, it refers to themoiety covalently bound to the surface.Ligand-exchange chromatography:A technique in which chelating ligandsare added to the mobile phase andundergo sorption onto a packing. Thesesorbed molecules can act as chelatingagents with certain solutes. For example,copper salt can be added to the mobilephase for the chelation and separation ofamino acids. Chelating resins functionin a similar manner: chelating groups arechemically bonded to the polystyrenebackbone.Linear elution adsorption chro-

matography: Refers to adsorption chro-

matography performed in the linear por-tion of an adsorption isotherm. A termcoined by Lloyd Snyder.Linear velocity (u): The velocity of

the mobile phase moving through thecolumn. Expressed in centimeters persecond. Related to flow rate by thecrosssectional area of the column. De-termined by dividing the column length(L) by the retention time of an unre-tained compound. See also void time.Liquid chromatography (LC): A sepa-

ration technique in which the mobilephase is a liquid. Most often performedin a column.Liquid–liquid chromatography: One

of the earliest separation modes ofHPLC; it gave way to chemically bondedphases in the early 1970s. Same as parti-tion chromatography.Liquid–solid chromatography: Same

as adsorption chromatography.Loadability: The maximum amountof analyte that can be injected onto acolumn that no longer permits the isola-tion of product at the desired level ofpurity or recovery level; important inpreparative chromatographyLoading (phase loading versus

sample loading): The amount of sta-tionary phase coated or bonded onto asolid support. In liquid–liquid chro-matography, the amount of liquid phasein milligrams of per gram of packing. Inbonded-phase chromatography, theloading may be expressed in micromolesper square meter or percentage carbon(w/w). Also called coverage or surfacecoverage. An alternate and unrelatedmeaning is the amount of sample massinjected on an analytical- or prepara-tivescale column; preparative-scalecolumns often are operated in an overloaded condition for throughputreasons.log kw: The extrapolated intercept of

a plot of log k versus volume fraction oforganic modifier in reversed-phase LC.See also S.Longitudinal diffusion: Same as

molecular diffusion term. B term invan Deemter equation. See also vanDeemter equation.Low pressure mixing: See high pres-

sure mixing.

GLOSSARY 31

M ��µ: See electrophoretic mobility.Macroporous resin (macroreticular):

Cross-linked ion-exchange resins thathave molecular-scale micropores andalso macropores of several hundredangstroms. These highly porous resinshave large internal surface areas that areaccessible to large molecules.Mass transfer (interphase): The

process of solute movement betweenthe moving and stationary zones. The Cterm of the van Deemter equation iscalled the interphase mass transfer term.The faster the mass transfer process, thebetter the column efficiency. In HPLC,slow mass transfer is the most importantfactor affecting column efficiency. Itsrate can be increased by using small-par-ticle packings, thin stationary-phase lay-ers, low-viscosity mobile phases, andhigh temperatures.Mean pore diameter: The average

diameter of the pore of a porous pack-ing. It most commonly is determined bythe BET method and is reported asfourfold the specific pore volume di-vided by the specific surface area (4V/A)based on the assumption of uniformcylindrical pores. The pore diameter isimportant in that it must allow free dif-fusion of solute molecules into and outof the pore so that the solute can interact with the stationary phase. Addi-tionally, the pores must be well-con-nected, with a minimum of dead ends,so many paths can allow a molecule toaccess any part of the pore space. InSEC, the packings have different porediameters; therefore, molecules of dif-ferent sizes can be separated. For a typi-cal substrate such as silica gel, 60- and100-Å pore diameters are most popular.Pore diameters greater than 300 Å areused for the separation of biomolecules.Pores usually are classified as micro(<20 Å), meso (20–500 Å), and macro(>500 Å).MECC: See micellar electrokinetic

capillary chromatography.Megapores: See perfusion chro-

matography.

MEKC: See micellar electrokineticcapillary chromatography.Metal-affinity chromatography: A

special form of ligand-exchange chro-matography used to separate biopoly-mers with a particular affinity for aspecific metal cation, typicallycopper(II), zinc(II), and iron(II).Metalophile: A compound that has

high affinity for active acidic silanolgroups on silicas surface. Usually astrongly basic amine or multifunctionalcarboxylate or phenol.Method development: A process for

optimizing the separation, including thesample pretreatment, to obtain a repro-ducible and robust separation. Usually, itemphasizes the search for the stationaryphase, eluent, and column temperaturecombination that provides an adequate,if not optimum, separation.Method validation: A process of

testing a method to show that it per-forms to the desired limits of precisionand accuracy in retention, resolution,and quantitation of the sample compo-nents of interest.Micellar chromatography: Adding

micelles to the mobile phase to causeseparation. The micelles may act as dis-placing or partitioning agents and pro-vide another parameter to changeselectivity. Surfactants at concentrationsgreater than their critical micelle concen-tration are used in micellar chromatogra-phy and in MEKC.Micro-LC: Refers collectively to tech-

niques in which a column of smallerthan conventional inner diameter is usedfor separation. The term micro-LC mostoften is used for HPLC in columns withinner diameters smaller than 0.5 mm;micro-LC is used in high-sensitivityanalysis when the sample amount is lim-ited and with certain ionization tech-niques in LC–MS in which the volumeof solvent flowing into the ionizationsource must be minimized.Microbore: Refers to the use of

smaller-than-usual inner diametercolumns in HPLC. Columns of 2 mmand less are considered to be microboresizes. Inner diameters of 0.5 mm and

smaller are considered micro-LCcolumns.Microchip devices: Microdevices

based on silicon, glass, and other typesof microfabricated chips in which exper-iments can be miniaturized into singleormultichannel microfluidic circuits.These devices can be used for CE andCEC. They should be low cost and dis-posable. Using microdevices for separa-tion currently is in its infancy, andapplications should expand with time.Microparticulate: Refers to the small

particles used in HPLC. Generally pack-ings with a particle diameter of less than10 mm that are totally porous are con-sidered microparticles.Microporous resin: Same as

microreticular resin.Microreticular resin: Cross-linked,

synthetic ion-exchange resins that havepores with openings that correspond tomolecular sizes. Diffusion into the nar-row pores can be impaired, and lowexchange rates and poor performancecan occur, especially for large molecules.Migration rate: See electrophoretic

mobility.Migration time (tm): The time it

takes for a charged molecule to movefrom the point of injection to the pointof detection in a CE capillary. Distinctfrom holdup time (tM).Minimum plate height: The mini-

mum of the van Deemter curve that re-sults from a plot of H versus v. Thisvalue represents the most theoreticalplates that can be obtained for a certaincolumn and mobile-phase system. Usu-ally occurs at excessively low flow rates.Also known as the optimum plateheight. It typically is two- to threefoldthe particle diameter of well-packedcolumns.Mixed-bed column: Combination of

two or more stationary phases in thesame column, used most often in IEC(mixed anion and cation resins) andSEC (mixture of different pore sizepackings). Its advantage in IEC is thetotal removal of both cationic andanionic compounds. Useful in SECbecause a wider molecular weight range


OCTOBER 2008


can be accommodated by the same column.Mixed-mode separation: A separa-

tion that occurs in a single columncaused by the retention and selectivityprovided by a dual-retention mechanism.For example, a reversed-phase columnwith residual silanols at intermediate-to-high pH values can separate by hy-drophobic interaction and ionicinteraction by the ionized silanols.Sometimes mixed-mode separations canbe quite beneficial to the selectivity(band spacing), but they can cause peakasymmetry, and the precise balance of interactions may be difficult to reproduce with subsequent packingbatches.Mobile phase: The solvent that

moves the solute through the column.In LC, the mobile phase interacts withboth the solute and the stationary phaseand, therefore, can have a powerful influence on the separation.Mobile-phase strength: See solvent

strength.Mobile-phase velocity (uM): The

velocity at which the mobile phase per-colates through the bed of particles; uM= L/tM, where L is column length andtM is holdup time. See also adjustedretention volume, holdup volume, anddead volume.Mobility: See electrophoretic

mobility.Modifier: An additive that changes the

character of the mobile phase. For ex-ample, methanol is the strong solvent inreversed phase and sometimes is calledthe modifier (water is the weak solvent);sometimes other additives — competingbases such as triethylamine or ion-pair-ing reagents — are referred to as modi-fiers, but they more correctly should becalled additives. See also additives.Molecular diffusion term (B term):

Refers to the B term (second term) ofthe van Deemter equation. Also calledlongitudinal or axial diffusion term. Itdominates band broadening only at verylow flow rates below the minimum plateheight at which the diffusion of individ-ual solutes can occur in a longitudinal(lengthwise) direction on the column.The contribution to the B term arisesfrom diffusion in the mobile phase and

is 2gDM, where g is the obstruction fac-tor (typically 0.6–0.8) and DM is thediffusion coefficient. See also vanDeemter equation.Molecular weight distribution: The

distribution of molecular weight ofmolecules in a polymer sample. Distri-bution can be defined as weight averageand number average.Molecularly imprinted phases: See

imprinted phases.Monodisperse particles: Particles

that fall into a narrow range of diame-ters. See also polydisperse particles.Monomeric phase: Refers to a

bonded phase in which single moleculesare bonded to a support. For silica gel,monomeric phases are prepared by thereaction of an alkyl- or aryl- mono-chloro-or alkoxysilane. Polymeric phasesgenerally are prepared from a di- ortrichlorosilane or an alkoxysilane reac-tant in the presence of water.Moving zone: To be distinguished

from the mobile phase, this zone is thefraction of the mobile phase in the col-umn that occupies the interstitial spaces.See also stationary phase.Multidimensional chromatography:

The use of two or more columns orchromatographic techniques to generatea better separation. It is useful for sam-ple cleanup, increased resolution,increased throughput, and increasedpeak capacity. It can be used off-line bycollecting fractions and reinjecting themonto a second column or on-line byusing a switching valve. Also called cou-pled column chromatography, columnswitching, multicolumn chromatography,and boxcar chromatography.

N ��n: See peak capacity.N: The number of theoretical plates;

N = 16(tR/wb)2, where tR is retentiontime and wb is the base width of thepeak. A measure of the efficiency of acolumn. Sometimes measured as N =5.54(tR/wh)2, where wh (or w1Y2) is thepeak width at half height. See also efficiency and theoretical plate.n: See reduced velocity.NanoLC: LC practiced with columns

less than 100-um in internal diameter;usually requires specialized instrumenta-tion; often used in proteomic studieswhere sample is limited and sensitivityis required.Narrow-bore column: Columns of

less than 2-mm i.d. used in HPLC. Alsocalled microbore.Neff: See effective theoretical plates.Noise: See Baseline NoiseNonaqueous reversed-phase chro-

matography: Refers to reversed-phasechromatography performed withoutwater as a component of the eluent on areversed-phase packing. Used for verynonpolar compounds that cannot beeluted or are difficult to elute from areversed-phase column with 100%methanol or acetonitrile. In these cases,solvent A should be acetonitrile, and sol-vent B should be a stronger solvent suchas tetrahydrofuran. Reversed-phase rulesapply to nonaqueous reversed-phasechromatography; that is, the more nonpolar the analyte, the greater the retention.Non-Polar: A non-polar molecule is

one that the electrons are distributedmore symmetrically and thus does nothave an abundance of charges at theopposite sides. The charges all cancelout each other. Non-polar compounds,solvents or bonded phases readily dis-solve in organic solvents, such ashexane, or prefer such solvents in placeof water. Non-polar substances do notreadily dissolve in water.Nonporous packing: Particles similarto porous-layer bead but with particlediameters in the sub-5-µm range; parti-cles often are in the sub-2-µm dp range.Used for high-speed separations in shortcolumns. Common column abbrevia-tions include NPS, which refers to non-porous silica; NPR, which refers tononporous resins; and NPZ, whichrefers to nonporous zirconia.Nonporous particle: Refers to a solid

particle used as a support for a porouscoated or bonded phase; pellicular parti-cles are nonporous particles of largeparticle diameter (;40 µm). Nonporoussilicas and resins with small particlediameters of less than 3 µm usually aremicrobeads with thin porous outer coat-ings of silica gel, bonded silica gel, or

GLOSSARY 33

polymeric phase.Normal-phase chromatography: A

mode of chromatography performedwhen the stationary phase is more polarthan the mobile phase. A typical normal-phase system would be adsorption chro-matography on silica gel or aluminausing mixtures of less polar eluents suchas hexane–diethethyl ether as a mobilephase. Also refers to the use of polarbonded phases such as cyano and alu-mina. Sometimes called straight-phasechromatography.

O ��Octadecylsilane: The most popular

reversed phase in HPLC. Octadecylsi-lane phases are bonded to silica or poly-meric packings. Both monomeric andpolymeric phases are available. Abbrevi-ated in column names as C18 and ODS.Octylsilane: A popular stationary

phase in reversed-phase chromatogra-phy. Usually provides slightly less reten-tion than the more popular C18. Bothmonomeric and polymeric phases areavailable. Abbreviated in column namesas C8.ODS: See octadecylsilane.On-column detection: The column

itself serves as the flow cell in HPLC orCE–CEC. Generally, the term used withfused-silica capillary applications. Outerpolyimide layer is removed, an opticalbeam is directed through the capillary,and a measuring device such as a photomultiplier tube is located on theopposite side of the capillary.On-line preconcentration: A precol-

umn is placed in front of the separationcolumn to concentrate analytes beforetheir separation. Different mechanisms— hydrophobic interaction, adsorption,or enzymatic reaction — may be used toretain analyte as a function of time.Then concentrated analytes are trans-ferred to the separation column by a displacement process such as solventelution or pH change.Open tubular columns: Small

inner diameter columns (less than 100µm) currently being investigated for use

in HPLC, supercritical fluid chromatog-raphy (SFC), and CE. Stationary phasescan be bonded on the internal walls ofthese small columns. The most fre-quently used column material is fusedsil-ica tubing. Used very little in routineHPLC or SFC but frequently in CE.Optically active resin: Incorporationof optically active groups into an ionex-change resin to allow separation ofoptically active isomers. Few commer-cially available resins for HPLC applications.Organic modifier: Water-miscible

organic solvent added to an aqueousmobile phase to obtain separations inreversed-phase HPLC. Common organic modifiers are acetonitrile,methanol, isopropanol, and tetrahydrofuran.Outlet Check Valve: See Check ValveOverload: In preparative chromatog-

raphy the overload is defined as thesample mass injected onto the column at which efficiency and resolution begins to be effected if the sample size is increased further. See also sample capacity.

P ��Dp: See head pressure.Pa: See pascal.Packing: The adsorbent, gel, or solid

support used in an HPLC column. Mostmodern analytical HPLC packings areless than 10 µm in average diameter,and 5 µm is the current favorite.Paired-ion chromatography: Same as

ion-pair chromatography.Particle size (dp): The average parti-

cle size of the packing in the LC col-umn. A 5-µm dp column would bepacked with particles with a definite par-ticle-size distribution because packingsare never monodisperse. See alsomonodisperse particles, particle size distribution, and polydisperse particles.Particle-size distribution: A measure

of the distribution of the particles usedto pack the LC column. In HPLC, a

narrow particle-size distribution is desir-able. A particle-size distribution of dp ±10% would mean that 90% of the parti-cles fall between 9 and 11 µm for anaverage 10-µm dp packing.Particulates: Generally refers to a

small particles found in the mobilephase that can cause back pressureproblems by lodging in frits; it can alsorefer to the small particles packed intoHPLC columnsPartition chromatography: Separa-

tion process in which one of two liquidphases is held stationary on a solid sup-port (stationary phase) while the other isallowed to flow freely down the column(mobile phase). Solutes partition them-selves between the two phases based ontheir individual partition coefficients.Liquid–liquid chromatography is anexample; modern bonded-phase chro-matography can be considered to be aform of partition chromatography inwhich one of the liquid phases is actu-ally bonded to the solid support. Mecha-nistically partition chromatographyimplies that the solute becomes at leastpartially embedded within the stationaryphase, which is impregnated, coated, orbonded to the substrate. In contrast toan adsorption process in which thesolute does not penetrate into the reten-tive surface or interphase.Partition coefficient (K): The ratio of

the equilibrium concentration of solutein the stationary phase relative to theequilibrium concentration of solute inthe mobile phase. Also called distribu-tion coefficient, KD, and distributionconstant (Kc).Pascal (Pa): A unit of pressure. 1 MPa

is approximately 10 bar (atm) or 150psi.Peak: The profile of an analyte com-

pound as it elutes from a columnthrough a detector; usually depicted on avisual output on a recorder or printerbased on the detector’s electricalresponse.Peak area: The area measured under a

chromatographic peak; usually measuredby an integrator or data system; the peakarea is related to the amount of sub-


OCTOBER 2008


stance eluted in a peak.Peak capacity (n): The number of

equally well-resolved peaks (n) that canbe fit in a chromatogram between theholdup volume and some upper limit inretention. For R = 1, n is given by theapproximation 1 + 0.25[(N)1Y2 ln(1 +kn)], where R is the resolution, N is thenumber of theoretical plates, and kn isthe retention factor for peak n.Peak dispersion: See band

broadening.Peak doublet: A split peak generally

caused by a column void. Could beclosely eluted compounds.Peak height: The height of a chro-

matographic peak as measured from thebaseline to the peak apex; the peakeight is related to the amount of sub-

stance eluted in a peak.Peak shape: Describes the profile

of a chromatography peak. Theory as-sumes a Gaussian peak shape (perfectlysymmetrical). Peak asymmetry factor describes shape as a ratio. See Figures 1and 2. See also asymmetry.Peak tracking: A way of matching

peaks that contain the same compoundbetween different experimental runsduring method development. Reliesupon detection parameters of each pureanalyte. Diodearray detectors and massspectrometers are among the best detec-tors for peak tracking because of theirspecificity.Peak variance (s2): The second cen-

tral moment of the peak about the re-tention time. For a Gaussian peak, thevariance is the fundamental parametercontrolling peak width. See Figure 2. Seealso Gaussian peak.Peak volume: The total volume occu-

pied by a chromatographic peak as itpasses through the detector; VR= F xwb (see Figure 2).Peak width (wb): Same as band-

width. See Figure 2.Pellicular packing: See porous-layer

bead.Percent B solvent (% B solvent):

Refers to the stronger solvent in a bi-nary solvent mixture. % A solventwould be the weaker solvent analog.Perfusion chromatography: Refers to

chromatography performed using parti-

cles with very large pores (4000–8000Å) called throughpores (megapores orgigapores). Eluent flows between thelarge pores and through the particles’300–1000 Å interconnecting pores,called diffusive pores. Best suited for the preparative separation of macromol-ecules.Permeability (Bo): Also called col-

umn permeability and specific perme-ability. A term expressing the resistanceof the packed column to the flow ofmobile phase. For a packed column, Bo 'dp 2«3/[180(1 2 «)2] 5 dp 2/1000. Acolumn with high permeability gives alow pressure drop.Permeation: Refers to the SEC

process in which a solute can enter amobile-phase-filled pore of the packing.Phase ratio (b): The relative amount

of stationary to mobile phase in the col-umn. In partition chromatography, b =VS/VM, where VS and VM are the vol-ume, of stationary and mobile phase inthe column, respectively. The retentionfactor is the product of the phase ratioand the partition coefficient.Phenyl phase: A popular nonpolar

bonded phase prepared by the reactionof dimethylphenylchloro- or alkoxysi-lanewith silica gel. Reportedly has affin-ity for aromatic-containing compoundsand does impart a different selectivitycompared with alkyl-bonded phases.Pirkle column: Chiral, brush-type

stationary phases based on 3,5-dini-trobenzoylphenylglycine silica used inthe separation of a wide variety ofenantiomers. Named after its developer,William Pirkle of the University of Illinois.Planar chromatography: A separa-

tion technique in which the stationaryphase is present as or on a plane(IUPAC). Typical forms are paper andthin-layer chromatography.Plate height (H): See HETP.Plate number: See column plate

number.Plate or plate number: Refers to the-

oretical plates in a packed column(IUPAC). See also theoretical plate.Polar: A polar molecule may be polar

as a result of polar bonds or as a resultof an asymmetric arrangement of non-

polar bonds and non bonding pairs ofelectrons; Polar molecules are generallyable to dissolve in water (H2O) due tothe polar nature of water; polar mole-cules do not prefer non-polar organicsolvents such as hexane. Polar moleculeshave slightly positive and slightly nega-tively charged ends.Polyacrylamide gel: Neutral

hydrophilic polymeric packings used inaqueous SEC. Prepared by the copoly-merization of acryl-amide with N,N9-methylenebisacrylamide.Polydisperse particles: Particles that

have a substantial range of diameters(>10%).Polyethyleneimine: An anionic poly-

meric phase used to coat or bond ontosilica or a polymeric packing. Most often used for separating proteins andpeptides.Polymeric packings: Packings based

on polymeric materials, usually in theform of spherical beads. Typical polymers used in LC are polystyrene–di-vinylbenzene (PS–DVB), polydivinyl-benzene, polyacryl-amide,polymethylacrylate, polyethylene-oxide,polydextran, and polysaccharide.Polymeric phase: Refers to a chemi-

cally bonded phase in which a polymerspecies is bonded to silica-based particles.Polystyrene–divinylbenzene resin

(PS–DVB): The most common basepolymer for ion-exchange chromatogra-phy. Ionic groups are incorporated byvarious chemical reactions. Neutral PS–DVB beads are used in reversed-phasechromatography. Porosity and mechani-cal stability can be altered by varying thecross-linking through the DVB content.Poppe Plot: A kinetic plot named

after Prof. Hans Poppe [J. Chromatogr.A 778, 3 (1997)], University of Amster-dam, the Netherlands, where the platetime [log (t0/N) is depicted as a func-tion of the number of theoretical plates(N) in order to assess the limits of col-umn performances as a function of par-ticle size, column pressure drop, etc.Pore diameter: Same as mean pore

diameter.Pore size: The average size of a pore

in a porous packing. Its value is

GLOSSARY 35

expressed in angstroms or in nanome-ters. The pore size determines whether amolecule can diffuse into and out of thepacking. See also mean pore diameter.Pore volume: The total volume of the

pores in a porous packing, usuallyexpressed in milliliters per gram. Moreappropriately called the specific porevolume. It is measured by the BETmethod of nitrogen adsorption or bymercuryintrusion porosimetry in whichmercury is pumped into the pores underhigh pressure.Porosity: For a porous substrate, the

ratio of the volume of the pores in aparticle to volume occupied by the parti-cle. The pore volume is a measure ofthe porosity and is expressed in milli-liters per gram.Porous-layer bead: A small glass bead

coated with a thin layer of stationaryphase. The thin layer can be an adsor-bent, resin, or a phase chemicallybonded onto the adsorbent. These pack-ings were among the first to be used inHPLC. They had 20–40 µm particlesizes, which were larger than themicroparticulate packings of today, butwere easy to pack and provided adequateefficiency. Also called controlled surfaceporosity supports and pellicularmaterials.Porous particle: Refers to column

packing particles that possess intercon-necting pores of specified diameter andpore volume. For HPLC applications,analysts generally use porous particleswith diameters less than 10 mm. Largerparticles are used in preparative-scalechromatography because of lower costand higher column permeability.Porous polymer: A packing material,

generally spherical, that is based onorganic polymers or copolymers. Popu-lar examples include PS–DVB, polyacry-lates, polydextrans, polyacrylamides, andpolybutadienes.Precolumn: A small column placed

between the pump and the injector. Itremoves particulate matter that may bepresent in the mobile phase, presaturatesthe mobile phase with stationary phaseor with dissolved substrate to prevent a

loss of stationary phase or dissolutionof the analytical column, and chemicallyabsorbs substances that might interferewith the separation. Its volume has littleeffect on isocratic elution but con-tributes a delay to the gradient in gradi-ent elution.Precolumn Filter: A filter used

between the injector and the column (orguard column) to keep unwanted samplecomponents from reaching the column;sometimes called in-line filter, occasion-ally inlet filter.Preconcentration: See trace

enrichment.Preparative chromatography: Refers

to the process of using LC as a tech-nique for the isolation of a sufficientamount of material for other experi-mental or functional purposes. For pharmaceutical or biotechnological pu-rifications, large columns of several feetin diameter can be used for multiplegrams of material. For isolating a fewmicrograms of valuable natural productan analytical column with a 4.6-mm i.d.can be used. Based on the intended needof the chromatographer, both size ofcolumns are preparative chromato-graphic approaches.Pressure (pressure drop) (Dp): See

head pressure.Pressure injection: Pressure-induced

injection in CE. Using pressure or vac-uum to inject nanoliter-level volumes ofsample into a capillary column. Best fornarrow-bore capillaries that have innerdiameters less than 10 µm. A version ofhydrostatic injection.Process-scale chromatography:

Refers to the use of LC at the industrial-scale level outside of laboratories. Gen-erally requires specially designedcolumns (usually with diameters > 5cm), recoverable solvents, low-costpackings (larger and irregular-shapedparticles), and overloaded operating conditions compared with laboratory-scale HPLC.Programmed-temperature chro-

matography: Varying temperature dur-ing a chromatographic run. Seldom usedin LC.

PS–DVB: See polystyrene–divinylben-zene resin.Pulsating flow: Flow originating from

a reciprocating pump. Normally, thepulses are dampened by a pulse damper,an electronic pressure feedback circuit,or an active damper pump head. Detec-tors such as electrochemical and refrac-tive index detectors are greatly affectedby flow pulsations.

Q ��Quaternary methyl amine: A stronganion-exchange functionality popular inresin-based packings. Usually supplied inchloride form.Quaternary mobile phase: A mobilephase comprising four solvents orbuffers.Quaternary-Solvent Mobile Phase: Amobile phase consisting of four separatesolvents which allow for fine tuningmobile phase composition; most oftenthis mobile phase is delivered by a low-pressure quaternary pump.

R ��r: See relative retention.Radial compression: Using radial

pressure applied to a flexible wall col-umn to reduce wall effects.Radial diffusion–dispersion: Diffu-

sion– dispersion across the LC columnin a radial direction. If the sample is in-jected into the exact center of a column,it will spread not only in a longitudinaldirection as it moves down the columnbut also radially, which allows the soluteto reach the wall region where the eluentvelocity is different than in the center ofthe column.Re: See Reynolds number.Recovery: The amount of solute or

sample that is eluted from a column rel-ative to the amount injected. Excellentrecovery is important for good quantita-tion, preparative separations, especiallybiomolecules, and good peak shape andresolution. Reasons for inadequate re-covery can be solute interaction with ac-


OCTOBER 2008

OCTOBER 200836 GLOSSARY

tive sites on the packing, column frits,and column tubing. Compound decom-position during the separation processalso can affect recovery.Recycling chromatography: A tech-

nique in which the column effluent isrecirculated onto the head of the col-umn to take advantage of extended col-umn length. Can be performed on asingle column by passing the effluentthrough the pump again. An alternativetechnique uses two columns connectedby a switching valve where the effluentof one column is directed onto the headof the other column. Very seldom usedin HPLC and then only in exclusionchromatography.Reduced plate height (h): Used to

compare efficiencies of differentcolumns; h = H/dp, where H is theheight equivalent to a theoretical plateand dp is the particle diameter. An hvalue of 2 or less at the optimum veloc-ity is considered to be a well-packedHPLC column.Reduced velocity (n): Used with the

reduced plate height to compare differ-ent packed chromatographic columns. Itrelates the solute diffusion coefficient(DM) in the mobile phase to the particlesize of the column packing (dp); n =udp/DM, where u is the average intersti-tial mobile-phase linear velocity. See alsoKnox equation.Refractive index peak: A pseudo-

peak normally found near the dead vol-ume that results from the refractiveindex sensitivity of absorbance andother detectors. See also vacancy peak.Regeneration: Regenerating the pack-

ing in the column to its initial state aftera gradient elution. Mobile phase ispassed through the column stepwise orin a gradient. The stationary phase isrestored or solvated to its initial condi-tion. In ion exchange, regenerationinvolves replacing ions taken up in theexchange process with the original ions,which occupied the exchange sites.Regeneration also can refer to bringingany column back to its original state; forexample, removing impurities with astrong solvent.Relative retention (r): Retention rela-

tive to a standard; r = tR9/tR(st)9 =k/kst, where tR9 is the adjusted reten-

tion time of the component of interest,tR(st)9 is the adjusted retention time ofthe standard, k and kst are the corre-sponding retention factors. For two ad-jacent peaks, a expresses the relativeretention and is called separation factor(formerly called selectivity or selectivityfactor); calculated as a 5 tR29/tR19 5k2/k1, where tR29 and tR19 are theadjusted retention times of peaks 2 and1, respectively, and k2 and k1 are thecorresponding retention factors.Residual silanols: The silanol

(–Si–OH) groups that remain on thesurface of a packing after chemicallybonding a phase onto its surface. Thesesilanol groups, which may be present invery small pores, may be inaccessible toa reacting bulky organosilane such asoctadecydimethylchlorosilane) but maybe accessible to small polar compounds.Often they are removed by endcappingwith a small organosilane such astrimethylchlorosilane. See also endcapping.Resin: A solid polymeric packing used

in ion-exchange separations. The mostpopular resins are PS–DVB copolymerswith particle sizes less than 10 µm. Ionicfunctionality is incorporated into theresin.Resolution (Rs): Ability of a column

to separate chromatographic peaks; Rs [(tR2 – tR1)/[(wb1 + wb2)/2], wheretR2 and tR1 are the retention times ofthe two peaks and wb is the baselinewidth of the peaks. It usually is ex-pressed in terms of the separation oftwo peaks. A value of 1 is considered tobe the minimum for a measurable sepa-rationto occur and to allow good quanti-tation. A value of 0.6 is required todiscern a valley between two equal-height peaks. A value of 1.5 is consid-ered sufficient for baseline resolution fortwo peaks of equal height. Values of 1.7or greater generally are desirable forrugged methods. See Figure 2.Resolution equation: Also called the

general resolution equation and the Pur-nell equation; R 5 4N1Y2[(a 2 1)/a][k/(11 k)], where N is the efficiency, a is theseparation factor, and k is the retentionfactor.Retention factor (k): The period of

time that the sample component resides

in the stationary phase relative to thetime it resides in the mobile phase. It iscalculated from the adjusted retentiontime divided by the holdup time; k =(tR – tM)/tM, where tR is retentiontime for the sample peak and tM is theretention time for an unretained peak.(Formerly, k9 was used, and it was calledthe capacity factor or the capacity ratio.)Retention time (tR): Also called the

total retention time. The time betweeninjection and the appearance of the peakmaximum. The total retention volume(VR) is determined by multiplying theretention time by the flow rate. Theadjusted retention time (tR9) adjusts forthe column void volume; tR9 = tR –tM. It usually is measured from thepoint of injection to the apex of thepeak, but it should be measured to thecenter of gravity of the peak for asym-metric peaks.Retention volume (VR): The volume

of mobile phase required to elute a sub-stance from the column; VR = F tR orVR = VM + KDVS, where VM is thevoid volume, KD is the distribution co-efficient, and VS is the stationary-phasevolume. See also retention time.Reversed-phase chromatography:

The most frequently used mode inHPLC. Uses low-polarity packings suchas octadecyl- or octylsilane phasesbonded to silica or neutral polymericbeads. The mobile phase usually is wateror water-miscible organic solvents suchas methanol or acetonitrile. Elutionusually occurs based on the relativehydrophobicity or lipophilicity of thesolutes. The more hydrophobic, thestronger the retention. The greater thewater solubility of the analyte, the less itis retained. The technique has manyvariations in which various mobile-phaseadditives impart a different selectivity.For example, adding a buffer and atetraalkylammonium salt to an anionanalysis would allow ion-pairing to occurand generate separations that rival thoseof ion-exchange chromatography. Morethan 90% of HPLC analysts usereversed-phase chromatography.Reynolds number (Re): The ratio of

viscous to inertial energy of the movingfluid. A measurement of flow in asmooth unpacked pipe; Re 5 ud/(h/r),

OCTOBER 2008 GLOSSARY 37

where u is the average velocity (in centi-menters per second), d is the pipe diam-eter, h is the viscosity (in grams percentimeter seconds), and r is the density(in grams per cubic centimeters). At lowRe, viscous friction dominates and con-trols fluid motion, making it slow andsteady. In an unpacked tube, flow be-comes fully turbulent when Re exceeds4200. In a packed bed, u is replaced withthe average interstitial velocity and dwith the average particle diameter. Flowbecomes turbulent in a packed bed at Revalues greater than approximately 10 butis not fully turbulent until Re exceeds100–200.Rs: See resolution.

S ��S: The solvent-strength parameter in

reversed-phase chromatography. Thesolute-dependent slope of a plot oflog10 k versus volume fraction of or-ganic modifier. S varies with modifiertype, stationary phase, and temperature.s2: See peak variance.Salting-out effect: Using a highcon-

centration salt buffer in the mobilephase to cause a low-polarity analyte tohave a decreased solubility in water andtherefore precipitate or come out ofsolution. Most often used for thehydrophobic interaction chromatogra-phy of proteins when proteins are pre-cipitated first at high salt concentrationsand then eluted by gradual dilutionusing reversed-gradient elution.Sample capacity: Refers to the

amount of sample that can be injectedonto an LC column without overloading.Often expressed as grams of sampleper gram of packing. Overloading isdefined as the sample mass injectedwhen the column efficiency decreases by10% from its normal value; sometimescalled sample loading.Sampling Rate: See Data Acquisition

RateSaturator column: See precolumn.Scaleability: In going from analytical

to preparative chromatography, refers to

the reproducibility of results oncolumns of different internal diameterswhen using the same particle size andbonded phase; normally a larger diame-ter column is used to increase capacity;a linear scaleup process minimizes timerequired to optimize preparative separations.SEC: See size-exclusion

chromatography and steric exclusionchromatography.Sedimentation: A technique used for

the sizing of resins for ion-exchangechromatography. A broad distribution ofbeads are placed in a solvent, oftenwater, in a container that is affixed to astationary surface. Based on particle sizeand particle density, the beads will settleat different velocities into a gradient ofsizes, and the fraction of interest isremoved. Workers can obtain very narrow cuts of particle size by sedimentation.Selectivity or selectivity factor (a):

Old term replaced by the separationfactor. Sometimes called relative retention.Selectivity coefficient (kA/B): In

ionexchange chromatography, the equi-librium coefficient obtained by applyingthe law of mass action to an ionexchanger and characterizing the abilityof an ion exchanger to select two ionspresent in the same solution using elec-troosmotic flow. For example, theexchange of Na+ for H+ kNa/H =([Na]S[H]M)/([Na]M[H]S).Semipreparative chromatography:

Refers to preparative LC performed onanalytical (4 –5 mm i.d.) or slightlylarger (6–10 mm i.d.) columns. Normalinjection size would be milligram- tolow-gram-size samples.Separation factor (a): A thermody-

namic factor that is a measure of relativeretention of two substances. Formerlycalled selectivity or selectivity factor. Therelative retention; a 5 tR29/tR19 5k2/k1, where tR29 and tR19 are theadjusted retention times of peaks 2 and1, respectively, and k2 and k1 are thecorresponding retention factors.Separation impedance (E): A figure

of merit developed by John Knox tocompare the efficiency of two chro-matographic systems that normalize forboth analysis time and pressure drop; E = tRDp/N2n(1 1 k), where tR is theretention time, Dp is the pressure drop,N is the efficiency, n is the reducedvelocity, and k the retention factor. Thelower the value of E , the better the system.SFC: See supercritical fluid chro-

matography.Silanol: The Si–OH group found on

the surface of silica gel. Silanols vary instrength depending upon their location,relationship to each other, and the metalcontent of the silica. The strongestsilanols are acidic and often lead toundesirable interactions with basic com-pounds during chromatography.Silanophile: A compound that has

high affinity for active or acidic silanolgroups on a silica surface. Usually astrongly basic amine.Silica gel: The most widely used

HPLC packing. It has an amorphousstructure, is porous, and is composed ofsiloxane and silanol groups. It is used inall modes of LC as a bare packing foradsorption, as the support for liquid–liq-uid chromatography or for chemicallybonded phases, and as an SEC packingwith various pore sizes. Microparticulatesilicas of 3-, 5-, and 10-µm average par-ticle diameter are used in HPLC. Com-pared with irregular silicas, sphericalsilicas are preferred in modern analyticalHPLC columns because of their packingreproducibility and lower pressure drops.Sometimes called silica.Siloxane: The Si–O–Si bond. A prin-

cipal bond found in silica gel or a silylat-edcompound or bonded phase. Stable,except at high pH values. Has littleeffect on the HPLC separation.Silylation: The reaction process of an

organochloro- or organoalkoxysilanewith a compound that contains an reac-tive group. In LC, it refers to the processof derivatizing the solute before chro-matography to make it detectable or toprevent unwanted stationary-phase inter-actions. It also can refer to the process



of adding a chemically bonded phase to a solid support or deactivating thepacking to reduce surface activity.Simulated moving bed: A chromato-

graphic system involving a series ofcolumns and valves set up to simulatethe countercurrent movement of themobile and stationary phases and enablethe continuous removal of product andreapplication of sample. A complexform of recycle chromatography used inpreparative-scale chromatography.Size-exclusion chromatography

(SEC): Same as steric exclusion chromatography.Slurry packing: The technique most

often used to pack HPLC columns withmicroparticles. The packing is sus-pended in a slurry of approximately10% (w/v) and rapidly pumped into the empty column using special high-pressure pumps.Snyder «o: Solvent-strength parame-

terin adsorption chromatography. Theenergy of solvent adsorption per unitsurface area occupied by the solvent.Soap chromatography: The earliername for ion-pair chromatography.Long-chain soaps or detergents wereused as the mobile-phase additives.Sol gel: Silica gel formed by the ag-

gregation of silica sol. Generates Type Bsilica gel with lower surface acidity, lowertrace metal, lower surface area andporosity, and greater high-pH stabilitythan older Type A silica gels.Solid-phase extraction (SPE): A tech-

nique for sample preparation using a20–40 µm dp solid-phase packing con-tained in a small plastic cartridge, disk,or in the wells of a 96-well flowthroughplate. The solid stationary phases usedare identical to HPLC packings.Although related to chromatography, theprinciple of SPE is different and issometimes called digital chromatogra-phy. The process as most often practicedrequires four steps: conditioning the sor-bent, adding the sample, washing awaythe impurities, and eluting the sample inas small a volume as possible with astrong solvent.Solid support: Same as support.Solute: See also analyte.Solvent: The liquid used to dissolve a

sample for injection into an HPLC col-umn or CE capillary. Sometimes refersto the mobile phase used. See also eluent.Solvent demixing: Occurs when two

solvents with very different strengths —A is the weak solvent, and B is thestrong solvent — are used with unmodi-fied silica or alumina. The strong solvent(B) will be adsorbed preferentially by theactive surface of the stationary phaseuntil it is saturated; until this occurs, theweak solvent (A) will be enriched ordemixed as it travels down the column.Eventually, when the entire column issaturated with solvent B, this solvent willbe eluted, mixed with solvent A at theinitial strength, and sample componentswill be eluted with the sudden change insolvent strength.Solvent selectivity: Ability of a sol-

ventto influence selectivity. For example,a change in solvent strength from 5% to10% solvent B or a change frommethanol to acetonitrile as the reversed-phase organic modifier will affect bandspacing.Solvent-selectivity triangle: A useful

guide for choosing among different sol-vents for changing band spacing. Solventselectivity is dependent on dipolemoment, acidity, and basicity of the solvent molecule. See reference 4 fordetails.Solvent strength: Refers to the ability

of a solvent to elute a particular soluteor compound from a column. Snyderdescribed this quality for linear elutionadsorption chromatography (liquid–solidchromatography) on alumina and quan-titatively rated solvents in an eluotropicseries. Less-extensive data are availablefor silica and carbon adsorbents. Seealso Snyder «o.Sorb: The process of being retained

by a stationary phase when the retentionmechanism — adsorption, absorption,or partitioning — is unclear.Sorbent: Refers to a packing used in

LC. Common sorbents are polymers, sil-ica gel, alumina, titania, zirconia, andchemically modified materials.SPE: See solid-phase extraction.

Specific surface area: The surfacearea of an LC packing based on meas-urement by an accepted technique such

as the BET method using nitrogenadsorption.Spherical packing: Refers to spheri-

cal, solid packing materials. In analyticalHPLC, spherical packings generally arepreferred over irregular particles, butirregular particles often are used inpreparative work because of their lower cost.Standards: A sample which contains

known quantities of the compounds ofinterest. Standards are used to help iden-tify sample peaks by comparing the timein which they elute to the retentiontimes obtained through the injection ofthe sample under the same conditions.For quantitation, external standards arecompounds that are used to constructcalibration curves of detector output(peak area or peak height) vs. concentra-tion; the concentration of unknowns aredetermined by fitting the detector out-put to the calibration curve. Internalstandards are compounds of knownconcentration with different retentiontimes that are added to the sample andrelative detector responses between theinternal standard and the unknown arecompared in order to quantitativelymeasure unknown compounds.Stagnant mobile phase: The fraction

of the mobile phase contained withinthe pores of the particle.Stationary phase: The chromato-

graphically retentive immobile phaseinvolved in the chromatographicprocess. The stationary phase in LC canbe a solid, a bonded, an immobilized ora coated phase on a solid support or awall-coated phase. The stationary phaseoften characterizes the LC mode. Forexample, silica gel is used in adsorptionchromatography and octadecylsilanebonded phase is used in reversed-phasechromatography.Stationary zone: To be distinguished

from the stationary phase. The station-ary zone includes the stagnant mobilephase and the chromatographicallyactive stationary phase.Stepwise elution: Using eluents of

different compositions during a chro-matographic run. These eluents areadded in a stepwise manner with apump or a selector valve. Gradient elu-tion is the continuous version of chang-

GLOSSARY 39

ing solvent composition.Steric exclusion chromatography: A

major mode of LC in which samples areseparated by virtue of their size in solution. Also known as size-exclusionchromatography, gel-permeation chromatography, gel-filtrationchromatography, and gel chromatogra-phy. Steric exclusion chromatography isused most often for polymer separationand characterization.Sterically protected bonded phase:

Bonded phase that has sterically protect-ing bulky functional groups such as isopropyl and isobutyl surrounding asiloxane covalent surface bond. Preventsattacks on siloxane bond, catalyzedhydrolysis, and loss of bonded phase at pH levels less than 3.Straight-phase chromatography:

Same as normal-phase chromatogra-phy.Strong anion exchanger: Anionex-

change packing with strongly basic iono-genic groups such as tetraalkylam-monium groups.Strong cation exchanger: Cationex-

change packing with strongly acidicionogenic groups such as sulfonategroups.Strong solvent: In general, refers to a

solvent which is a good solvent for achemical compound; in chromatography,refers to the mobile phase constituentthat provides a higher solvent strengththat causes an analyte to elute morequickly from the column; in a water-acetonitrile binary solvent system for reversed-phase LC, acetonitrile wouldbe considered to be the strongsolvent.Sub-2-µm: A term that refers to the

use of porous packings below 2-µmaverage particle diameter; current prod-ucts vary from 1.5- to 2.0-µmSulfonyl cation exchanger: A strong

cation-exchange functionality found inresin-based packings, usually propyl-SO3H. May come in cationic formssuch as sodium, ammonium, silver, andcalcium.Supercritical fluid chromatography

(SFC): A technique that uses a supercriti-cal fluid as the mobile phase. The tech-nique has been applied to the separationof substances that cannot be handledeffectively by LC (because of detectionproblems) or GC (because of the lack ofvolatility). Examples include separationsof triglycerides, hydrocarbons, and fattyacids. GC detectors and HPLC pumpshave been used together in SFC.Superficial velocity (us): The hypo-

thetical velocity that a mobile phasewould have if the same column wereoperated unpacked but with the sameflow rate; us = F/Ac, where F is the flowrate and Ac is the cross-sectional area ofthe tube.Superficially porous packing: Same

as porous-layer bead.Support: Refers to solid particles. A

support can be naked, coated, or have achemically bonded phase in HPLC.Normally the solid support doesn’t con-tribute to the chromatographic process.Suppressor column: Refers to the col-

umn placed after the ion-exchange col-umn. Its purpose is to remove orsuppress the ionization of buffer ions sothat sample ions can be observed in aweakly conducting background with aconductivity detector. Sometimes mem-brane suppressors are used rather than acolumn.Surface area: Refers to the total area

of the solid surface in an adsorbent asdetermined by an accepted measurementtechnique such as the BET method,which uses nitrogen adsorption. Thesurface area of a typical porous adsor-bent such as silica gel can vary from lessthan 100 to 600 m2/g.Surface coverage: Usually refers to

the mass of stationary phase per unitarea bonded to an LC support. Oftenexpressed in micromoles per squaremeter of surface. Sometimes the per-centage of carbon is given as an indica-tor of surface coverage.Swelling–shrinking: Process in which

resins and gels increase or decrease theirvolume because of their solvent envi-ronment. Swelling is dependent uponthe degree of cross-linking; low-cross-

linking resins will swell and shrink morethan highly cross-linked resins. Ifswelling occurs in a packed columnblockage, increased back pressure canoccur, and column efficiency can be affected.

T ��Tailing: The phenomenon in which a

normal Gaussian peak has an asymmetryfactor greater than 1. The peak will havean extended trailing edge. Tailing iscaused by packing sites that have both astronger-than-normal retention for thesolute and slower desorption kinetics. Atypical example of a tailing phenome-non would be the strong adsorption ofamines on the residual silanol groups ofa low-coverage reversed-phase packingat intermediate pH values. Tailing alsocan result from injecting an excessivemass or sample, badly packed columns,excessive extracolumn volume, poor fit-tings, excessive detector volume, andslow detector response. See Figure 1.Tailing factor: U.S. Pharmacopeia

measure of peak asymmetry defined asthe ratio of the peak width at 5% of theapex to twofold the distance from theapex to the 5% height on the short timeside of the peak. Greater than unity fortailed peaks. See also asymmetry factor.Temperature programming: Chang-

ing column temperature as a function oftime during the separation. Rarely usedin HPLC; if so, usually in a stepwisemanner.Ternary mobile phase: Mobile phase

that is a mixture of three solvents orbuffers.Theoretical plate (N): A concept

described by Martin and Synge. Relateschromatographic separation to the the-ory of distillation. Length of columnrelating to this concept is called heightequivalent to a theoretical plate. See alsoHETP. Plates are calculated as N =16(VR/wb)2 = 16 (tR/wb)2, where VRis the retention volume, wb is the widthat the peak base, and tR is the retentiontime. See also N.Thermally tuned tandem column

chromatography: A form of LC in


OCTOBER 2008

OCTOBER 200840 GLOSSARY

which two columns with distinctly dif-ferent selectivities are placed in tandemand operated at two temperatures tooptimize the resolution or analysisspeed. Both columns use a common elu-ent, and the entire sample passesthrough both columns and is detectedwith a single detector. It is not a two-di-mensional technique because each sam-ple component provides only one peak.Titania: An uncommon adsorbent

used in adsorption chromatography.tm: See migration time.tM: Holdup time.Tortuosity or tortuosity factor: A

packed-column property that controlsthe inhibition of longitudinal diffusionof the solute as it diffuses along the col-umn axis. The B term in the vanDeemter equation is proportional to thetortuosity. See also B term, g, andmolecular diffusion term.Total mobile-phase volume (Vt):

The total volume of mobile phase in anSEC column. Also known as totally in-cluded volume. Same as VM.Total permeation volume (Vp): The

retention volume of an SEC packing inwhich all molecules smaller than thesmallest pore will be eluted. In otherwords, all molecules totally permeate allof the pores at Vp and are eluted as asingle peak. Same as VM.Total porosity («T): Ratio of the total

volume of mobile phase in the columnto the total column volume; « 5 VM/Vc5 «e 1 «i(1 2 «e); where VM is themobile-phase volume, Vc is the columnvolume, «e is the interstitial porosity,and «i is the intraparticle porosity.Totally porous packing: The station-

ary phase is a porous matrix, and solutespenetrate the porous matrix to interactwith the stationary phase.tR: See retention time.tR9: See adjusted retention time and

retention time.Trace enrichment: Technique in

which trace amounts of compounds areretained on an HPLC or precolumnpacking out of a weak mobile phase orsolution and then are eluted by adding astronger mobile phase in a concentratedform. The technique has been appliedmost successfully in the concentration

oftrace amounts of hydrophobic com-pounds such as polynuclear aromatic hydrocarbons from water using a reversed- phase packing. A strong solvent such as acetonitrile will elute the enriched compounds.Triethylamine: A very common addi-

tive used to block silanol groups inreversed-phase chromatography whenseparating basic analytes.Trifluoroacetic acid: A very common

additive in reversed-phase chromatogra-phy for peptides and proteins.Tryptic digestion: A method for se-

lectively and reproducibly dissectingpeptide chains of proteins to yield acharacteristic pattern of smaller unitsthat enables analysis of the parent protein by gradient elution reversed-phase LC.Turbulence: The state in which fluid

velocity fluctuates randomly at a point.See also Reynolds number and turbu-lent flow.Turbulent flow: A form of fluid

motion in which the flow ceases to besmooth and steady and becomes chaoticand fluctuates with time. It is character-ized by a pressure drop significantlyhigher than what would be extrapolatedfrom the laminar region to achieve thesame volumetric flow rate.Turbulent flow chromatography:

Chromatography performed at very highlinear velocities with large particlesunder conditions using high Reynoldsnumbers. At these conditions, the H ver-sus n curves show a decrease in H as nincreases. See Figure 2.tw: See bandwidth.Two-dimensional chromatography:

A procedure in which part or all of theseparated sample components are sub-jected to additional separation steps. Itcan be performed by conducting a par-ticular fraction eluted from the first col-umn into a second column or systemthat has a different separation character-istic. It includes techniques such astwodimensional TLC using two eluentsystems in which the second eluent isapplied after rotating the plate through90°. It also includes LC followed by GCand one LC mode followed by a differ-ent mode such as reversed-phase chro-

matography followed by SEC. See alsomultidimensional chromatography.Type A silica: Silica gel formed by

gelling soluble silicates. Generally hashigher acidity, higher surface area andporosity, more trace metals, and poorerhigh-pH stability than Type B silicas.Type B silica: See sol gel.t0: See void time.

U ��u: See linear velocity and velocity.ue: See interstitial velocity.uM: See mobile-phase velocity.us: See superficial velocity.uz: See zone velocity.UHPLC: Refers to Ultra High Pressure

Liquid Chromatography; often looselyused for any separation performed overthe pressures of conventional pumps(400 bar); original meaning was for sepa-rations in the 20,000 psi+ range.

V ��Vacancy chromatography: Technique

in which a mobile-phase additive causesa positive detector signal output. Whena solute is eluted from the column, itdilutes the signal and generates a nega-tive peak or vacancy. The technique hasbeen applied primarily to single-columnion chromatography in which mobilephases such as citrate and phthalatebuffers absorb in the UV region. Whena nonabsorbing anion is eluted, it dilutesthe UV-absorbing background andcauses a negative peak; the detector out-put leads usually are reversed so that thechromatogram looks normal. It also hasbeen used in CE for detection.van Deemter equation: An equation

used to explain band broadening inchromatography. The equation repre-sents the height of a theoretical plate(HETP) and has three terms. The Aterm describes eddy dispersion or diffu-sion that results from axial velocity het-erogeneity. The B term is for thecontribution of molecular diffusion orlongitudinal diffusion of the solutewhile passing through the column. TheC term is the contribution from inter-phase mass transfer, which allows for

OCTOBER 2008 GLOSSARY HPLC 41

the finite rate of transfer of the solutebetween the stationary phase and mobilephase. In its simplest representation, h 5A 1 B/n 1 Cn. See also reduced plateheight and reduced velocity.Vc: See column volume.Vd: See dead volume.Ve: See interstitial volume.Velocity (u): Same as linear velocity.veo: See electroosmotic flow.Vi: See intraparticle volume.Viscosity (h): Also called mobile-

phase viscosity. The viscosity of the mo-bile phase varies with the temperatureof the column. Low-viscosity mobilephases generally provide better effi-ciency than less-viscous ones becausediffusion coefficients are inversely re-lated to solvent viscosity. For example,column efficiency is higher in reversed-phase chromatography with acetonitrileas an organic modifier than with iso-propanol, which is more viscous. Col-umn back pressure is directlyproportional to solvent viscosity.VM: See holdup volume. Also mobile-

phase volume.Void: The formation of a space or

gap, usually at the head of the column,caused by a settling or dissolution of thecolumn packing. A void in the columnleads to decreased efficiency and loss ofresolution. Even a small void can be dis-astrous for small-particle microparticu-late columns. The void sometimes canbe filled with glass beads or the sameporous packing used in a column.Void time (t0): The elution time of an

unretained peak; also called the deadtime and the holdup time (tM). Thevoid volume is determined by multiply-ing the void time and the flow rate.Void volume (VM): The total volume

of mobile phase in the column; theremainder of the column is taken up bypacking material. This volume can bedetermined by injecting an unretainedsubstance. Also called dead volume. Thesymbol V0 is often used to denote thevoid volume. This is valid only for a col-umn packed with nonporous particles.V0 is valid when used to denote the

excluded volume (Ve) in SEC.Vp: See total permeation volume.VR: See retention volume and elution

volume.VR9: See adjusted retention volume.Vt: See total mobile-phase volume.Vo: See exclusion volume.

W ��Wall effect: The consequence of a

looser packing density near the walls ofa rigid HPLC column. The mobile phasehas a tendency to flow slightly fasternear the wall because of the increasedlocal permeability. The solute moleculesnear the wall are carried along fasterthan the average of the solute band,and,consequently, band spreading resultsand the column loses efficiency.wb: See peak width.Weak anion exchanger: Anionex-

change packing with weakly basic iono-genic groups such as aminodiethylamino ethyl groups.Weak cation exchanger: Cation

exchange packing with weakly acidicionogenic groups such as carboxylgroups.Weak solvent: In general, refers to a

solvent which is a poor solvent for achemical compound; in chromatography,refers to the mobile phase constituentthat provides a low solvent strength thatcauses an analyte to elute more slowlyfrom the column in a water-acetonitrilebinary solvent system for reversed-phaseLC, water would be considered to be theweak solvent.Wilke–Chang equation: A semiem-

pirical equation used to estimate diffu-sion coefficients in liquids as a functionof solute molecular size and solventviscosity.

X ��Xerogels: Gels used in SEC that swell

and shrink in different solvents. Alsorefers to silica-based packings that areprepared from acidification of solublesilicates to generate an amorphous, high-

surface area, high-porosity, rigid particle.

Z ��Zero dead volume: Any fitting or

component that has no volume that isunswept by the eluent.Zirconia: Porous zirconium oxide.

Used as a chromatographic sorbent,usually coated or bonded with polymericorganic phase.Zone: See band.Zone velocity (uz): The velocity at

which the solute zone travels; uz =uM/(1 + k) = L/tR, where uM is themobile-phase velocity, k is the retentionfactor, L is the column length, and tR isthe retention time.

Zwitterions: Compounds that carryboth positive and negative charges insolution.

References(1) R.E. Majors and P.W. Carr, LCGC

19(2) 124-162 (2001).(2) “Nomenclature for Chromatogra-

phy” In Pure and Appl. Chem. 65 (4), 819-872 (1993).

Peter W. Carr

Peter W. Carr is a professor of chemistry in

the Department of Chemistry, University

of Minnesota, 207 Pleasant Street SE,

Minneapolis, MN 55455-0431, and is a

member of LCGC’s editorial advisory board.

Ronald E. Majors

Ronald E. Majors, “Column Watch” and

“Sample Prep Perspectives” Editor

Ronald E. Majors is Senior Chemist, Columns

and Supplies Division, Agilent Technologies,

Life Sciences Chemical Analysis, Wilmington,

Delaware, and is

also a member of

LCGC’s editorial

advisory board.


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HPLCAPPLICATIONAND EDUCATIONNOTEBOOK - · PDF fileTable 1: LC ... Curr. Opin. Chem. Biol. 8; 418-423; 2004. ... Here we look closely at the problem and propose calculations that improve