Agilent HPLC Application Notebook & Terms PDF

HPLC Application Guide and Glossary of Terms
HPLC APPLICATION AND

EDUCATION NOTEBOOK
Your Must Have Guide for Optimal HPLC Separations
APPLICATIONS
Applications for Optimal HPLC Separation
EDUCATION
Complimentary ChemStation and LC Online Courses
Glossary of Liquid-Phase Separation Terms
2 HPLC ADVERTISING SUPPLEMENT
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OCTOBER 2008 HPLC 3
LC SOLUTIONS THAT REVOLVE AROUND YOUR NEEDS
Achieving more with less, confidence in analytical results, and cost-effectiveness are key
objectives in todays 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 as
life science research and quality control. With our leadership in liquid chromatography
solutions, Agilent continues its commitment to taking scientific discovery from dream to reality
and 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 worldwide
since the introduction of the 1100 and subsequent 1200 liquid chromatography portfolios, customers
have recognized the value derived from Agilents 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 and
development organizations have been the major contributors to achieving the high standards we set
for performance. Agilents 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, the
most significant advancement in liquid chromatography in the last eight years has been the utilization
of small particles for faster, higher resolution separations. In 2000, it was Agilent who led the
industry with our introduction of the Rapid Resolution High Throughput columns packed with sub
2-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.
Its all about you and getting the performance, efficiency, resolution, selectivity, and produc-
tivity youre looking for from your liquid chromatography investment.
We hope you enjoy the Agilent HPLC Application and Education Notebook.
Sincerely,
Helmut Schulenburg-Schell
Worldwide Liquid Chromatography Marketing Manager
Agilent Technologies Inc.
4 HPLC RESOLUTION OCTOBER 2008
HPLC APPLICATION AND EDUCATION NOTEBOOK
This notebook will both inspire and inform you by highlighting how Agilent
liquid chromatography products and expertise effectively enhance your
labs progress. Whether selectivity, resolution, speed or sensitivity are most
important to you, Agilent solutions revolve around your labs needs. The
applications selected in this compendium represent just a sampling of the
successes in analyses that are achieved using Agilent LC systems.
HPLC Applications That Revolve Around Your Needs
Page 5 RESOLUTION Agilent 1200 Series Rapid Resolution LC system

and the Agilent 6210 TOF MS Highest data content with
highest throughput
Page 10 SPEED Improving the Effectivenss of Method Translation for

Fast 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, Pharmaceutical

Impurity 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' eSeminar

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

eSeminar Series
OCTOBER 2008 RESOLUTION HPLC 5
RESOLUTION Agilent 1200 Series Rapid Resolution

LC system and the Agilent 6210 TOF MS
Highest data content with highest throughput
Michael Frank,
Agilent Technologies, Waldbronn, Germany
Fast and unambiguous determination of purity and

identity of compounds derived from screening
libraries is a common task for many analytical labs in
the pharmaceutical industry. The method of choice to
determine the identity of compounds is mass spec-
trometry, preferably with accurate mass. As yet, data
quality was usually compromised by gaining higher
throughput. This Application Note demonstrates how
a daily throughput of far more than 1000 samples can
be achieved together with full spectral data acquisi-
tion and accurate mass information with close to
FT-MS mass accuracy.
I n the quest to achieve highest throughput in LC/MS analyses,

the quality of the data is often compromised. There are certain
approaches to increase the throughput of LC/MS systems. One
approach is to do flow injection analysis. This probably delivers
the highest possible throughput, however since no chromato-
graphic separation occurs, the probability to lose compounds by
the ion suppression effect during the ionization process is high.
Orthogonal detection methods like UV detection do not succeed
at all in flow injection analysis as all compound signals are over-
laid. Approaches to achieve at least minimal chromatographic sep- dispersion heat exchangers consisting of 1.6 L internal volume
aration by using very short columns with 5 m particles and have been used as well as the high pressure rated
ballistic gradients are an improvement in view of data quality, 2-position/10-port valve.
however, not state-of-the-art. Some manufactures have established The instrument set-up is shown in figure 1:
parallel working instrumentation with a shared mass spectrome- Two Agilent 1200 Series binary pumps SL with the new Agi-
ter and shared UV detector. Obviously, this also compromises lent 1200 Series micro vacuum degasser placed between the
data quality as the full acquisition rate of each instrument has to two pumps eliminates the need for long tubing to the pumps.
be shared on each LC channel1. Agilent 1200 Series high performance autosampler SL.
With the introduction of an LC/MS system which facilitates An Agilent 1200 Series thermostatted column compartment
the use of columns with sub two micron particles it is now pos- SL, equipped with a high pressure, 2-position/10-port valve,
sible to achieve short analyses times as well as high chromato- facilitating alternating column regeneration.
graphic resolution. Furthermore the system is able to acquire full An Agilent 1200 Series diode-array detector SL allowing a data
UV spectral data and mass spectral data with accurate masses. acquisition rate of 80 Hz and equipped with a 500 nano liter
flow cell with 0.12-mm id connecting capillaries.
Experimental Agilent 6210 Time-of-Flight mass spectrometer allowing a
The Agilent 1200 Series Rapid Resolution LC system is set up maximum data acquisition rate of 40 Hz and equipped with
for alternating column regeneration (ACR)2 using 2.1-mm id a dual ESI source for parallel ionization of the analyte and a
columns. The pumps are in the low delay volume configuration reference mixture.
with an internal volume of only ca. 120 L. All other modules Two ZORBAX SB C18, 2.1 mm id x 50 mm, 1.8 m
are optimized for lowest delay volumes by using the low delay columns
volume capillary kit (G1316-68744) and the alternating column As mobile phase gradient grade water with 0.1 % trifluoro
regeneration kit (G1316-68721). Consequently, from the injec- acetic acid and acetonitrile with 0.08 % trifluoro acetic acid-
tion valve on only capillaries of 0.12 mm id are used. In the ther- was used. No additional filtering of the solvents was made.
mostatted column compartment the newly introduced low Instrument control and data acquisition was done by the
Agilent TOF-software A02.01 running on a Hewlett-Packard tings. Saturation of the MS detector would produce incorrect re-
xw 4300 workstation with an Intel dual core Pentium sults in mass determination. The solution is to intentionally de-
D840 CPU at 3.2 GHz. sensitize the TOF MS. This can be done quite easily by applying
the functionality of the TOF software to alter the MS parameters
from one run to the other, simply by adding one or more MS-
parameter columns to the worklist (figure 2). Select add
columns from the worklist and then chose MS-parameter and
the desired parameter. As the reference mixture is also affected by
these settings, the concentration of the reference mixture was in-
creased. Only the capillary voltage, the fragmentor voltage and
the skimmer voltage were varied. The optimal conditions deter-
mined by this approach can be found in the method parameters
in table 1.
Figure 1: Agilent 1200 Series Rapid Resolution LC system with Agi-

lent 6210 TOF-MS with low delay volume for high speed applications
using 2.1-mm id columns with lengths ranging from 20 to 50 mm.
Results and Discussion

By applying elevated temperatures the viscosity of the solvent can
be reduced which allows higher flow rates and therefore shorter Figure 2: Feature of the TOF software to modify the MS parameter
gradient times. A maximum temperature of 80 C was applied, from run to run.
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 m).
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 high
organic content solvent and then re-equilibrated again with the
starting composition of the gradient while on the second column
the separation of a sample occurs. After this sequence the 10-port
valve is switched and both columns are exchanged in the flow
path. Details of alternating column regeneration and the correct
setting of time points are described in another Application Note2.
Despite the high flow rate (1.8 mL/min), the column effluent was
not split prior to reaching the mass spectrometer. The standard
ESI source specifies a maximum flow rate of up to 1 mL/min,
however even these higher flows are tolerated if the drying gas
temperature and flow rate are set to maximum and little conden-
sation occurs. Condensation of water is practically eliminated
when using ACR because equilibration is done on the column
which is not connected to the detector. Generally the use of an Figure 3: Comparison of corresponding peaks in the UV (red trace)
Agilent multi mode source with a specified flow rate up to 2 and the MS detection (black trace).
mL/min even with pure water is recommended. The chromato-
graphic conditions in table 1 were used to achieve gradient times In figure 3 the total ion chromatogram and the UV chro-
of 0.5 min. Under these conditions, the peak capacity for the MS matogram achieved with conditions above (80 Hz DAD, 30 Hz
detection is in the range of >40 in 39 s. With the use of a 5-m TOF data acquisition rate) is shown for a five-component sample
particle size column of the same dimension the peak capacity (58 ng/L atenolol, 85 ng/L primidon, 62 ng/L metoprolol,
would only be half! 125 ng/L verapamil and 75 ng/L beclomethasone-dipropi-
The detector of the Agilent 6210 TOF MS would be saturated onat). The peaks of the total ion chromatogram are inherently
if the compound concentrations used here to give also significant broader than the peaks of the UV chromatogram because of ad-
UV signals would be injected into the MS without special set- ditional extra column volume from the flow cell and also from
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.
Method:
Solvent: A = water (0.1% TFA), B = ACN (0.08% TFA)
Temperature: 80 C
Flow: 1.8 mL/min
Gradient: 0.00 min 5%B Regeneration: 0.00 min 5%B
0.50 min 90%B 0.01 min 95%B
0.51 min 5%B 0.20 min 95%B
0.65 min 5%B 0.21 min 5%B
0.65 min 5%B
Stoptime: 0.65 min no limit
Posttime: off off
DAD: Wavelength: 210 nm (8), ref. off
Peak width: >0.0025 min (0.05s responsetime), 80 Hz
Spectra: no
Slit: 8 nm
Balance: pre-run
MS: Scan range: 100-1000 m/z
Acquisition rate: 5, 20, 30 and 40 cycles/s
Data type: profile data
Capillary voltage: 3000 V
Fragmentor: 180 V
Skimmer: 40V
Gas temperature: 350 C
Gas flow: 13 L/min
Injection volume: 1L
Injector: Overlapped injection, Automatic delay volume reduction,
Sample flush out factor = 10
Valve position: Next position
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 height
of 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 to
0.42 s. The chromatograms shown in figure 5 were produced
under the same chromatographic conditions, but with different
data acquisition rates of the time-of-flight MS. The peak form
and resolution are improved by having high data acquisition rates
in the MS which shows clearly in figure 5. The effect is nicely
demonstrated on the little side peak next to the primidon peak
with 40-Hz data acquisition rate it is obvious that an additional
compound shows up but with 5 Hz data acquisition rate this
could 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 TOF
centroid data, a cycle time of 49 s was achieved. The achievable Figure 4: MS total ion chromatogram of highest speed LC-TOF-MS
cycle time is not only dependent on the used run time (that is the analysis (40 Hz TOF data acquisition rate).
gradient time plus additional flush and re-equilibration times, or
in Agilent terminology the stop time plus post time) but also very
much dependent on the instrument overhead time. This is usu- are constantly written to the hard disc during data acquisition,
ally caused by communication between the data system and the whereas the UV data are buffered and added to the data file after
individual LC/MS modules as well as the data system writing data the stop time of the method, the cycle time depends more on the
to the hard disc and initiating certain processes. The overhead UV data amount than on the MS data amount. The cycle time
time caused by the data system can be significant if the computers was calculated from the time stamp each file gets assigned from
performance is not sufficient to handle the data amount or if the WindowsXP operating system after closing the file fol-
other software programs or processes are consuming the power lowing data acquisition.
available. To decrease the cycle time it might be worth decreasing
If using a TOF MS the attention is certainly focused on the ac-
the amount of data acquired.
curate mass. The question may arise if the possibility to obtain
Table 2 shows the cycle times and the possible daily through-
low mass accuracy errors might suffer from these high speed con-
put depending on the DAD and MS settings. Since the MS data
ditions. Figure 6 shows the achieved mass accuracy errors of the

analysis of 140 members of a chemical library used in a screening
campaign by a pharmaceutical company. The shown error-values
have been extracted from an automated empirical formula con-
firmation report and involved no manual interference. Sixteen of
the compounds could not be ionized under positive ESI condi-
tions and two compounds showed large mass errors of 11 and 15
ppm, probably caused by co-eluting isobaric impurities. The cycle
time was 90 s and was determined by a required injector program
which allowed an on-line dilution of the samples directly prior to
the analysis. Chromatographic conditions applied a 5-100 %
water-acetonitrile (0.1 % TFA) gradient in 0.7 min at a flow rate
of 1.5 mL/min and 60 C column temperature. UV data acqui-
sition to determine purity was done in the wavelength range of
210 to 500 nm with an acquisition rate of 80 Hz. The MS data
acquisition rate was at 8 Hz to reduce the file size. The scan range Figure 5: Total ion chromatograms recorded with varying data
was 120 1200 Da, capillary voltage 4000 V and the fragmen- acquisition rates dependence of the MS peak shape and resolution
tor voltage at 215 V. No ACR was applied and the flow to the MS on the data acquisition rate.
was 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 ionizable
compounds (outliers included) have a mass accuracy error in the
range of 2.0 ppm. Excluding the outliers even 93 % of the an-
alyzed samples lie in-between the 2.0 ppm range. In the 1.0
ppm range which is FT-MS-like mass accuracy 71 % of the sam-
ples can be found (72 % excluding the outliers).
Figure 6: Mass accuracy errors of the analyses of a set of chemical

library 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.
DAD (80 Hz) TOF (100 1000 Da) Cycletime Throughput

Type Wavelength Profile Data rate [Hz] [s] [Samples/day]
spectral 190-900 (1) x 20 62 1394
spectral 190-900 (1) 20 62 1394
spectral 190-400 (2) x 20 59 1464
spectral 190-400 (2) x 40 59 1464
spectral 190-400 (2) x 30 58 1490
signal 210/254 x 20 50 1728
signal 210 30 49 1763
References
1. 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.
Figure 7: Histogram of the mass accuracy errors of the analyses

of a set of chemical library members under fast LC conditions.
The given populations of the 1.0 ppm and 2.0 ppm range
include the outliers.
Conclusion
The Agilent 1200 Series Rapid Resolution LC system together
with the Agilent 6210 Time-of-Flight mass spectrometer allows
acquisition of a wealth of data to unambiguously determine the
purity and identity of compounds in samples as they are typical
for 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 190-900
nm wavelength in a band width of 1 nm at an 80 Hz acquisition
rate 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 with
highest data acquisition rates and most importantly stable and
easy-to-use accurate mass, this system outperforms other high
throughput LC/MS techniques used as yet in throughput and/or
data quality. The linear velocities achieved were in the range of 11
mm/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 m, the UV peak
capacities were still in the range of fifty and even the MS peak
capacities were in the range of forty for a gradient time of 39 s.
Order your Agilent Rapid Resolution Liquid Chromatography Kit today www.agilent.com/chem/futureLCnow | 800 227 9770
10 HPLC SPEED OCTOBER 2008
SPEED Improving the Effectivenss of Method

Translation 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) The combined effect of reduced length and diameter con-
columns and increased demand for methods friendly tributes to a reduction in solvent consumption and, again as-
to mass spectrometers has led to strong trend toward suming the same analyte mass can be injected on the smaller
conversion of existing HPLC methods to smaller column, a proportional increase in peak response. We normally
diameter and smaller particle size columns. While the scale the injection mass to the size of the column, though, and a
conversion is a simple mathematical exercise requir- proportional injection volume would be calculated from the ratio
ing the scaling flow rates, gradient times and injec- of the void volumes of the two columns, multiplied by the injec-
tion volumes, many users observe less than perfect tion volume on the original column.
results. Here we look closely at the problem and
propose calculations that improve the speed and/or
resolution in a more predictable and
beneficial way.
M ethods developed on older columns packed with large 5-

or 10-m particles are often good candidates for mod-
ernization by replacing these columns with smaller dimension For isocratic separations, the above conditions will normally
columns packed with smaller particle sizes. The potential benefits result in a successful conversion of the method with little or no
include reduced analysis time and solvent consumption, improved change in overall resolution. If one wishes to improve the out-
sensitivity and greater compatibility with mass spectrometer ion- come of the method conversion, though, there are several other
ization sources. parameters that should be considered. The first of these parame-
Simplistically, a column of 250-mm length and containing 5- ters is the column efficiency relative to flow rate, or more cor-
m particles can be replaced by a 150-mm length column packed rectly efficiency to linear velocity, as commonly defined by van
with 3-m particles. If the ratio of length to particle size is equal, Deemter [1] and others, and the second is the often overlooked
the two columns are considered to have equal resolving power. effect of extracolumn dispersion on the observed or empirical ef-
Solvent consumption is reduced by L1/L2, here about 1.6-fold ficiency of the column.
reduction in solvent usage per analysis. If an equal mass of ana- Van Deemter observed and mathematically expressed the rela-
lyte can then be successfully injected, the sensitivity should also tionship of column efficiency to a variety of parameters, but we
increase by 1.6-fold due to reduced dilution of the peak as it trav- are most interested here in his observations that there is an opti-
els through a smaller column of equal efficiency. mum linear velocity for any given particle size, in a well-packed
LC/MS (Liquid Chromatography/Mass Spectrometry) ioniza- HPLC column, and that the optimum linear velocity increases as
tion sources, especially the electrospray ionization mode, have the particle size decreases. Graphically, this is often represented
demonstrated greater sensitivity at lower flow rates than typically in van Deemter plots as shown in Figure 1, a modified version of
used in normal LC/UV (UltraViolet UV/VIS optical detection) the original plot [2].
methods, so it may also be advantageous to reduce the internal di- In Figure 1 we observe that the linear velocity at which 5-m
ameter of a column to allow timely analysis at lower flow rates. materials are most efficient, under the conditions used by the au-
The relationship of flow rate between different column thors, is about 1 mm/sec. For 3.5-m materials the optimum lin-
diameters is shown in Equation 1. ear velocity is about 1.7 mm/sec and has a less distinct optimum
value, suggesting that 3.5-m materials would give a more con-
sistent column efficiency over a wider flow range. For the 1.8-m
materials, the minimum plate height, or maximum efficiency, is
a broad range beginning at about 2 mm/sec and continuing past
the range of the presented data. The practical application of this
information 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. This
increase in elution speed will decrease absolute peak width and
may require the user to increase data acquisition rates and reduce
OCTOBER 2008 SPEED HPLC 11
Note that the use of % change per column volume rather than
% change per minute frees the user to control gradient slope by
altering gradient time and/or gradient flow rate. A large value for
gradient 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 reduced
sensitivity. 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 the
gradient time.
Resolution increases with shallow gradients because the effec-
tive capacity factor, k*, is increased. Much like in isocratic sepa-
Figure 1: van Deemter plot with various flow rates and particle rations, where the capacity term is called k', a higher value directly
sizes. increases resolution. The effect is quite dramatic up to a k value
of about 5 to 10, after which little improvement is observed. In
signal filtering parameters to ensure that the chromatographic the subsequent examples, we will see the results associated with
separation is accurately recorded in the acquisition data file. the calculations discussed above.
The second important consideration is the often overlooked
effect of extracolumn dispersion on the observed or empirical ef- Experimental Conditions
ficiency of the column. As column volume is reduced, peak elu-
tion volumes are proportionately reduced. If smaller particle sizes System
are also employed there is a further reduction in the expected peak Agilent 1200 Series Rapid Resolution LC consisting of:
volume. The liquid chromatograph, and particularly the areas G1379B micro degasser
where the analytes will traverse, is a collection of various con- G1312B binary pump SL
necting capillaries and fittings which will cause a measurable G1367C autosampler SL, with thermostatic temperature control
amount of bandspreading. From the injector to the detector flow G1316B Thermostatted column compartment SL
cell, the cumulative dispersion that occurs degrades the column G1315C UV/VIS diode array detector SL, flow cell as indicated in
performance and results in observed efficiencies that can be far individual chromatograms
below the values that would be estimated by purely theoretical ChemStation 32-bit version B.02.01
means. It is fairly typical to see a measured dispersion of 20 to
100 L in an HPLC system. This has a disproportionate effect Columns
on the smallest columns and smallest particle sizes, both of which Agilent ZORBAX SB-C18, 4.6 mm, 250 mm, 5 m
Agilent ZORBAX SB-C18, 3.0 mm, 150 mm, 3.5 m
are expected to yield the smallest possible peak volumes. Care
must be taken by the user to minimize the extracolumn volume
Mobile phase conditions
and to reduce, where practical, the number of connecting fittings
Organic solvent: Acetonitrile
and the volume of injection valves and detector flow cells.
Aqueous solvent: 25 mm phosphoric acid in Milli-Q water
For gradient elution separations, where the mobile phase com-
position increases through the initial part of the analysis until the Gradient Conditions
analytes of interest have been eluted from the column, successful Gradient slope: 7.8% or 2.3% per column volume, as
method conversion to smaller columns requires that the gradient indicated. See individual chromatograms
slope be preserved. While many publications have referred to gra- for flow rate and time
dient slope in terms of % change per minute, it is more useful to
express it as % change per column volume. In this way, the change Sample
in column volume during method conversion can be used to ac- Standard mixture of chlorinated phenoxy acid herbicides,
curately render the new gradient condition. If we think of each 100 g/mL in methanol
line of a gradient table as a segment, we can express the gradient
by the following equation:
12 HPLC SPEED OCTOBER 2008
Conditions Conditions:
EPA Method 555 with ZORBAX SB-C18 columns and fast EPA Method 555 with ZORBAX SB-C18 columns and fast
DAD detector DAD detector
ZORBAX SB-C18 4.6 mm x 250 mm, 5 m ZORBAX SB-C18 3.0 mm x 150 mm, 3.5 m
Column temp: 25 C Column temp: 25 C
Gradient: 10% to 90% ACN vs. 25 mM H3PO4 Gradient: 25 mm H3PO4/ACN, 0% to 90%
Gradient slope: 7.8% ACN/column volume ACN in 18 minutes
Analysis flow rate: 1 mL/min Gradient slope: 7.8% ACN/column volume
Analysis flow rate: 0.43 mL/min
Group A Compounds
Detection: UV 230 nm, 3-mm 2-L flow cell,
Total analysis time: 60 min
filter 0.2 seconds
Detection: UV 230 nm, 10-mm 13-L flow cell,
Total analysis time: 36 min.
filter 2 seconds (default)
Figure 2: Gradient separation of herbicides on 4.6 250 mm 5-m Figure 3: Gradient separation of herbicides on 3.0 150 mm,
ZORBAX SB-C18. 3.5-m ZORBAX SB-C18.
Results
The separation was initially performed on a standard 4.6 250 In Table 1 the column has been replaced with a low dead vol-
mm, 5-m ZORBAX SB-C18 column thermostatted to 25 C ume connecting union in a system fitted with 0.12-mm id capil-
(Figure 2) using conditions referenced in US EPA Method 555. lary tubing at all points of sample contact. A 1-L injection of
The method was then scaled in flow and time for exact transla- dilute actone is made to determine the bandspreading contribu-
tion to a 3.0 150 mm, 3.5-m column (Figure 3). Solvent con- tion of the system, with various flow cells. Multiple flow cells were
sumption is reduced from 60 mL to 15.5 mL per analysis. tested, and the average result reported, where possible. The elu-
The separation was then re-optimized for faster separation with tion volume summarizes the total volume of all tubing in the sys-
the identical slope, 7.8%, by increasing the flow rate from 0.43 tem. While the absolute volume from the 2-L to the 13-L flow
to 1.42 mL/min, and proportionately reducing the gradient time cells is 11 L, we observe an increase of 15 to 16 L because of
(Figure 4). Finally, increased resolution is demonstrated by keep- the larger diameter inlet tubing integral to the larger volume flow
ing the original times used in Figure 3 with the increased flow cells.
rate (Figure 5). This yields a gradient with identical time but a re-
duced slope of 2.3%. The increased resolution of peaks 4 and 5 Conclusion
is readily apparent. Careful analysis of the existing gradient conditions, coupled with
The conditions in Figure 4, 7.8% slope at increased linear ve- an awareness of the need to accurately calculate new flow and gra-
locity on 3.0 150 mm, 3.5-m material, yield a separation with dient conditions can lead to an easy and reliable conversion of
comparable resolution to the original 4.6 250 mm method, but existing methods to new faster or higher resolution conditions. In
with only a 12-minute total analysis time. This is excellent for addition, awareness of extracolumn dispersion, especially with
high throughput screening and quantitation of a large number of small and high resolution columns, will ensure good column ef-
samples. Figure 5, with the gradient slope reduced to 2.3%, re- ficiency which is critical to a successful translation of the method.
sults in a high-resolution separation with a calculated R value of
3.3 vs. the standard 3.0 150 mm separation value of 1.9, for the
critical pair seen in Figure 5 at 7.5 to 8 minutes.
OCTOBER 2008 SPEED HPLC 13
Conditions Conditions
EPA Method 555 with ZORBAX SB-C18 columns and fast EPA Method 555 with ZORBAX SB-C18 columns and fast
DAD detector DAD detector
ZORBAX SB-C18, 3.0 mm x 150 mm, 3.5 m ZORBAX SB-C18, 3.0 mm x 150 mm, 3.5 m
Column temp: 25 C Temp: 25 C
Gradient: 25 mM H3PO4/ACN, 10% to 90% Gradient: 25 mM H3PO4/ACN, 10% to 90%
ACN in 5.4 min. ACN in 18 min.
Gradient slope: 7.8% ACN/column volume Gradient slope: 2.3% ACN/column volume
Analysis flow rate: 1.42 mL/min Analysis flow rate: 1.42 mL/min
Detection: UV 230 nm, 3-mm 2-L flow cell, Detection: UV 230 nm, 3-mm 2-L flow cell,
filter 0.2 seconds filter 0.2 seconds
Total analysis time: 12 min. Total analysis time: 36 min.
Figure 4: High speed gradient separation of herbicides on 3.0 Figure 5: Reduced slope gradient separation of herbicides on 3.0
150 mm, 3.5-m ZORBAX SB-C18. 150 mm, 3.5-m ZORBAX SB-C18.
References
Table 1: Volumetric Measurements of Various Flow Cells
1. J. J. van Deemter, F. J. Zuiderweg, A. Klinkenberg,
Chemical Engineering Science 1956, 5, 271289
Elution Half height 5 Sigma
2. The Influence of Sub-Two Micron Particles on HPLC Flow cell volume (L) width (L) width (L)
Performance, Agilent Technologies, application note New SL 11 5 12
5989-9251EN, May 2003 2 L 3 mm
Micro 14 6 18
6 mm 1.7 L
(n = 2)
Semi-micro 13 6.5 18.5
6 mm 5 L
(n = 2)
Standard 26 11 26
10 mm 13 L
New SL 27 11 25
10 mm 13 L
14 HPLC SELECTIVITY OCTOBER 2008
SELECTIVITY Unique Selectivity and High-Throughput

Applications 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 employing ful for the analysis of aromatic-containing compounds. The
ZORBAX SB-Phenyl Rapid Resolution High Through- unique selectivity for the phenyl phase is derived from an inter-
put (RRHT) columns are shown. SB-Phenyl possesses action of the pi electrons found in the phenyl groups. Phenyl
useful selectivity for compounds containing phenyl- columns are typically used in applications involving pharmaceu-
type moieties. Using RRHT columns, several combi- ticals such as analgesics and other aromatic compounds
nations of stationary and mobile phase were quickly
investigated to determine the best selectivity and res- Experimental
olution for analgesics and steroids. In both cases, An Agilent 1200 HPLC system was used throughout these ex-
ZORBAX SB-Phenyl yielded excellent peak shape in periments. This system included an autosampler, binary pump,
acetonitrile, but in methanol even more selectivity temperature-controlled column compartment, and a diode array
was obtained. detector at 254 nm. Samples were purchased from Sigma-Aldrich
(St. Louis, MO USA) and include the analgesics in Figure 1: tol-
C urrently, much HPLC method development is being driven

by the need for faster analysis times, resulting in higher pro-
ductivity. This trend to faster analysis is reflected in the configu-
metin, naproxen, diflusinal, ibuprofen, and diclofenac. The
steroids in Figure 2, hydrocortisone, prednisone, betamethasone,
dexamethasone, and corticosterone were also purchased from
ration of HPLC columns currently used for method development. Sigma- Aldrich. The analgesics and steroids were prepared indi-
Whereas several years ago, a 250-mm long column packed with vidually in methanol at approximately 1 mg/mL. The individual
5-m particles was standard, todays methods often involve 3.5- components were then mixed at 1 part each tolmetin, naproxen
m particles packed in column lengths of 100 mm and shorter. and diclofenac with 0.5 parts diflusinal and 2 parts ibuprofen.
In addition, contemporary HPLC column technology is focused Columns used in this work include StableBond SB-C18, SB-C8,
on even smaller particles (< 2 m) delivering higher theoretical SB-CN, and SB-Phenyl, all 4.6 50 mm with 1.8-m particles.
plate counts and with the capability of running at very fast flow The part numbers for these rapid-resolution high-throughput
rates. The high-throughput separations thus obtained are highly (RRHT) columns are listed in Table 1.
dependent on column efficiency alone with the assumption that
if sufficient resolution can be obtained on a standard column,
Table 1: Columns Used in This Work
the use of smaller particles, higher flow rates, and optimized in-
strumentation will make the separation faster. 1 ZORBAX SB-Phenyl RRHT
Increasing column efficiency is one way to increase resolution. 1.8 m, 4.6 50 mm, Part Number 827975-912
It can be accomplished by increasing column length or by de- 2 ZORBAX SB-CN RRHT
creasing particle size. Resolution, however, is a function of the 1.8 m, 4.6 50 mm, Part Number 827975-005
square root of N as shown: 3 ZORBAX SB-C8 RRHT
1.8 m, 4.6 50 mm, Part Number 827975-006
4 ZORBAX SB-C18 RRH
1.8 m, 4.6 50 mm, Part Number 827975-002
Large changes in N result in small changes in chromatographic

resolution. Another approach, which can be far more effective, is
to work with selectivity, (a). Selectivity can be altered by chang- Results and Discussion
ing bonded phase (column) or changing the mobile phase. Because of the high resolution and short length of these RRHT
An accepted standard for equilibrating an HPLC column is 10 columns, evaluations of all column and mobile phase combina-
column volumes. This can be a rather time-consuming task on tions were accomplished in less than 1 day. Short high-resolution
conventional columns (4.6 150 mm, 1 mL/min), but by using columns can allow many column options to be examined with
short columns (4.6 50 mm, 2 mL/min), this can be accom- very little time, typically equilibrating to most mobile phases in
plished in less than 5 minutes. By taking advantage of this fast fewer than 10 mL or about 5 minutes.
equilibration, columns and mobile phases can be used for selec- Analgesics such as ibuprofen and others depicted in Figure 1 re-
tivity improvements in less time. duce pain, fever, and inflammation. They are categorized as non-
Phenyl is an alternative to ODS phases and is particularly use- steroidal anti-inflammatory drugs or NSAIDs. As can be seen in
OCTOBER 2008 SELECTIVITY HPLC 15
Figure 3, these compounds can be readily separated at pH 2.5 in

acetonitrile. Since these are small-particle columns, in many cases
higher flow rates can be used to speed the separation without loss
of efficiency. The optimum flow rate for maximum efficiency is
dependent on a methods specific operating conditions. The best
flow rate, therefore, should be determined experimentally. In this
mobile phase, the optimal efficiency was achieved at 1.85
mL/min.
In Figure 4, several columns from the StableBond family are
compared. Using the optimized mobile phase conditions from
Figure 1, some interesting selectivity is revealed. StableBond C18
shows the longest retention of these analgesics, followed by SB-
C8, phenyl, and Cyano (CN). An interesting elution order change
between C18 and C8 for the last two eluting peaks was observed
for ibuprofen and diclofenac. On the C18 column we observe
ibuprofen as the latest eluter, while on the C8, it can be seen to
partially co-elute with diclofenac. Phenyl yields a similar elution
order to C8, but in this case we have complete resolution of the
last two components. CN shows another selectivity change mov-
ing the third component, diflusinal, to the last position. The peak
shape for diflusinal is better, however, on the phenyl and the C8
column than on the C18 or CN.
Comparing methanol and acetonitrile mobile phases in Figure
5 show some interesting results. Since acetonitrile is a stronger
solvent than methanol, a higher concentration of methanol is used
Figure 1: Steriods used in this study. to achieve an iso-elutropic comparison. In the methanol com-
parison, one quickly observes the co-elution of tolmetin and
naproxen, but also the longer retention of these phenyl-like struc-
tures. Longer retention of analytes can be advantageous in cases
where 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 of
these compounds on StableBond C8 and Phenyl columns yields
similar chromatograms; the peaks are all well shaped and well re-
solved. However, the more highly aliphatic C18 is found to be
slightly less retentive than the C8. The StableBond CN column
has 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 separate
on the phenyl column, no complete separation is achieved on the
other columns. Methanol again shows more retention and better
selectivity. A recently published work proposes that acetonitrile
overwhelms or blocks the pi-pi (-) interactions between the an-
alytes and phenyl stationary phase[1]. This work supports that
proposal, using steroids and analgesics as examples.
Conclusions
Figure 2: Structures of analgesic compounds used in this study. While the initial column offerings of sub-two-micron columns
were primarily C18, selectivity advantages offered by alternative
phases such as C8, phenyl, and cyano can yield improvements in
resolution and speed. Short, 1.8-micron columns quickly equili-
16 HPLC SELECTIVITY OCTOBER 2008
Figure 5: Comparison of acetonitrile and methanol solvent

conditions.
Figure 3: Determining optimum flow rate for RRHT columns for

analgesics method.
Figure 6: Steroid separation on fast and selective SB RRHT

Figure 4: Analgesic separation on fast and selective family of finish of columns.
StableBond RRHT columns
OCTOBER 2008 SELECTIVITY HPLC 17
Figure 7: SB-Phenyl with methanol shows substantial differ-

ences in k and a.
brate, allowing selectivity to be quickly evaluated using many dif-

ferent columns or mobile phases. When choosing a stationary
phase, a separation mechanism that employs differences in the
chemical structures of the target analytes should be used. For
analyses in which the target analytes are structurally very similar,
this is especially critical. For analgesics and steroids, this includes
separations based on - 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 of
methanolic mobile phase should be considered when using a
phenyl column.
References
1. M. Yang, S. Fazio, D. Munch, and P. Drumm, Impact of
methanol and acetonitrile on separations based on -
interactions with reversed phase phenyl column, J. of
Chromatography, 1097, 124-129. 2005.
18 OCTOBER 2008
Food and Flavor Analysis Pharmaceutical Impurity Profiling

Rapid Screening and Analysis of Components in Analysis
Nonalcoholic Drinks Impurity Profiling with the Agilent 1200 Series LC System
Publication Number 5989-5178EN Part 1: Structure Elucidation of Impurities with LC/MS
Publication Number 5989-5617EN
Multiresidue Analysis of 301 Pesticides in Food Samples
by LC Triple Quadrupole Mass Spectrometry Impurity Profiling with the Agilent 1200 Series LC
Publication Number 5989-8614EN System Part 3: Rapid Condition Scouting for Method
Development
Environmental Analysis Publication Number 5989-5619EN
EPA Method 1694: Agilents 6410A LC/MS/MS Solution Impurity Profiling with the Agilent 1200 Series LC System
for Pharmaceuticals and Personal Care Products in Water, Part 5: QA/QC Application Example Using a Fast LC
Soil, Sediment, and Biosolids by HPLC/MS/MS Method for Higher Sample Throughput
Publication Number 5989-9665EN Publication Number 5989-5621EN
Rapid Separation and Identification of Carbonyl

Compounds by HPLC
Pharmaceutical Drug Discovery Analysis
Publication Number 5989-7483EN Developing a fast, generic method for rapid resolution
LC with quadrupole MS detection Combining the
Agilent 6140 quadrupole MS and multimode source
Specialty Chemical Analysis with the Agilent 1200 Series Rapid Resolution LC system:
Fast, generic LC/MS method enables drug analysis in less
Analysis of Phenolic Antioxidant and Erucamide Slip than one minute
Additives in Polymer by Rapid-Resolution LC Publication Number 5989 -7592EN
Achieving fastest analyses with the Agilent 1200 Series
A Total Solution for the Analysis of Melamine and Rapid Resolution LC system and 2.1-mm id columns
Cyanuric Acid in Pet Food by GC/MS and Aqueous Publication Number 5989-4502EN
Normal-Phase LC/MS/MS
Publication Number 5989-7546EN Improve Peak Shape and Productivity in HPLC analysis of
Pharmaceutical compounds with Eclipse Plus C8 Columns
Forensic Analysis Publication Number 5989-5803EN
Determination of Benzodiazepines in Oral Fluid
Using LC/MS/MS
Access the complete applications online by visiting

www.agilent.com/chem/library
and entering the publication number specified.
OCTOBER 2008 GLOSSARY 19
Glossary of Liquid-Phase
Separation Terms
Ronald E. Majors and Peter W. Carr
n 2001, the second glossary of com- A IIIIIIIIIIIIIIII Adsorbent: Packing used in adsorp-
a: See separation factor. tion chromatography. Silica gel and alu-
I mon and not-so-common terms and
buzz words for reference to HPLC A solvent: Usually the weaker solvent mina are the most frequently used
in a binary eluent or gradient elution adsorbents in high performance liquid
columns and column technology was
separation. In reversed-phase liquid chromatography (HPLC).
published (1). It is time for an update
chromatography (LC), the A solvent typ- Adsorption: The process of retention
since new terms have arisen or, in some
ically is water or a water-rich mixture. in which the interactions between the
cases, their original meanings have ex-
A term: The first term in the van solute and the surface of an adsorbent
panded or changed. We have also de-
Deemter equation. See eddy dispersion dominate. The forces can be strong
cided to expand the terms dealing with
term and van Deemter equation. forces (hydrogen bonds) or weak (van
HPLC and LC to cover some of
der Waals forces). For silica gel, the
the common terms that we neglected to Absorption: The process of retention
silanol group is the driving force for
include in the earlier glossary. To make in which the solute partitions into a
adsorption, and any solute functional
room for this expansion, we have de- liquid-like coating.
group that can interact with this group
cided to separate the terms referring to Activity: The relative strength of the can be retained on silica. The term
capillary electrophoresis (CE) since this surface of the packing in adsorption adsorption places emphasis on the sur-
technique is rather specialized and not chromatography. For silica gel, the more face versus penetration or embedding in
all liquid chromatographers are also per- available the silanol groups, the more the stationary phase coated or bonded
forming the various forms of CE. We active the surface. Activity can be con- to a surface.
will also stick to the conventions of the trolled by adding water or other polar
International Union of Pure and Ap- Adsorption chromatography: One
modifier that hydrogen bonds to the
plied Chemistry (IUPAC) in their of the basic LC modes that relies upon
active sites, thereby reducing the surface
Nomenclature for Chromatography adsorption to the surface of an active
activity.
that provides guidance and changes in solid to effect the separation. Silica gel
Additive: A substance added to the and alumina are the most frequently
some of the more commonly accepted
mobile phase to improve the separation used normal-phase adsorbents, and mol-
terms (2). Since there is still widespread
or detection characteristics; for example, ecules are retained by the interaction of
usage of nomenclature that is not in
a competing base to negate the effects their polar function groups with the sur-
alignment with the IUPAC definitions,
of silanols, a chelating agent to block face functional groups; for example,
those terms specifically recommended
metal sites, or a UV-absorbing com- silanols of silica. Carbon also is used as
by them will be followed by a (IUPAC)
pound to perform indirect photometric an adsorbent in a reversed-phase mode.
in parens.
detection.
Adsorption isotherm: A plot of the
Adjusted retention time (tR): A equilibrium concentration of sample in
The article is not intended to be an
in-depth listing or highly theoretical cov-
erage. For example, we have elected not measure of the retention time adjusted the mobile phase per unit volume versus
to cover the myriad of terms used in in- for the holdup time; tR = tR tM, the concentration in the stationary phase
strumentation, detection, data handling, where tR is the retention time and tM is per unit weight in adsorption chro-
and validation associated with HPLC the holdup time (the time it takes for a matography. The shape of the adsorp-
analysis but have chosen to use terms small, unretained compound that com- tion isotherm can determine the
that may be encountered in everyday pletely permeates the pores to be eluted chromatographic behavior of the solute;
laboratory work around columns, from the chromatographic column). for example, peak tailing, peak fronting,
phases, method development, and gen- Adjusted retention volume (VR): and column overload.
eral usage. The listing should be helpful Adjusts the retention volume for the Aerogel: A packing prepared when
to those just starting in HPLC but it can holdup volume; VR = VR VM, the dispersing agent is removed from a
also serve as a refresher for longtime where VR is the retention volume of the gel system without collapsing the gel
users in the field. peak of interest and VM is the holdup structure. Silica gels and glass beads used
The entire listing, including the removed volume (the volume corresponding to
for size-exclusion chromatography (SEC)
the total volume of mobile phase in the
CE terms, can also be found on the LCGC are examples of aerogels that can retain
column). See also dead volume and
website at www.chromatographyonline.com. their structures even at the high pres-
holdup volume.
20 GLOSSARY OCTOBER 2008
sures used in HPLC. See also xerogels. separation of carbohydrates with a peak along the horizontal line (distance
Affinity chromatography: A tech- wateracetonitrile mobile phase. It is a B) divided by the distance along the hor-
nique in which a biospecific adsorbent is relatively unstable phase. izontal line to the front of the peak (dis-
prepared by coupling a specific ligand tance A) produces a ratio called the peak
such as an enzyme, antigen, or hor- asymmetry factor (see Figure 1). The
Amphoteric ion-exchange resin:
Ionexchange resins that have both posi-
mone for the macromolecule of in- tive and negative ionic groups. These ratio is 1 for a symmetrical peak, less
terest to a solid support (or carrier). resins are most useful for ion retardation than 1 for a fronting peak, and greater
This immobilized ligand will interact in which all ionic materials can be re- than 1 for a tailing peak. The higher the
only with molecules that can selectively moved from solution because the an- value, the less symmetrical the peak; val-
bind to it. Molecules that will not bind ionic and cationic functionalities coexist ues greater than 2 are unacceptable.
will be eluted unretained. The retained on the same material. Atmosphere (atm): A measure of the
compound later can be released in a pu- Analyte: The compound of interest to pressure drop across an HPLC column;
rified state. Affinity chromatography is be analyzed by injection into and elution 1 atm = 14.7 lb/in.2 (psi). See also bar
normally practiced as an onoff separa- from an HPLC column. and pascals.
tion technique.
Analytical Column: An HPLC col-
Agarose: High molecular weight poly- umn used for qualitative and quantitia-
saccharide used as a separation medium tive analysis; a typical analytical column B IIIIIIIIIIIIIIII
in biochromatography. It is used in bead will be 4.6-mm I.d. X 50-250 cm in b: See phase ratio.
form, often in gel-filtration chromatog- length but columns with smaller diame- Bo: See permeability.
raphy, with aqueous mobile phases. ters (down to 0.05-mm I.d.) can also be B solvent: Usually the stronger sol-
considered as analytical columns; can be vent in a binary eluent or gradient sepa-
constructed of stainless steel , glass, ration; typically the organic modifier or
glass-lined SS, PEEK and other metallic modifier-rich binary mixture with water
and non-metallic materials. in reversed-phase LC.
Anion exchange: The ion-exchange B term: The second term of the van
procedure used for the separation of Deemter equation. See also longitudi-
anions. Synthetic resins, bonded-phase nal diusion and molecular diusion
silicas, and other metal oxides can be
analyzed in this mode. A typical an-
term.
Backflushing: A column-switching
ionexchange functional group is the technique in which a four-way valve
tetraalkylammonium, which makes a placed between the injector and the col-
Figure 1: Example of a tailing peak: (Mod- strong anion exchanger. An amino umn allows mobile-phase flow in either
ified with permission from reference 3.)
group on a bonded stationary phase is direction. Backflushing is used to elute
an example of a weak anion exchanger. strongly held compounds at the head of
Alkoxysilane: A reactant used for the
preparation of chemically bonded Asymmetry: Factor describing the a column. It can be used for analyzing
phases. It will react with silica gel as shape of a chromatographic peak. Chro- these compounds or merely removing
follows: R3SiOR + [SiOH matographic theory assumes a Gaussian them from the column.
[SiOSiR3 1 ROH, where R is an shape and that peaks are symmetrical. A Back Pressure: Same as head pres-
alkyl group. quantitative measure is the peak asym- sure, column pressure.
metry factor, which is the ratio of the
Alumina: A normal-phase adsorbent Back Pressure Regulator: A device
distance from the peak apex to the back
used in adsorption chromatography. placed on-line after the detector to
side of the chromatography curve over
Aluminum oxide is a porous adsorbent maintain a positive pressure on the flow
the distance from the peak apex to the
that is available with a slightly basic sur- cell minimizing solvent outgassing prob-
front side of the chromatography curve
face; neutral and acidic modifications lems in the detector.
at 10% of the peak height. Other meas-
also can be made. Basic alumina can Band: Refers to the chromatographic
ures of asymmetry are commonly used,
have advantages over silica, which is peak as it moves down and is eluted
especially the U.S. Pharmacopeia (USP)
considered to have an acidic surface. from the column.
method. See Figure 1. See also
Amino phase: A propylamino phase Band broadening: The process of
used in normal bonded-phase chro- increasing width and concomitant dilut-
FoleyDorsey equation.
Asymmetry factor: A factor that
matography. It is somewhat reactive for ing of the chromatographic band as it
denotes band shape. The asymmetry
solute molecules such as aldehydes or moves down the column. The peak is
factor is calculated from the chromato-
mobile-phase additives that can react injected as a narrow slug and, ideally,
graphic peak by dropping a perpendicu-
with amines. The amino phase has each separated component would be
lar at the peak apex and a horizontal line
found some applications as a weak anion eluted as a narrow slug of pure com-
at 10% of the peak height; at the inter-
exchanger, and it also is used for the pound if not for the process of band
section, the distance to the tail of the
broadening. The measure of band or ceramic surfaces and components. CS: See Langmuir isotherm.
broadening is bandwidth (tw) or, more Capacity: See sample capacity.
correctly, the number of theoretical
Bonded-phase chromatography:
The most popular mode in LC in which Capacity factor (k9): Old term for a
plates (N) in the column. Sometimes a phase chemically bonded to a support chromatographic parameter that meas-
called band dispersion or band spread- is used for separation. The most popular ures the degree of retention. Now
ing. See Figure 2. support for bonded-phase chromatogra- defined as the retention factor (k) by
Bandwidth (tw): The width of the phy is microparticulate silica gel, and the the International Union of Pure and
chromatographic band during elution most popular type of bonded phase is Applied Chemistry (IUPAC). See also
from the column. It usually is measured organosilanesuch as octadecyl for re- retention factor for method of
at the baseline by drawing tangents to versedphase chromatography. Approxi- calculation.
the inflection points on the sides of the mately 70% of all HPLC applications
Capillary column: Refers to columns
Gaussian curve that represents the peak. are performed using chemically bonded
with inner diameters less than 0.5 mm.
Small bandwidths usually represent effi- phases.
cient separations; also called peak width. Bonded-phase concentration: See
Capillary electrochromatography
See Figure 2. (CEC): A hybrid technique in which cap-
illary columns are packed with chro-
coverage.
Bar: A unit of pressure measurement Boxcar chromatography: See matographic sorbents and electroosmotic
in HPLC equal to 1 atm, ;15 lb/in.2,
flow rather than pressure moves mobile
or 0.1 MPa.
column switching.
Breakthrough volume: The volume phase through the column; technique
Baseline: The baseline is the line at which a particular solute pumped has the surface-mediated selectivity
drawn by the recording device repre- continuously through a column will potential of HPLC and the high effi-
senting the signal from the detector begin to be eluted. It is related to the ciency of capillary electrophoresis (CE).
when only mobile phase is passing column volume and the retention factor Capillary LC: Generally refers to
through. It also represents the point of the solute. It is useful to determine HPLC performed in a fused-silica or
from which calculations are often made the total sample capacity of the column other type of capillary column; the inner
on peaks to determine peak area or peak for a particular solute. diameters typically are less than 0.5 mm;
height. Buffer: A solution that maintains con- has also been called micro-LC.
Baseline Noise: Irregular variations stant pH by resisting changes in pH
(short term) in the chromatographic from dilution or addition of small
Capillary micellar electrochro-
matography: The CEC version of mi-
baseline due to electrical noise or tem- amounts of acids and bases. cellar electrokinetic capillary
perature fluctuations, outgassing in the Buffer capacity: A quantitative meas- chromatography (MEKC).
flow cell, or poorly mixed mobile phase ure of the potential of a buffer solution
solvents. Capillary tubing: Tubing to connect
(defined as the number of equivalents various parts of a chromatograph and
BET method: Developed by Bruner, of strong acid or base to cause a one pH direct flow to the proper places. Most
Emmett, and Teller (BET), a method for unit change in 1 L of a buffer solution) capillary tubing used in HPLC is less
measuring surface area that uses nitro- or simply the ability of a buffer to with- than 0.020 in. in inner diameter. The
gen adsorptioncondensation in pores stand injections of a buffered sample smallest useful inner diameter is approx-
at liquid nitrogen temperature. Pore vol- solution without changing mobile-phase imately 0.004 in.
ume and pore size distribution also can pH; capacity determined by pH, buffer
be obtained from BET method calcula- Capping: Same as endcapping.
pKa, and buffer concentration.
tions. Carbon Load: For a bonded phase
Buffer Strength: See Ionic Strength.
Bidentate silane: A specific type of silica, term usually used to describe the
bonded phase in which a short hydro- surface coverage or the degree to which
the available silanols on the column
carbon bridge connects two silicon C IIIIIIIIIIIIIIII packing's surface have reacted and been
atoms in a silane that is bound to the C term: The interphase mass transfer
surface through two siloxane groups. replaced with the bonded phase; the
term of the van Deemter equation. See higher the carbon load, the lower num-
Binary mobile phase: Mobile phase also mass transfer and van Deemter ber of residual silanols. The carbon load
comprising two solvents or buffers. equation. is normally expressed as a % carbon
Biocompatible: A term to indicate C8: See octylsilane. (e.g. 12% carbon). In reversed-phase
that the column or instrument compo- C18: See octadecylsilane. LC, the higher the carbon load, the
nent will not irreversibly or strongly greater the analyte retention.
C4, C8, C18, etc.: Refer to the alkyl-
adsorb or deactivate biomolecules such
chain length of a reversed bonded Carrier: A term most often used in
as proteins. Frequently means metal-free
phase. affinity chromatography; refers to the
versible and usually occur on packings

with reactive functional groups such as
silanol or bonded amino phases.
Chemisorption is common with metal
oxide phases that have strong Lewis acid
sites.
Chiral recognition: The ability of a
chiral stationary phase to interact differ-
ently with two enantiomers leading to
their HPLC separation.
Chiral stationary phases: Stationary
phases that are designed to separate
enantiomeric mixtures. The phases can
be coated or bonded to solid supports,
created in situ on the surface of the
solid support, or exist as surface cavities
that allow specific interactions with one
enantiomeric form.
Chlorosilane: A chemical reagent used
to prepare siloxane bonded phases; reac-
tivity changes from a monochlorosilane
< dichlorosilane < trichlorosilane; the
alkyl portion (octadecyl, octyl, etc.) will
dictate the hydrophobicity of the result-
ing bonded phase; alkoxysilanes can be
used but are less reactive.
Chromatogram: A plot of detector
Figure 2: Widths of a Gaussian peak at various heights as a function of the standard devia-
signal output or sample concentration

tion (?????) of the peak. (Modified with permission from reference 2.)
versus time or elution volume during the

upport that binds the active ligand, CGE: See capillary gel electrophoresis. chromatographic process.
usually by a covalent bond; can also refer CZE: See capillary zone electrophoresis. Chromatograph: As a noun: a device
to the support in other chromatography used to implement a chromatographic
Chain length: The length of carbon
modes such as liquidliquid chromatog- separation. As a verb (IUPAC): the act
chain in the hydrocarbon portion of a
raphy. of separating by elution through a chro-
reversed-phase packing. It is expressed
Carrier gas: The mobile phase in gas as the number of carbon atoms (C8, matographic bed.
chromatography (GC). C18, etc.). It specifically excludes the Chromatographic Conditions: Those
Cartridge column: A column type short chains typically methyl, iso- chromatographic method experimental
that has no endfittings and is held in a propyl, and sec-butyl groups that also parameters that describe how an analysis
cartridge holder. The column comprises are attached to the silane. was performed. Sufficient information
a tube and packing contained by frits in Channeling: Occurs when voids cre- must be presented so that the analysis
each end of the tube. Cartridges are easy ated in the packing material cause can be duplicated for verification pur-
to change and are less expensive and mobile phase and accompanying solutes poses.
more convenient than conventional to move more rapidly than the average Classification: The process of sizing
columns with endfittings. flow velocity, which in turn allows band column packing particles; generally in
Cation-exchange chromatography: broadening to occur. The voids are cre- HPLC, small particle-size distribution
The form of ion-exchange chromatog- ated by poor packing or erosion of the provides better efficiency and a greater
raphy that uses resins or packings with packed bed. permeability because of the absence of
functional groups that can separate Check Valve: A device inserted into a fines. Classification can be performed by
cations. An example of a strong cation moving liquid stream that allows flow of sedimentation, elutriation, and centrifu-
functional group would be a sulfonic the stream in only one direction; most galair techniques.
acid; a weak cation-exchange functional often used on the inlet and outlet sides Column back pressure: See head
group would be a carboxylic acid. of an HPLC pump. pressure.
CE: Capillary electrophoresis. Chemisorption: Sorption caused by a Column chromatography: Any form
CEC: See capillary electrochromatogra- chemical reaction with the packing. of chromatography that uses a column
phy. Most of these interactions are irre- or tube to hold the stationary phase.
Open-column chromatography, HPLC, dimethyloctylamine at 1050 mM con- divinylbenzene is a typical cross-linking

and open-tubular capillary chromatogra- centration to the mobile phase in agent for the production of polystyrene
phy all are forms of column chromatog- reversed-phase chromatography to ion-exchange resins. The swelling and
raphy. Most often refers to open-column inhibit basic analytes from interacting diffusion characteristics of a resin are
chromatography used for prepara- with residual silanols; works by the law governed by its degree of cross-linking.
tivescale work. of mass action because concentration of Cyano Phase: A chemically bonded
Column Dead Time: The time associ- competing base is much greater than phase that terminates with the -CN
ated with the dead volume; determined analyte. See also additive. functional group; it can be used in nor-
by the dead volume divided by the flow Comprehensive two-dimensional mal phase as a moderate polarity sorbent
rate; in reversed phase LC, uracil is often chromatography: Two-dimensional and in reversed-phase as a short chain
used to measure dead volume and dead chromatography applied to every frac- bonded phase.
times. tion. See also two-dimensional chro- Cyclodextrins: Cyclic oligomers of
Column length (L): The length of matography. several D-(+)-glucopyranose units used
chromatography column in HPLC or in chiral HPLC and CE separations;
capillary in CE used to perform the
Controlled surface porosity support:
Same as porous-layer bead and pellicu- popular ones are named a-, b-, and g-
liquid-phase separation. lar packing. cyclodextrins; they have a truncated
Column Packing: The solid material, Counterion: The ion in solution used cone shape, a relatively hydrophobic
usually a porous solid with or without a to displace the ion of interest from the cavity, and primary and secondary hy-
chemically interactive surface, placed ionic site in an ion-exchange process. In droxyl groups at their ends; they sepa-
inside of the column used to differen- ion pairing, it is the ion of opposite rate on the basis of differential inclusion
tially retain analytes; referred to as the charge added to the mobile phase to of enantiomers; modified cyclodextrins
stationary phase; common packings are form a neutral ion pair in solution. with derivatized hydroxyl groups also are
unbonded and bonded silica, resins, Coupled columns: A form of column used for selectivity modification.
inorganic-organic hybrids, graphtized switching that uses a primary column
carbon connected to two secondary columns by
Column performance (N): Refers to a selector valve. Fractions from the first D IIIIIIIIIIIIIIII
the efficiency of a column; the number column can be selectively transferred to Data Acquisition Rate: A term refer-
of theoretical plates for a given test the second and third columns for addi- ring to the rate of sampling of a detec-
compound. tional separations. This term also is used tor output. To characterize a
Column plate number (N): Denotes to describe two or more columns con- chromatographic peak at least 20-30
the column efficiency; the larger the nected in series to provide an increased data points must be collected. The data
plate number, the more theoretical plates number of plates. acquisition rate, usually measured in
the column possesses; a typical well- Coverage: Refers to the amount of Hertz, defines how many data points per
packed column with a 5-m dp porous bonded phase on a silica support in second are collected while the peak is
packing in a 15 cm x 4.6 mm column bonded-phase chromatography. Cover- moving through the detector. For fast
should provide 10,00012,000 plates. age usually is described in micromoles chromatography, the data acquisition
Column switching: Using multiple per square meter or in terms of percent- rate must be sufficiently rapid to charac-
columns connected by switching valves age carbon (w/w). terize a narrow peak. Modern detectors
for better chromatographic separations Critical micelle concentration: The have data rates up to 80 Hz; also known
or sample cleanup. Fractions from a pri- concentration of an ionic surfactant as data rate and sampling rate.
mary column can be switched to two or above which a micelle is formed by Dead volume (VM): The column dead
more secondary columns, which in turn aggregation; micelles added to a mobile volume comprises the entire space ac-
can be further diverted to additional phase improve the separation of non- cessible to a small molecule that can
columns or to detectors; sometimes ionic substances in HPLC and CE fully permeate all pores of a packing
called multidimensional chromatogra- (MEKC) by a partitioning mechanism. material. It includes the interstitial vol-
Cross-linking: During the process of ume and the unoccupied pore volume. It
is denoted as VM. The system dead vol-
phy.
Column volume (Vc): The volume of copolymerization of resins to form a
the unpacked column; Vc = Ac L, where three-dimensional matrix, a difunctional ume includes the additional volume in
Ac and L are the cross-sectional area of monomer is added to form cross-link- the tubing that connects the injector and
the tube and the tube length, respec- ages between adjacent polymer chains. detector to the column. The system
tively. The degree of cross-linking is deter- dead volume usually is approximated by
mined by the amount of the monomer injecting a small, essentially unretained
Competing base: Adding a small
added to the reaction. For example, species. Uracil, acetone and thiourea are
basic compound such as triethylamine or
most commonly used species in 105 cm2/s. solvent composition are made so that
reversed-phase chromatography. See also Diol phase: A hydrophilic phase that the column experiences the composition
adjusted retention volume, holdup vol- is useful in normal and reversed phase. change in the shortest possible time.
ume, and void volume. It is a diol structure (two OH groups Low-pressure mixing systems generally
DEAE: See diethylaminoethyl. on adjacent carbon atoms in an aliphatic have larger dwell volumes than highpres-
Degassing: The process of removing chain). In normal-phase work, it is less sure mixing systems.
dissolved gas from the mobile phase polar than silica. It has been used to sep- Dynamic coating: The formation of
before or during use. Dissolved gas may arate proteins and polypeptides in in-situ coatings on the packing in HPLC
come out of solution in the detector cell reversed-phase chromatography. or on capillary walls in CE by adding a
and cause baseline spikes and noise. Dis- Displacement chromatography: A substance to the mobile phase that
solved air can affect detectors such as chromatographic process in which the adsorbs onto (or absorbs into) the pack-
electrochemical (by reaction) or fluores- sample is placed onto the column head ing or at the wall surface. The purpose
cence (by quenching) detectors. Dis- and then is displaced by a compound of a dynamic coating is to generate a
solved gases also can cause pumps to that is more strongly sorbed than the new stationary phase or to deactivate the
lose their prime. Degassing is performed compounds of the original mixture. packing material or capillary wall to pre-
by heating the solvent, helium sparging, Sample molecules then are displaced by vent unwanted interactions. One simple
or using vacuum (in a vacuum flask) or each other and by the more strongly example is the adjustment of the mobile
online evacuation from a tube made of a sorbed compound. The result is that the phase or running buffer to less than pH
gas-permeable substance such as polyte- eluted sample solute zones may be 3 to protonate silanols and negate their
trafluoroethylene (PTFE). sharpened; displacement techniques effect. Another example is coating the
Denaturing HPLC: Using reversed- have been used mainly in preparative- phase with a hydrophilic polymeric
phase HPLC to investigate genetic scale HPLC applications. material to prevent adsorption of
mutations by the investigation of DNA Distribution constant (coefficient) proteins.
base pairs. (Kc): The total equilibrium concentra-
Desalting: Technique in which low tion of a component in all forms or on
molecular weight salts and other com- the stationary phase divided by the total E IIIIIIIIIIIIIIII
pounds can be removed from nonionic equilibrium concentration of the com- E: See separation impedance.
and high molecular weight compounds. ponent in the mobile phase; also called : See interparticle porosity.
An example is using a reversed-phase the distribution coefficient or the parti-
tion coefficient in partition chromatog-
Eddy dispersion (diffusion) term (l):
packing to retain sample compounds by The A term in the van Deemter equa-
hydrophobic effects yet allowing salts to raphy. In partition chromatography, Kc tion. It is the contribution to plate
pass through unretained. Using an SEC is used when the concentration in the height from the heterogeneity in axial
column to exclude large molecules and stationary phase is expressed per unit velocities as a result of the particle size
retain lower molecular weight salts is volume of the phase and geometry of the packing, as well as
another example. (VR = VM + KcVS). In a solid station- wall effects; A 5 2ldp, where l is an
Dextran: Polydextran-based packing ary phase, KVg is used and is expressed empirical column constant. Typical val-
material primarily used for low-pressure per mass (weight) of the dry solid phase. ues of l for well-packed columns are
biochromatography; an example would In adsorption chromatography with a 0.81.0. Some theories of chromatogra-
be Sephadex (Amersham Pharmacia well characterized adsorbent of known phy indicate a velocity-dependent contri-
Biotech, Piscataway, New Jersey). surface area, the concentration in the bution to the height equivalent to a
stationary phase is expressed per unit theoretical plate (HETP) from this
Diethylaminoethyl (DEAE): A popu-
surface area. process. Also known as eddy diffusion,
lar weak anion-exchange functionality
(typically attached to cellulose or DM: See diusion coecient. flow-heterogeneity induced broadening,
Sepharose [Amersham Pharmacia dp: See particle size. and the multipath term. See also van
Biotech]) used for separating biomole- DS: See diusion coecient. Deemter equation.
cules. Dwell time: The time equivalent to e: See interstitial porosity.
Diffusion coefficient (DM or DS): A dwell volume; determined by the prod- Effective plate height (Heff): The
fundamental parameter of a molecule in uct of flow rate and the dwell volume. column length divided by the effective
gas, solution (DM), or the stationary Dwell volume: The volume between plate number.
phase (DS). Expressed in square cen- the point of mixing of solvents (usually Effective theoretical plates (Neff):
timeters per second. DM is dependent in the mixing chamber or at the propor- Also called the effective plate number by
on the molecular weight of the solute, tioning valves in the liquid chromato- IUPAC. The true number of plates in a
temperature, solvent viscosity, and molar graph) and the head of an LC column. column, because it corrects theoretical
volume of the solute. A typical value for Important in gradient elution or in iso- plates for dead volume. Neff =
a 100-Da molecule in reversed-phase cratic elution situations when changes in 16[(tR9/wb)2], where tR9 is the adjusted
chromatography at room temperature is
retention time and wb is the bandwidth Elution: The process of passing mo- used with reversed-phase packings to
of the peak (see Figure 2). It is a better bile phase through the column to trans- minimize undesirable adsorption of
figure of merit than simple plate num- port solutes down a column. basic, ionizable, and ionic compounds.
ber for comparing devices of very dif- Elution chromatography: The most Endcapping reactions also are used to
ferent geometries and phase ratios. commonly used chromatographic remove terminal silanol groups from
Efficiency (N or H ): A measure typi- method in which a sample is applied to polymeric phases.
cally determined by the number of theo- the head of the column as a narrow Endfitting: The fitting at the end of
retical plates (N) calculated from the zone and individual analytes are sepa- the column that permits connection to
equation N 5 16(VR/wb)2 5 16 rated and eluted from the end of the the injector or detector. Most HPLC
(tR/wb)2, where wb is the peak width column. Compare with displacement endfittings have frits to contain the
measured at the base (see Figure 2). If chromatography and frontal analysis. packing and low dead volumes for mini-
the peak width is measured at half Elution volume (VR): Refers to the mum band spreading. They usually are
height, the following equation is used: N volume of mobile phase necessary to constructed of stainless steel, but poly-
5 5.545 (VR/wh)2. The plate height (H) elute a solute from a column. It is the etherether ketone (PEEK) and other
or HETP is determined by H 5= L/N. volume from the point of injection to polymeric materials also are used.
The efficiency of asymmetric peaks is the volume at maximum concentration T: See total porosity.
better determined from the peak cen- (apex) for a symmetrical peak; VR = Exchange capacity: See ion-exchange
troid and variance by mathematical FtR, where F is the flow rate and tR is
analysis of the peak shape. See also
capacity.
the retention time of the peak of Excluded volume: See interstitial
FoleyDorsey equation. interest.
Effluent: The mobile phase leaving
volume.
Elutriation: A technique used to frac- Exclusion chromatography: See
the column; same as eluate. tionate packing particles by size based ionexclusion chromatography and
i: See intraparticle porosity. on the difference in their Stokes termi-
Eluate: Combination of mobile phase nal velocities. It most often is used for
steric exclusion chromatography.
Exclusion limit: The upper limit of
and solute exiting the column; also the separation of ion-exchange resins molecular weight (or size) beyond which
called euent. that require a particularly narrow size molecules will be eluted at the same
Eluent: The mobile phase used to per- range, such as amino acid resins. The retention volume, called the exclusion
forma separation. technique involves the upward flow of volume. Many SEC packings are known
Eluite: The species being eluted, the water into a large tube. The unsized by their exclusion limit. For example, a
analyte, or the sample. beads are added to the moving water, 105 column of porous silica gel will
and the particles seek their own level, exclude any compounds with a molecu-
Eluotropic series: A series of sol-
depending upon their density and parti- lar weight greater than 100,000, based
vents (eluents) with an increasing degree
cle size. They are removed at certain on a polystyrene calibration standard.
of solvent strength generally used in liq-
levels in the tube. High-purity spherical
uid solid or adsorption chromatogra- Exclusion volume (V0, Vei): The
silica gels sometimes are sized by
phy. In normal-phase chromatography, a minimum retention volume of a mole-
elutriation.
nonpolar solvent such as pentane would cule on an SEC packing in which all
be at the low end of the scale, an inter- Enantiomeric Compound: Chemical molecules larger than the size of the
mediate solvent such as methylene chlo- compounds that display chiral activity; largest pore are totally excluded. These
ride would be in the middle of the scale, such compounds will require a separa- molecules are incapable of penetrating
and a strongly polar solvent such as tion mechanism that can differentiate the pores and are eluted at the interstitial
methanol would be near the upper end between the R- or S-enantiomer and (interparticle) volume of the column.
of the scale. In reversed-phase chro- specialty columns are available for this
matography, the reverse order of purpose.
Exponentially modified Gaussian
peak: An asymmetric peak resulting
strength would be observed; water Endcapping: A technique used to from passing a Gaussian peak through a
would be weak and acetonitrile strong. remove silica gel silanol groups that may detector that is excessively slow or has
Thus, when developing a method or remain after reaction with a large sily- an excessive volume. Frequently used to
running a gradient, an eluotropic series latin agent such as octadecyltrichlorosi- model peak tailing arising from the col-
is useful for selecting solvents. See also lane. The column is said to be umn per se. The basis for the
Snyder o. endcapped when a small silylating FoleyDorsey equations. See also
Elute: To chromatograph by elution reagent (such as trimethylchlorosilane
or dichlorodimethylsilane) is used to
FoleyDorsey equation.
chromatography. The term elute is pre- Extracolumn effects: The total band
ferred over develop, which was used in bond residual silanol groups on a silica-
broadening effects of all parts of the
older nomenclature. gelbased packing surface. Most often
chromatographic system outside of the have different selectivities than hydro- the isotherm and from using poorly
column itself. Extracolumn effects must carbon phases. packed columns.
be minimized to maintain the efficiency FoleyDorsey equation: A correction
of a column. Sources of band broaden- of the plate count and retention time
ing can include the injector design, for peak tailing from extracolumn G IIIIIIIIIIIIIIII
injection volume, connecting tubing, sources of broadening. See reference 3. g: The obstruction or tortuosity fac-
endfittings, frits, detector cell volume, FPLC: See fast protein LC. tor. Molecular diffusing term. See also
and internal detector tubing. The vari-
Fractionation range: Refers to the
ances of all of these contributions are
tortuosity.
operating range of a gel or packing in Gaussian curve: A standard error
additive. curve, based on a mathematical func-
SEC. This range is where a packing can
Extracolumn volume: The volume separate molecules based on their size. tion, that is a symmetrical, bell-shaped
between the effective injection point and At one end of the range, molecules that band or peak. Most chromatographic
the effective detection point, excluding are too large to diffuse into the pores theory assumes a Gaussian peak. Using
the part of the column containing the are excluded. At the other end of the the peak maximum position as a meas-
stationary phase. It comprises the vol- range, molecules that can diffuse into all ure of retention and the efficiency equa-
umes of the injector, connecting lines of the pores totally permeate the pack- tions mentioned above assume Gaussian
and frits, and the detector. It determines ing and are eluted (unseparated) at the peak shape. See Figure 2.
the extracolumn eects. permeation volume. Gaussian peak: A peak whose shape
Frit: The porous element at either end conforms closely to the equation: C =
of a column that contains the column Cmax exp[-(t - tR)2/2s2].
F IIIIIIIIIIIIIIII packing. It is placed at the very ends of Gel: The solid packing used in gel
F: See ow rate. the column tube or, more commonly, in chromatography or gel-permeation
F: See ow resistance parameter. the endfitting. Frits can be stainless steel chromatography (GPC). An actual gel
Fast LC: Use of HPLC of short or other inert metal or plastic such as consists of two parts: the dispersed
columns (1.57 cm) with conventional porous PTFE or polypropylene. The frit medium (solid portion) and the dispers-
inner diameters (26 mm) packed with porosity must be less than the smallest ing medium (the solvent). Also defined
small particles (3- or 5-m dp). Separa- particle in the HPLC column; otherwise as a colloidal dispersion of a solid and
tion times in the range of minutes, or particles will pass through the frit, and liquid in which the solid is the continu-
even seconds, are common. the packing will be lost. ous phase.
Fast protein LC (FPLC): A termed Frontal analysis: A chromatographic Gel-filtration chromatography
coined to cover the specific use of technique that involves continuous addi- (GFC): Also called aqueous size-exclu-
HPLC for separating proteins. Gener- tion of sample to the column with the sion chromatography. Performed with
ally, glass columns, moderate pressure, result that only the least sorbed com- aqueous mobile phases. Generally refers
and spherical microbeads are used for pound, which moves at the fastest rate, to molecular size separation performed
FPLC. is obtained in a pure state. The sec- on soft gels such as polydextrans, but
Flash chromatography: A very fast ondleast-sorbed compound is eluted analysts also can use highly cross-linked
form of classic LC used by synthetic with the first-eluted compound, the polymers, silica gels, and other porous
organic chemists for rapid purification. thirdleast-sorbed compound with the media. Most gel-filtration separations
Performed primarily in the normal- first and second compound and so on involve biopolymers and water-soluble
phase mode, sometimes with reversed- until the original sample is eluted at the polymers such as polyacrylic acid.
phase chromatography. column exit. Frontal analysis is seldom Gel-permeation chromatography
Flow rate (F): The volumetric rate of used and is mainly a preparative tech- (GPC): SEC performed with organic
flow of a mobile phase through an LC nique. mobile phases used for the separation
column. Typical flow rates are 12 Frontal chromatography: Same as and characterization of polymers. SEC
mL/min for a conventional 4.6-mm i.d. frontal analysis. with aqueous mobile phases is called
HPLC column. Fronting: Peak shape in which the aqueous GPC, GFC, or aqueous SEC.
Flow resistance parameter (F): F 5 front part of the peak (before the apex) GFC: See gel-ltration chromatogra-
dp 2/Bo, where Bo is permeability. See in a chromatogram tapers in advance of phy.
also permeability. the remainder of the peak; that is, the Gigapores: See perfusion chromatog-
Fluoro phase: One of a family of front is less steep than the rear. The
peak has an asymmetric distribution
raphy.
aliphatic and aromatic reversed-phase GPC: See gel-permeation chromatog-
materials in which a substantial fraction with a leading edge. The asymmetry fac-
tor for a fronting peak has a value of
raphy.
of the bonded phase is fluorinated. Gradient: A process to change solvent
Sometimes called fluorous phases or less than one. Tailing is the opposite ef-
strength as a function of time (normally
perfluoro phases. Typically these phases fect. Fronting can result at high sample
solvent strength increases) thereby elut-
loads because of positive curvature in
ing progressively more highly retained equivalent to a theoretical plate and dp is and high pressures. Sometimes called
analytes. Typically gradients can be the particle diameter. See also reduced high-pressure LC.
binary, ternary, and quaternary solvent High Pressure Mixing: A configura-
mixtures in which solvents are blended
plate height.
H: Same as HETP. See also eciency. tion of a gradient HPLC system where
to achieve the proper strength. h: See viscosity. the solvents are mixed on the high pres-
Gradient Delay Volume: See dwell Head pressure (Dp): The difference sure side of multiple pumps (usually 2,
volume in pressure between the inlet and outlet binary); such a system offers a lower
Gradient elution: Technique for of a column measured in pounds per gradient delay volume than low pressure
decreasing separation time by increasing square inch. Governed by the following mixing systems where the solvents are
the mobile-phase strength over time approximate equation for a column mixed by proportioning valves prior to a
during the chromatographic separation. packed with spherical particles of typical single pump.
Also known as solvent programming. internal porosity (0.5): Dp = Holdup volume (VM): The total vol-
Gradients can be continuous or step- 3000Lh/tMdp 2, where L is the column ume of mobile phase in the column
wise. Binary, ternary, and quaternary sol- length in centimeters, h is the mobile- regardless of where it exists; VM = Ve
vent gradients have been used routinely phase viscosity in centipoise, tM the col- + Vi, where Ve is the interstitial volume
in HPLC. umn holdup time in minutes, and dp is and Vi is the intraparticle volume. Also
Graphitized carbon: Graphitized car- the particle diameter in micrometers. called the column void volume. IUPAC
bon is a graphitic carbon with more or Pressure can be expressed in pounds indicates that use of the term dead vol-
less perfect three-dimensional hexagonal per square inch, bars, atmospheres, or ume should be eliminated for this con-
crystalline order prepared from non- pascals. cept. The use of dead volume is limited
graphitic carbon by graphitization heat Heart cutting: Refers to collection of to regions not swept by the flowing
treatment; this packing material has a the center of the peak at which purity mobile phase system. Holdup volume is
strong affinity for polar compounds in should be maximum in preparative LC. measured by injecting an unretained
aqueous samples and water miscible The term also is used in column species that fits in all the pores. See also
organic extracts. Commonly used in pes- switching. interstitial porosity and intraparticle
ticide analysis of food samples Heff: See eective plate height. porosity.
Graphitized carbon packing: A Helium sparging: See degassing. HPLC: See high performance liquid
reversed-phase packing material consist- Helium has a very low solubility in most chromatography.
ing of pure graphitic carbon. Possesses common liquids. Hybrid silica: Silica gel comprising
interesting sorbent properties such as HETP: Height equivalent to a theoreti- both organic and inorganic moieties
preferential separation of geometric iso- cal plate. A carryover from distillation with hybrid properties of polymeric
mers such as o-, m- and p-aromatics and theory; a measure of column efficiency; packings and silica packings. Synthesized
cistrans isomers. HETP = L/N, where L is column from silanes containing organic func-
Guard column: A small column length and N is the number of theoreti- tionality. Different selectivity but better
placed between the injector and the ana- cal plates. HETP should be approxi- high-pH stability than bare or uncoated
lytical column. It protects the analytical mately 23 dp for 5-m particles with a silica gel.
column from contamination by sample typical well-packed HPLC column, Hydrodynamic volume: The molecu-
particulates and strongly retained HETP (or H) values usually are in the lar volume defined by the effective di-
species. The guard column usually is range of 0.010.03 mm. See also e- ameter of a molecule in free solution at
packed with the same material as that in ciency and h. which the hydrodynamic sphere would
the analytical column and is often of the High performance CE: A technique in be a sphere defined by the molecule as it
same inner diameter. It is much shorter, which small-diameter capillaries, revolves around its central axis in solu-
costs less, and usually is discarded when buffered conducting solutions, and high tion. Term used in SEC to define molec-
it becomes contaminated. Integrated voltages (as much as 30,000 V) separate ular shape and to explain why molecules
guardanalytical column systems often ionic molecules based on their differen- with the same molecular weight often
are preferred to minimize extracolumn tial electrophoretic mobilities. Nonionic have different elution volumes. Meas-
effects caused by connecting tubing with (neutral) molecules can be separated by ured by determining the Stokes radius.
separate guard and analytical columns. MEKC. High performance liquid chro- Hydrophilic: Greek word for water
matography loving. Refers to stationary phases that
(HPLC): The modern, fully instrumen- are fully compatible with water and to
IIIIIIIIIIIIIIII water-soluble molecules in general.
tal form of liquid-phase chromatogra-
H
h: Reduced plate height. Defined as phy technique that uses small particles Many columns used to separate proteins
HETP/dp, where HETP is the height such as ion-exchange, SEC, and
affinity columns are hydrophilic in phase in hydrophobic interaction chro- lyte. When an analyte acts to increase
nature and should not irreversibly sorb matography. the concentration of the indicating
or denature protein in an aqueous envi- Hydroxyapatite: A porous calcium species, it produces a positive peak.
ronment. hydroxy phosphate solid that chemically When a negative signal is detected, the
resembles bone and tooth. Used as a detector signals are reversed to the out-
put device.
Hydrophilic interaction chromatog-
raphy: An alternative technique to packing material in biochromatography
reversed-phase HPLC (RPC) for the for nucleic acid constituents, mono- Infinite diameter column effect: At
separation of highly polar analytes that clonal antibodies, and proteins. a certain column length, a sample in-
may be only slightly retained by RPC, Hyphenated techniques: Refers to jected into center of a packed bed
HILIC requires a high percentage of a the family of techniques best known by spreads by radial diffusion but never
nonpolar mobile phase and a polar sta- their acronyms, including LCmass reaches column wall, where wall effects
tionary phase, similar to the require- spectrometry (MS), LCFourier trans- can cause band broadening. Phenome-
ments in normal phase chromatography form IR spectroscopy (FTIR), and LC non observed by John Knox, who
(NPC). However, unlike NPC which MSMS. See also multidimensional showed that a sample peak collected in
uses nonpolar solvents such as hexane the exact center of the column exit dis-
and methylene chloride and tries to ex- played a higher efficiency than a sample
chromatography.
clude water from the mobile phase, peak collected near the wall. The infinite
HILIC requires some water in the mo- I IIIIIIIIIIIIIIII diameter effect depends on column
bile phase to maintain a stagnant en- IC: See ion chromatography. length, internal diameter, particle size,
riched water layer on the surface into and mobile-phase properties. Very sel-
which analytes may selectively partition. dom applied in HPLC.
Immobilized metal-affinity chro-
matography: See metal-anity chro-
In addition, watermiscible organic sol- Injection Solvent: Solvent used to
vents are used instead of the water-im- inject sample into an HPLC column;
matography.
miscible solvents used in NPC. With Immunoaffinity Chromatography: A
solvent should be of equal or lower
HILIC, sorbents such as bare silica, specific form of separation where an
strength than the mobile phase to pre-
bonded diol, and polyhdroxyethylaspar- antibody is bonded or immobilized
vent premature movement down the
tamide are used. Polar analytes are well onto the surface of an HPLC support
column due to the presence of a
retained and elute in order of increasing material. Based a molecular recognition
stronger solvent.
hydrophilicity, just the inverse of RPLC. mechanism, analytes that are specifically
targetted by the antibody can be selec- Inlet: The initial part of the column
Hydrophobic: Greek word for water where the solvent and sample enter. An
tively retained via antibody-antigen
fearing. Refers to stationary phases that inlet frit usually holds the packing in
interactions from a complex mixture.
are incompatible with water or to mole- place and, in some cases, protects the
After interferences are washed away,
cules that in general have little affinity packed bed.
retained analytes can be released by
for water. Hydrophobic molecules have Inlet/Outlet Check Valves: The check
changing the mobile phase conditions
few polar functional groups. Most have valve(s) on an LC pump that allow(s)
such that the strong binding is dis-
a high content of hydrocarbon (aliphatic mobile phase to flow in one direction
rupted.
and aromatic) functionality. but not in the reverse direction. The
Imprinted phases: Polymer and silica
Hydrophobic interaction chro-
phases generated in the presence of a inlet check valve allow flow from the
matography: A technique in which reservoir into the pump and the outlet
template or printing molecule. These
weakly polar (nonhydrocarboneous) check valve allows mobile phase to flow
phases have enhanced selectivity for the
packings are used to separate molecules to the column from the pump.
templating molecule.
by the interactions of their hydrophobic Inlet Filter: Filtration devices attached
moieties and the hydrophobic sites on Included volume: Also known as
totally included volume. The volume at to the inlet lines of the pump that
their packing surface. High concentra- removes particulate matter from the
tions of salt solutions are used in the which a small molecule that explores the
entire pore space of a column is eluted. mobile phase before the solvent reaches
mobile phases, and separations are gen- the pump; reservoir filters are an inlet
erated by changing the salt concentra- See also size-exclusion chromatogra-
filter that resides in the solvent bottle.
tion. The technique is analogous to
In-line filter: A device that prevents
phy.
salting-out molecules from solution. Indirect detection: Used for non-UV
absorbing or nonfluorescing analytes. A particulate matter from damaging the
Gradients are run by decreasing the
UV-absorbing or fluorescent compound column. Modern low-volume, in-line fil-
salt concentration. The technique often
added to the mobile phase maintains a ters can be placed between the injector
is used to separate proteins that are sen
high background signal; when a nonab- and the column without major contribu-
sitive to denaturization by the organic
sorbing or nonfluorescing analyte is tions to band broadening. A filter in this
solvents used in regular reversed-phase
eluted, the background is diluted and a position prevents sample particles from
chromatography. Usually little or no
negative peak is observed for that ana- entering the packed bed or column
organic solvent is used in the mobile
inlet frit.
Interparticle porosity (e): The inter- sensitivity. In nonsuppressed IC, low- highly charged. The higher the ionic
particle volume of a packed column per concentration, weakly conducting strength of a mobile phase the more the
unit column volume; e 5= Ve/Vc, buffers are carefully selected, the entire mobile phase competes with the analyte
where Ve is the interstitial volume and effluent is passed through the detector, for ionic or adsorptive sites.
Vc is the total column volume. See also and ions are detected above the back-
ground signal.
Ion-moderated partitioning chro-
interstitial porosity. matography: A technique used for
Interparticle volume (Vo): The vol- Ion-exchange capacity: The number separating carbohydrates using strong
ume of mobile phase located outside the of ionic sites on the packing that can cation exchange packings that are in
particles. participate in the exchange process. The specific cationic form (for example,
Interstitial porosity (e): The frac- exchange capacity is expressed in mil12 calcium, hydrogen, silver). The separa-
tion of the volume in the column lo- liequivalents per gram. A typical tion mechanism is complexation rather
cated in the interparticle (interstitial) styrenedivinylbenzene strong anionex- than ion exchange.
space; e = Ve/Vc. change resin may have 35 mequiv/g Ion-pair chromatography: Form of
capacity. Exchangers for IC have very chromatography in which ions in solu-
Interstitial velocity (ue): The actual
low capacity. Capacity of weak anion tion can be paired or neutralized and
velocity of the eluent as it moves
and cation exchangers varies dramati- separated as an ion pair on a reversed-
through the column flowing around the
cally with pH. phase column. Ion-pairing agents usually
particles; ue = F/Ace. The interstitial
velocity is the basis for computing the Ion-exchange chromatography: A are ionic compounds that contain a
reduced velocity. mode of chromatography in which ionic hydrocarbon chain, which imparts a cer-
Interstitial volume (Ve): The volume substances are separated on cationic or tain hydrophobicity so that the ion pair
between the particles. It does not in- anionic sites of the packing. The sample can be retained on a reversed-phase col-
clude the volume in the pores of the ion, usually with a counterion, will umn. Retention is proportional to the
particles. Also called the excluded vol- exchange with ions already on the iono- length of the hydrophobic chain and the
ume (see SEC) and interparticle volume. genic group of the packing. Retention is concentration of the ion-pair additive.
Measured by injecting a molecule that based on the affinity of different ions Ion pairing also can occur in normal-
does not permeate any pores and does for the site and other solution parame- phase chromatography when one
not interact with the surface of the ters such as pH, ionic strength, and part of the pair is dynamically loaded
particles. In SEC, this volume is counterion type. Ion chromatography onto a sorbent, but this technique is not
denoted Vo. basically is an ion-exchange technique. as popular as reversed-phase chromatog-
Ion exclusion: The process in which raphy. Also known as ion-interaction
Intraparticle porosity (i): The frac-
ionized solutes can be separated from chromatography or dynamic ionex-
tion of the particle volume that is the
un-ionized or partially ionized solutes change chromatography, which stresses
pore volume; i = Vpore/Vparticle.
using ion-exchange resins. Separation that users sometimes do not know the
Intraparticle volume (Vi): The vol- precise mechanistic details of how the
results from Donnan potential in which
ume inside the pores of the particles. additive controls retention.
ionic solutes exist at a higher concentra-
Also called the internal and included
tion in solution than in the stationary Ion retardation: Refers to using
volume. Can be measured by the
phase, whereas nonionic solutes are amphoteric ion-exchange resins, which
BET method or mercury-intrusion
evenly distributed between the mobile retard ionic molecules and allow non-
porosimetry.
phase and resin. Therefore, ionic solutes ionic molecules or nonelectrolytes to be
Ion chromatography (IC): An ionex- will move faster down the column than eluted preferentially.
change technique in which low concen- nonionic solutes. Ion exclusion occurs in Ion suppression: Buffering in an
trations of organic and inorganic reversed-phase chromatography when aqueous mobile phase at a particular pH
anions or cations are determined using anions are separated at pH values at to suppress solute ionization. For exam-
ion exchangers of low ion-exchange which the silanol groups are ionized. ple, weak carboxylic acids can have their
capacity with dilute buffers. Conductiv-
Ionic Strength: Ionic strength is a ionization suppressed by the adjustment
ity detectors often are used. IC is prac-
characteristic of an electrolyte solution. of the pH below their pKa value. Useful
ticed in two forms: In suppressed IC, a
It is typically expressed as the average for improving peak shape of weak acids
second column or a membrane separa-
electrostatic interactions among an elec- and bases in reversed-phase chromatog-
tor is used to remove the buffer counter
trolytes ions. It is related to electrolyte raphy.
ion from the analyte and simultaneously
concentration but the main difference Irregular packing: Refers to the shape
replace it with a hydrogen or hydroxide
between ionic strength and electrolyte of a column packing. Irregular packings
ion that concomitantly converts the
concentration is that the former is are available in microparticulate sizes.
buffer to an uncharged species thereby
higher if some of the ions are more The packings are obtained from grind-
suppressing background and enhancing
ing solid materials into small particles velocity. Usually written in terms of the matography performed in the linear por-
and sizing them into narrow fractions dimensionless or reduced plate height tion of an adsorption isotherm. A term
using classification machinery. Spherical (h) and reduced velocity (v) as h = Av1Y3 coined by Lloyd Snyder.
packings are used more often than irreg- + B/v + Cv. See also van Deemter Linear velocity (u): The velocity of
ular packings in analytical HPLC, but equation. the mobile phase moving through the
the less-expensive, irregular packings are column. Expressed in centimeters per
still widely used in preparative-scale LC. second. Related to flow rate by the
Irreversible adsorption: When a com- L IIIIIIIIIIIIIIII crosssectional area of the column. De-
pound with a very strong affinity for an L: See column length. termined by dividing the column length
adsorbent is injected onto a column, it Laminar flow: The smooth timein- (L) by the retention time of an unre-
can be adsorbed so strongly that it can- variant flow that develops when a liquid tained compound. See also void time.
not be eluted from the column. A chem- is moving under conditions in which Liquid chromatography (LC): A sepa-
ical reaction between the sample and the viscous forces dominate inertial forces. ration technique in which the mobile
surface of the adsorbent is an example Laminar flow is characterized by a low phase is a liquid. Most often performed
of irreversible adsorption. See also Reynolds number (see Reynolds in a column.
chemisorption. number). In a cylindrical tube, fluid Liquidliquid chromatography: One
Isocratic: Using a time invariant streams in the center flow faster than of the earliest separation modes of
eluent composition in LC. those at the tube wall, which results in a HPLC; it gave way to chemically bonded
Isotherm: See adsorption isotherm. radially parabolic distribution in axial phases in the early 1970s. Same as parti-
Isothermal chromatography: Using fluid velocity. This nonuniformity of
axial velocities in the interstices in a
tion chromatography.
conditions of constant temperature. The Liquidsolid chromatography: Same
vast preponderance of all LC is per- packed bed also causes substantial peak as adsorption chromatography.
formed under isothermal conditions. broadening in packed columns.
Loadability: The maximum amount
Langmuir isotherm: A specific form of analyte that can be injected onto a
of an isotherm; CS = N0CM/(Kd + column that no longer permits the isola-
K IIIIIIIIIIIIIIII CM), where CS and CM are the equilib- tion of product at the desired level of
k: See retention factor. rium stationary and mobile-phase con- purity or recovery level; important in
k9: An old term that has been re- centrations of the solute, N0 the total preparative chromatography
placed by the IUPAC-approved term, number of surface sites available for
retention factor (k). sorption, and Kd the sorption binding
Loading (phase loading versus
sample loading): The amount of sta-
constant.
K: See partition coecient. tionary phase coated or bonded onto a
LC: See liquid chromatography. solid support. In liquidliquid chro-
kA/B: See selectivity coecient.
Ligand: In ligand-exchange chromatography, the amount of liquid phase
Kc: See distribution constant
matography, it refers to the analyte that in milligrams of per gram of packing. In
undergoes ligand exchange with the sta- bonded-phase chromatography, the
(coecient).
Kieselguhr: A diatomaceous earth tionary phase. In affinity chromatogra- loading may be expressed in micromoles
used in column chromatography and phy, it refers to the biospecific material per square meter or percentage carbon
also as a sample cleanup media. Only enzyme, antigen, or hormone (w/w). Also called coverage or surface
weakly adsorptive, it can be used as a coupled with the support (carrier) to coverage. An alternate and unrelated
support in liquidliquid chromatogra- form the affinity column. In bonded- meaning is the amount of sample mass
phy. Rarely used in HPLC. phase chromatography, it refers to the injected on an analytical- or prepara-
Kinetic Plot: Kinetic plots are meth- moiety covalently bound to the surface. tivescale column; preparative-scale
ods to characterize the practical limits of Ligand-exchange chromatography: columns often are operated in an
column performance, where theoretical A technique in which chelating ligands overloaded condition for throughput
plates (H) and separation impedance (E) are added to the mobile phase and reasons.
are plotted as a function of the pressure- undergo sorption onto a packing. These log kw: The extrapolated intercept of
drop limited plate number (N). The sorbed molecules can act as chelating a plot of log k versus volume fraction of
kinetic plot retains the information agents with certain solutes. For example, organic modifier in reversed-phase LC.
shown in van Deemter plots but com- copper salt can be added to the mobile See also S.
pletes it with the information on the bed phase for the chelation and separation of
permeability. See Poppe Plot. Longitudinal diffusion: Same as
amino acids. Chelating resins function
molecular diusion term. B term in
Knox equation: A modification of the in a similar manner: chelating groups are
van Deemter equation. See also van
van Deemter equation developed by chemically bonded to the polystyrene
John Knox in which the A term that backbone.
Deemter equation.
represents eddy dispersion multiplied by Low pressure mixing: See high pres-
u1Y3, where u is the interstitial eluent
Linear elution adsorption chro-
matography: Refers to adsorption chro-
sure mixing.
M IIIIIIIIIIIIIIII MEKC: See micellar electrokinetic smaller are considered micro-LC

columns.
: See electrophoretic mobility. capillary chromatography.
Metal-affinity chromatography: A Microchip devices: Microdevices
special form of ligand-exchange chro- based on silicon, glass, and other types
Macroporous resin (macroreticular):
Cross-linked ion-exchange resins that
matography used to separate biopoly- of microfabricated chips in which exper-
have molecular-scale micropores and
mers with a particular affinity for a iments can be miniaturized into singleor
also macropores of several hundred
specific metal cation, typically multichannel microfluidic circuits.
angstroms. These highly porous resins
copper(II), zinc(II), and iron(II). These devices can be used for CE and
have large internal surface areas that are
Metalophile: A compound that has CEC. They should be low cost and dis-
accessible to large molecules.
high affinity for active acidic silanol posable. Using microdevices for separa-
Mass transfer (interphase): The
groups on silicas surface. Usually a tion currently is in its infancy, and
process of solute movement between
strongly basic amine or multifunctional applications should expand with time.
the moving and stationary zones. The C
carboxylate or phenol. Microparticulate: Refers to the small
term of the van Deemter equation is
Method development: A process for particles used in HPLC. Generally pack-
called the interphase mass transfer term.
optimizing the separation, including the ings with a particle diameter of less than
The faster the mass transfer process, the
sample pretreatment, to obtain a repro- 10 mm that are totally porous are con-
better the column efficiency. In HPLC,
ducible and robust separation. Usually, it sidered microparticles.
slow mass transfer is the most important
factor affecting column efficiency. Its emphasizes the search for the stationary Microporous resin: Same as
rate can be increased by using small-par- phase, eluent, and column temperature microreticular resin.
ticle packings, thin stationary-phase lay- combination that provides an adequate, Microreticular resin: Cross-linked,
ers, low-viscosity mobile phases, and if not optimum, separation. synthetic ion-exchange resins that have
high temperatures. Method validation: A process of pores with openings that correspond to
Mean pore diameter: The average testing a method to show that it per- molecular sizes. Diffusion into the nar-
diameter of the pore of a porous pack- forms to the desired limits of precision row pores can be impaired, and low
ing. It most commonly is determined by and accuracy in retention, resolution, exchange rates and poor performance
the BET method and is reported as and quantitation of the sample compo- can occur, especially for large molecules.
fourfold the specific pore volume di- nents of interest. Migration rate: See electrophoretic
vided by the specific surface area (4V/A) Micellar chromatography: Adding mobility.
based on the assumption of uniform micelles to the mobile phase to cause Migration time (tm): The time it
cylindrical pores. The pore diameter is separation. The micelles may act as dis- takes for a charged molecule to move
important in that it must allow free dif- placing or partitioning agents and pro- from the point of injection to the point
fusion of solute molecules into and out vide another parameter to change of detection in a CE capillary. Distinct
of the pore so that the solute can selectivity. Surfactants at concentrations from holdup time (tM).
interact with the stationary phase. Addi- greater than their critical micelle concen- Minimum plate height: The mini-
tionally, the pores must be well-con- tration are used in micellar chromatogra- mum of the van Deemter curve that re-
nected, with a minimum of dead ends, phy and in MEKC. sults from a plot of H versus v. This
so many paths can allow a molecule to Micro-LC: Refers collectively to tech- value represents the most theoretical
access any part of the pore space. In niques in which a column of smaller plates that can be obtained for a certain
SEC, the packings have different pore than conventional inner diameter is used column and mobile-phase system. Usu-
diameters; therefore, molecules of dif- for separation. The term micro-LC most ally occurs at excessively low flow rates.
ferent sizes can be separated. For a typi- often is used for HPLC in columns with Also known as the optimum plate
cal substrate such as silica gel, 60- and inner diameters smaller than 0.5 mm; height. It typically is two- to threefold
100- pore diameters are most popular. micro-LC is used in high-sensitivity the particle diameter of well-packed
Pore diameters greater than 300 are analysis when the sample amount is lim- columns.
used for the separation of biomolecules. ited and with certain ionization tech- Mixed-bed column: Combination of
Pores usually are classified as micro niques in LCMS in which the volume two or more stationary phases in the
(<20 ), meso (20500 ), and macro of solvent flowing into the ionization same column, used most often in IEC
(>500 ). source must be minimized. (mixed anion and cation resins) and
MECC: See micellar electrokinetic Microbore: Refers to the use of SEC (mixture of different pore size
capillary chromatography. smaller-than-usual inner diameter packings). Its advantage in IEC is the
Megapores: See perfusion chro- columns in HPLC. Columns of 2 mm total removal of both cationic and
and less are considered to be microbore anionic compounds. Useful in SEC
because a wider molecular weight range
matography.
sizes. Inner diameters of 0.5 mm and
can be accommodated by the same c is 2gDM, where g is the obstruction fac- less than 100-um in internal diameter;
olumn. tor (typically 0.60.8) and DM is the usually requires specialized instrumenta-
Mixed-mode separation: A separa- diffusion coefficient. See also van tion; often used in proteomic studies
tion that occurs in a single column where sample is limited and sensitivity
is required.
Deemter equation.
caused by the retention and selectivity Molecular weight distribution: The
provided by a dual-retention mechanism. distribution of molecular weight of Narrow-bore column: Columns of
For example, a reversed-phase column molecules in a polymer sample. Distri- less than 2-mm i.d. used in HPLC. Also
with residual silanols at intermediate-to- bution can be defined as weight average called microbore.
high pH values can separate by hy- and number average. Neff: See eective theoretical plates.
drophobic interaction and ionic Molecularly imprinted phases: See Noise: See Baseline Noise
interaction by the ionized silanols.
Sometimes mixed-mode separations can
imprinted phases.
Monodisperse particles: Particles
Nonaqueous reversed-phase chro-
matography: Refers to reversed-phase
be quite beneficial to the selectivity that fall into a narrow range of diame- chromatography performed without
(band spacing), but they can cause peak ters. See also polydisperse particles. water as a component of the eluent on a
asymmetry, and the precise balance
Monomeric phase: Refers to a reversed-phase packing. Used for very
of interactions may be difficult to
bonded phase in which single molecules nonpolar compounds that cannot be
reproduce with subsequent packing
are bonded to a support. For silica gel, eluted or are difficult to elute from a
batches.
monomeric phases are prepared by the reversed-phase column with 100%
Mobile phase: The solvent that reaction of an alkyl- or aryl- mono- methanol or acetonitrile. In these cases,
moves the solute through the column. chloro-or alkoxysilane. Polymeric phases solvent A should be acetonitrile, and sol-
In LC, the mobile phase interacts with generally are prepared from a di- or vent B should be a stronger solvent such
both the solute and the stationary phase trichlorosilane or an alkoxysilane reac- as tetrahydrofuran. Reversed-phase rules
and, therefore, can have a powerful i tant in the presence of water. apply to nonaqueous reversed-phase
nfluence on the separation. chromatography; that is, the more
Moving zone: To be distinguished
Mobile-phase strength: See solvent from the mobile phase, this zone is the nonpolar the analyte, the greater the
strength. fraction of the mobile phase in the col- retention.
Mobile-phase velocity (uM): The umn that occupies the interstitial spaces. Non-Polar: A non-polar molecule is
velocity at which the mobile phase per- See also stationary phase. one that the electrons are distributed
colates through the bed of particles; uM more symmetrically and thus does not
= L/tM, where L is column length and have an abundance of charges at the
Multidimensional chromatography:
The use of two or more columns or
tM is holdup time. See also adjusted chromatographic techniques to generate opposite sides. The charges all cancel
retention volume, holdup volume, and a better separation. It is useful for sam- out each other. Non-polar compounds,
dead volume. ple cleanup, increased resolution, solvents or bonded phases readily dis-
Mobility: See electrophoretic increased throughput, and increased solve in organic solvents, such as
mobility. peak capacity. It can be used off-line by hexane, or prefer such solvents in place
Modifier: An additive that changes the collecting fractions and reinjecting them of water. Non-polar substances do not
character of the mobile phase. For ex- onto a second column or on-line by readily dissolve in water.
ample, methanol is the strong solvent in using a switching valve. Also called cou- Nonporous packing: Particles similar
reversed phase and sometimes is called pled column chromatography, column to porous-layer bead but with particle
the modifier (water is the weak solvent); switching, multicolumn chromatography, diameters in the sub-5-m range; parti-
sometimes other additives competing and boxcar chromatography. cles often are in the sub-2-m dp range.
bases such as triethylamine or ion-pair- Used for high-speed separations in short
ing reagents are referred to as modi- columns. Common column abbrevia-
fiers, but they more correctly should be N IIIIIIIIIIIIIIII tions include NPS, which refers to non-
called additives. See also additives. n: See peak capacity. porous silica; NPR, which refers to
N: The number of theoretical plates; nonporous resins; and NPZ, which
refers to nonporous zirconia.
Molecular diffusion term (B term):
Refers to the B term (second term) of N = 16(tR/wb)2, where tR is retention
the van Deemter equation. Also called time and wb is the base width of the Nonporous particle: Refers to a solid
longitudinal or axial diffusion term. It peak. A measure of the efficiency of a particle used as a support for a porous
dominates band broadening only at very column. Sometimes measured as N = coated or bonded phase; pellicular parti-
low flow rates below the minimum plate 5.54(tR/wh)2, where wh (or w1Y2) is the cles are nonporous particles of large
height at which the diffusion of individ- peak width at half height. See also particle diameter (;40 m). Nonporous
ual solutes can occur in a longitudinal eciency and theoretical plate. silicas and resins with small particle
(lengthwise) direction on the column. n: See reduced velocity. diameters of less than 3 m usually are
The contribution to the B term arises microbeads with thin porous outer coat-
NanoLC: LC practiced with columns
from diffusion in the mobile phase and ings of silica gel, bonded silica gel, or
polymeric phase. in HPLC, supercritical fluid chromatog- narrow particle-size distribution is desir-
Normal-phase chromatography: A raphy (SFC), and CE. Stationary phases able. A particle-size distribution of dp
mode of chromatography performed can be bonded on the internal walls of 10% would mean that 90% of the parti-
when the stationary phase is more polar these small columns. The most fre- cles fall between 9 and 11 m for an
than the mobile phase. A typical normal- quently used column material is fusedsil- average 10-m dp packing.
phase system would be adsorption chro- ica tubing. Used very little in routine Particulates: Generally refers to a
matography on silica gel or alumina HPLC or SFC but frequently in CE. small particles found in the mobile
using mixtures of less polar eluents such Optically active resin: Incorporation phase that can cause back pressure
as hexanediethethyl ether as a mobile of optically active groups into an ionex- problems by lodging in frits; it can also
phase. Also refers to the use of polar change resin to allow separation of refer to the small particles packed into
bonded phases such as cyano and alu- optically active isomers. Few commer- HPLC columns
mina. Sometimes called straight-phase cially available resins for HPLC Partition chromatography: Separa-
chromatography. applications. tion process in which one of two liquid
Organic modifier: Water-miscible phases is held stationary on a solid sup-
organic solvent added to an aqueous port (stationary phase) while the other is
O IIIIIIIIIIIIIIII mobile phase to obtain separations in allowed to flow freely down the column
Octadecylsilane: The most popular reversed-phase HPLC. Common (mobile phase). Solutes partition them-
reversed phase in HPLC. Octadecylsi- organic modifiers are acetonitrile, selves between the two phases based on
lane phases are bonded to silica or poly- methanol, isopropanol, and their individual partition coefficients.
meric packings. Both monomeric and tetrahydrofuran. Liquidliquid chromatography is an
polymeric phases are available. Abbrevi- Outlet Check Valve: See Check Valve example; modern bonded-phase chro-
ated in column names as C18 and ODS. Overload: In preparative chromatog- matography can be considered to be a
Octylsilane: A popular stationary raphy the overload is defined as the form of partition chromatography in
phase in reversed-phase chromatogra- sample mass injected onto the column which one of the liquid phases is actu-
phy. Usually provides slightly less reten- at which efficiency and resolution ally bonded to the solid support. Mecha-
tion than the more popular C18. Both begins to be effected if the sample s nistically partition chromatography
monomeric and polymeric phases are ize is increased further. See also implies that the solute becomes at least
available. Abbreviated in column names partially embedded within the stationary
phase, which is impregnated, coated, or
sample capacity.
as C8.
ODS: See octadecylsilane. bonded to the substrate. In contrast to
On-column detection: The column P IIIIIIIIIIIIIIII an adsorption process in which the
Dp: See head pressure. solute does not penetrate into the reten-
itself serves as the flow cell in HPLC or
tive surface or interphase.
CECEC. Generally, the term used with Pa: See pascal.
fused-silica capillary applications. Outer Partition coefficient (K): The ratio of
Packing: The adsorbent, gel, or solid
polyimide layer is removed, an optical the equilibrium concentration of solute
support used in an HPLC column. Most
beam is directed through the capillary, in the stationary phase relative to the
modern analytical HPLC packings are
and a measuring device such as a equilibrium concentration of solute in
less than 10 m in average diameter,
photomultiplier tube is located on the the mobile phase. Also called distribu-
and 5 m is the current favorite.
opposite side of the capillary. tion coefficient, KD, and distribution
Paired-ion chromatography: Same as constant (Kc).
On-line preconcentration: A precol-
umn is placed in front of the separation Pascal (Pa): A unit of pressure. 1 MPa
ion-pair chromatography.
Particle size (dp): The average parti- is approximately 10 bar (atm) or 150
column to concentrate analytes before cle size of the packing in the LC col-
their separation. Different mechanisms psi.
umn. A 5-m dp column would be
hydrophobic interaction, adsorption, Peak: The profile of an analyte com-
packed with particles with a definite par-
or enzymatic reaction may be used to pound as it elutes from a column
ticle-size distribution because packings
retain analyte as a function of time. through a detector; usually depicted on a
are never monodisperse. See also
Then concentrated analytes are trans- visual output on a recorder or printer
ferred to the separation column by a based on the detectors electrical
monodisperse particles, particle
size distribution, and polydisperse
displacement process such as solvent response.
elution or pH change. Peak area: The area measured under a
particles.
Particle-size distribution: A measure
Open tubular columns: Small chromatographic peak; usually measured
of the distribution of the particles used
inner diameter columns (less than 100 by an integrator or data system; the peak
to pack the LC column. In HPLC, a
m) currently being investigated for use area is related to the amount of sub-
stance eluted in a peak. cles with very large pores (40008000 polar bonds and non bonding pairs of
Peak capacity (n): The number of ) called throughpores (megapores or electrons; Polar molecules are generally
equally well-resolved peaks (n) that can gigapores). Eluent flows between the able to dissolve in water (H2O) due to
be fit in a chromatogram between the large pores and through the particles the polar nature of water; polar mole-
holdup volume and some upper limit in 3001000 interconnecting pores, cules do not prefer non-polar organic
retention. For R = 1, n is given by the called diffusive pores. Best suited for solvents such as hexane. Polar molecules
approximation 1 + 0.25[(N)1Y2 ln(1 + the preparative separation of macromol- have slightly positive and slightly nega-
kn)], where R is the resolution, N is the ecules. tively charged ends.
number of theoretical plates, and kn is Permeability (Bo): Also called col- Polyacrylamide gel: Neutral
the retention factor for peak n. umn permeability and specific perme- hydrophilic polymeric packings used in
Peak dispersion: See band ability. A term expressing the resistance aqueous SEC. Prepared by the copoly-
of the packed column to the flow of merization of acryl-amide with N,N9-
mobile phase. For a packed column, Bo ' methylenebisacrylamide.
broadening.
Peak doublet: A split peak generally
caused by a column void. Could be dp 23/[180(1 2 )2] 5 dp 2/1000. A Polydisperse particles: Particles that
closely eluted compounds. column with high permeability gives a have a substantial range of diameters
low pressure drop. (>10%).
Peak height: The height of a chro-
matographic peak as measured from the Permeation: Refers to the SEC Polyethyleneimine: An anionic poly-
baseline to the peak apex; the peak process in which a solute can enter a meric phase used to coat or bond onto
eight is related to the amount of sub- mobile-phase-filled pore of the packing. silica or a polymeric packing. Most
stance eluted in a peak. Phase ratio (b): The relative amount often used for separating proteins and
Peak shape: Describes the profile of stationary to mobile phase in the col- peptides.
of a chromatography peak. Theory as- umn. In partition chromatography, b = Polymeric packings: Packings based
sumes a Gaussian peak shape (perfectly VS/VM, where VS and VM are the vol- on polymeric materials, usually in the
symmetrical). Peak asymmetry factor ume, of stationary and mobile phase in form of spherical beads. Typical poly
describes shape as a ratio. See Figures 1 the column, respectively. The retention mers used in LC are polystyrenedi-
and 2. See also asymmetry. factor is the product of the phase ratio vinylbenzene (PSDVB), polydivinyl-
and the partition coefficient. benzene, polyacryl-amide,
Peak tracking: A way of matching
peaks that contain the same compound Phenyl phase: A popular nonpolar polymethylacrylate, polyethylene-oxide,
between different experimental runs bonded phase prepared by the reaction polydextran, and polysaccharide.
during method development. Relies of dimethylphenylchloro- or alkoxysi- Polymeric phase: Refers to a chemi-
upon detection parameters of each pure lanewith silica gel. Reportedly has affin- cally bonded phase in which a polymer
analyte. Diodearray detectors and mass ity for aromatic-containing compounds species is bonded to silica-based
spectrometers are among the best detec- and does impart a different selectivity particles.
tors for peak tracking because of their compared with alkyl-bonded phases.
Pirkle column: Chiral, brush-type
Polystyrenedivinylbenzene resin
specificity. (PSDVB): The most common base
Peak variance (s2): The second cen- stationary phases based on 3,5-dini- polymer for ion-exchange chromatogra-
tral moment of the peak about the re- trobenzoylphenylglycine silica used in phy. Ionic groups are incorporated by
tention time. For a Gaussian peak, the the separation of a wide variety of various chemical reactions. Neutral PS
variance is the fundamental parameter enantiomers. Named after its developer, DVB beads are used in reversed-phase
controlling peak width. See Figure 2. See William Pirkle of the University of chromatography. Porosity and mechani-
also Gaussian peak. Illinois. cal stability can be altered by varying the
Peak volume: The total volume occu- Planar chromatography: A separa- cross-linking through the DVB content.
pied by a chromatographic peak as it tion technique in which the stationary Poppe Plot: A kinetic plot named
passes through the detector; VR= F x phase is present as or on a plane after Prof. Hans Poppe [J. Chromatogr.
wb (see Figure 2). (IUPAC). Typical forms are paper and A 778, 3 (1997)], University of Amster-
thin-layer chromatography. dam, the Netherlands, where the plate
Peak width (wb): Same as band-
width. See Figure 2. Plate height (H): See HETP. time [log (t0/N) is depicted as a func-
Plate number: See column plate tion of the number of theoretical plates
Pellicular packing: See porous-layer
(N) in order to assess the limits of col-
umn performances as a function of par-
number.
Plate or plate number: Refers to the-
bead.
Percent B solvent (% B solvent):
oretical plates in a packed column ticle size, column pressure drop, etc.
Refers to the stronger solvent in a bi-
(IUPAC). See also theoretical plate. Pore diameter: Same as mean pore
nary solvent mixture. % A solvent
would be the weaker solvent analog. Polar: A polar molecule may be polar diameter.
as a result of polar bonds or as a result Pore size: The average size of a pore
Perfusion chromatography: Refers to
of an asymmetric arrangement of non- in a porous packing. Its value is
chromatography performed using parti-
expressed in angstroms or in nanome- loss of stationary phase or dissolution PSDVB: See polystyrenedivinylben-
ters. The pore size determines whether a of the analytical column, and chemically
molecule can diffuse into and out of the absorbs substances that might interfere
zene resin.
Pulsating flow: Flow originating from
packing. See also mean pore diameter. with the separation. Its volume has little a reciprocating pump. Normally, the
Pore volume: The total volume of the effect on isocratic elution but con- pulses are dampened by a pulse damper,
pores in a porous packing, usually tributes a delay to the gradient in gradi- an electronic pressure feedback circuit,
expressed in milliliters per gram. More ent elution. or an active damper pump head. Detec-
appropriately called the specific pore Precolumn Filter: A filter used tors such as electrochemical and refrac-
volume. It is measured by the BET between the injector and the column (or tive index detectors are greatly affected
method of nitrogen adsorption or by guard column) to keep unwanted sample by flow pulsations.
mercuryintrusion porosimetry in which components from reaching the column;
mercury is pumped into the pores under sometimes called in-line filter, occasion-
high pressure. ally inlet filter. Q IIIIIIIIIIIIIIII
Porosity: For a porous substrate, the Preconcentration: See trace Quaternary methyl amine: A strong
ratio of the volume of the pores in a enrichment. anion-exchange functionality popular in
particle to volume occupied by the parti- Preparative chromatography: Refers resin-based packings. Usually supplied in
cle. The pore volume is a measure of to the process of using LC as a tech- chloride form.
the porosity and is expressed in milli- nique for the isolation of a sufficient Quaternary mobile phase: A mobile
liters per gram. amount of material for other experi- phase comprising four solvents or
Porous-layer bead: A small glass bead mental or functional purposes. For buffers.
coated with a thin layer of stationary pharmaceutical or biotechnological pu- Quaternary-Solvent Mobile Phase: A
phase. The thin layer can be an adsor- rifications, large columns of several feet mobile phase consisting of four separate
bent, resin, or a phase chemically in diameter can be used for multiple solvents which allow for fine tuning
bonded onto the adsorbent. These pack- grams of material. For isolating a few mobile phase composition; most often
ings were among the first to be used in micrograms of valuable natural product this mobile phase is delivered by a low-
HPLC. They had 2040 m particle an analytical column with a 4.6-mm i.d. pressure quaternary pump.
sizes, which were larger than the can be used. Based on the intended need
microparticulate packings of today, but of the chromatographer, both size of
were easy to pack and provided adequate columns are preparative chromato- IIIIIIIIIIIIIIII
efficiency. Also called controlled graphic approaches.
R
r: See relative retention.
surfaceporosity supports and pellicular Pressure (pressure drop) (Dp): See
materials. Radial compression: Using radial
pressure applied to a flexible wall col-
head pressure.
Porous particle: Refers to column Pressure injection: Pressure-induced umn to reduce wall effects.
packing particles that possess intercon- injection in CE. Using pressure or vac-
necting pores of specified diameter and Radial diffusiondispersion: Diffu-
uum to inject nanoliter-level volumes of
pore volume. For HPLC applications, sion dispersion across the LC column
sample into a capillary column. Best for
analysts generally use porous particles in a radial direction. If the sample is in-
narrow-bore capillaries that have inner
with diameters less than 10 mm. Larger jected into the exact center of a column,
diameters less than 10 m. A version of
particles are used in preparative-scale it will spread not only in a longitudinal
hydrostatic injection.
chromatography because of lower cost direction as it moves down the column
and higher column permeability.
Process-scale chromatography: but also radially, which allows the solute
Refers to the use of LC at the industrial- to reach the wall region where the eluent
Porous polymer: A packing material, scale level outside of laboratories. Gen- velocity is different than in the center of
generally spherical, that is based on erally requires specially designed the column.
organic polymers or copolymers. Popu- columns (usually with diameters > 5
lar examples include PSDVB, polyacry- Re: See Reynolds number.
cm), recoverable solvents, low-cost
lates, polydextrans, polyacrylamides, and packings (larger and irregular-shaped Recovery: The amount of solute or
polybutadienes. particles), and overloaded operating sample that is eluted from a column rel-
Precolumn: A small column placed conditions compared with laboratory- ative to the amount injected. Excellent
between the pump and the injector. It scale HPLC. recovery is important for good quantita-
removes particulate matter that may be tion, preparative separations, especially
present in the mobile phase, presaturates biomolecules, and good peak shape and
Programmed-temperature chro-
matography: Varying temperature dur-
the mobile phase with stationary phase resolution. Reasons for inadequate re-
ing a chromatographic run. Seldom used
or with dissolved substrate to prevent a covery can be solute interaction with ac-
in LC.
tive sites on the packing, column frits, tion time of the component of interest, in the stationary phase relative to the
and column tubing. Compound decom- tR(st)9 is the adjusted retention time of time it resides in the mobile phase. It is
position during the separation process the standard, k and kst are the corre- calculated from the adjusted retention
also can affect recovery. sponding retention factors. For two ad- time divided by the holdup time; k =
Recycling chromatography: A tech- jacent peaks, a expresses the relative (tR tM)/tM, where tR is retention
nique in which the column effluent is retention and is called separation factor time for the sample peak and tM is the
recirculated onto the head of the col- (formerly called selectivity or selectivity retention time for an unretained peak.
umn to take advantage of extended col- factor); calculated as a 5 tR29/tR19 5 (Formerly, k9 was used, and it was called
umn length. Can be performed on a k2/k1, where tR29 and tR19 are the the capacity factor or the capacity ratio.)
single column by passing the effluent adjusted retention times of peaks 2 and Retention time (tR): Also called the
through the pump again. An alternative 1, respectively, and k2 and k1 are the total retention time. The time between
technique uses two columns connected corresponding retention factors. injection and the appearance of the peak
by a switching valve where the effluent Residual silanols: The silanol maximum. The total retention volume
of one column is directed onto the head (SiOH) groups that remain on the (VR) is determined by multiplying the
of the other column. Very seldom used surface of a packing after chemically retention time by the flow rate. The
in HPLC and then only in exclusion bonding a phase onto its surface. These adjusted retention time (tR9) adjusts for
chromatography. silanol groups, which may be present in the column void volume; tR9 = tR
Reduced plate height (h): Used to very small pores, may be inaccessible to tM. It usually is measured from the
compare efficiencies of different a reacting bulky organosilane such as point of injection to the apex of the
columns; h = H/dp, where H is the octadecydimethylchlorosilane) but may peak, but it should be measured to the
height equivalent to a theoretical plate be accessible to small polar compounds. center of gravity of the peak for asym-
and dp is the particle diameter. An h Often they are removed by endcapping metric peaks.
value of 2 or less at the optimum veloc- with a small organosilane such as Retention volume (VR): The volume
ity is considered to be a well-packed trimethylchlorosilane. See also of mobile phase required to elute a sub-
HPLC column. endcapping. stance from the column; VR = F tR or
Reduced velocity (n): Used with the Resin: A solid polymeric packing used VR = VM + KDVS, where VM is the
reduced plate height to compare differ- in ion-exchange separations. The most void volume, KD is the distribution co-
ent packed chromatographic columns. It popular resins are PSDVB copolymers efficient, and VS is the stationary-phase
relates the solute diffusion coefficient with particle sizes less than 10 m. Ionic volume. See also retention time.
(DM) in the mobile phase to the particle functionality is incorporated into the
resin.
Reversed-phase chromatography:
size of the column packing (dp); n = The most frequently used mode in
udp/DM, where u is the average intersti- Resolution (Rs): Ability of a column HPLC. Uses low-polarity packings such
tial mobile-phase linear velocity. See also to separate chromatographic peaks; Rs [ as octadecyl- or octylsilane phases
Knox equation. (tR2 tR1)/[(wb1 + wb2)/2], where bonded to silica or neutral polymeric
Refractive index peak: A pseudo- tR2 and tR1 are the retention times of beads. The mobile phase usually is water
peak normally found near the dead vol- the two peaks and wb is the baseline or water-miscible organic solvents such
ume that results from the refractive width of the peaks. It usually is ex- as methanol or acetonitrile. Elution
index sensitivity of absorbance and pressed in terms of the separation of usually occurs based on the relative
other detectors. See also vacancy peak. two peaks. A value of 1 is considered to hydrophobicity or lipophilicity of the
Regeneration: Regenerating the pack- be the minimum for a measurable sepa- solutes. The more hydrophobic, the
ing in the column to its initial state after rationto occur and to allow good quanti- stronger the retention. The greater the
a gradient elution. Mobile phase is tation. A value of 0.6 is required to water solubility of the analyte, the less it
passed through the column stepwise or discern a valley between two equal- is retained. The technique has many
in a gradient. The stationary phase is height peaks. A value of 1.5 is consid- variations in which various mobile-phase
restored or solvated to its initial condi- ered sufficient for baseline resolution for additives impart a different selectivity.
tion. In ion exchange, regeneration two peaks of equal height. Values of 1.7 For example, adding a buffer and a
involves replacing ions taken up in the or greater generally are desirable for tetraalkylammonium salt to an anion
exchange process with the original ions, rugged methods. See Figure 2. analysis would allow ion-pairing to occur
which occupied the exchange sites. Resolution equation: Also called the and generate separations that rival those
Regeneration also can refer to bringing general resolution equation and the Pur- of ion-exchange chromatography. More
any column back to its original state; for nell equation; R 5 4N1Y2[(a 2 1)/a][k/(1 than 90% of HPLC analysts use
example, removing impurities with a 1 k)], where N is the efficiency, a is the reversed-phase chromatography.
strong solvent. separation factor, and k is the retention Reynolds number (Re): The ratio of
Relative retention (r): Retention rela- factor. viscous to inertial energy of the moving
tive to a standard; r = tR9/tR(st)9 = Retention factor (k): The period of fluid. A measurement of flow in a
k/kst, where tR9 is the adjusted reten- time that the sample component resides smooth unpacked pipe; Re 5 ud/(h/r),
where u is the average velocity (in centi- the reproducibility of results on of merit developed by John Knox to
menters per second), d is the pipe diam- columns of different internal diameters compare the efficiency of two chro-
eter, h is the viscosity (in grams per when using the same particle size and matographic systems that normalize for
centimeter seconds), and r is the density bonded phase; normally a larger diame- both analysis time and pressure drop;
(in grams per cubic centimeters). At low ter column is used to increase capacity; E = tRDp/N2n(1 1 k), where tR is the
Re, viscous friction dominates and con- a linear scaleup process minimizes time retention time, Dp is the pressure drop,
trols fluid motion, making it slow and required to optimize preparative N is the efficiency, n is the reduced
steady. In an unpacked tube, flow be- separations. velocity, and k the retention factor. The
comes fully turbulent when Re exceeds SEC: See size-exclusion lower the value of E , the better the
4200. In a packed bed, u is replaced with chromatography and steric exclusion system.
the average interstitial velocity and d SFC: See supercritical uid chro-
with the average particle diameter. Flow
chromatography.
Sedimentation: A technique used for
becomes turbulent in a packed bed at Re
matography.
the sizing of resins for ion-exchange Silanol: The SiOH group found on
values greater than approximately 10 but chromatography. A broad distribution of the surface of silica gel. Silanols vary in
is not fully turbulent until Re exceeds beads are placed in a solvent, often strength depending upon their location,
100200. water, in a container that is affixed to a relationship to each other, and the metal
Rs: See resolution. stationary surface. Based on particle size content of the silica. The strongest
and particle density, the beads will settle silanols are acidic and often lead to
at different velocities into a gradient of undesirable interactions with basic com-
S IIIIIIIIIIIIIIII sizes, and the fraction of interest is pounds during chromatography.
S: The solvent-strength parameter in removed. Workers can obtain very Silanophile: A compound that has
reversed-phase chromatography. The narrow cuts of particle size by high affinity for active or acidic silanol
solute-dependent slope of a plot of sedimentation. groups on a silica surface. Usually a
log10 k versus volume fraction of or- strongly basic amine.
ganic modifier. S varies with modifier
Selectivity or selectivity factor (a):
Old term replaced by the separation Silica gel: The most widely used
type, stationary phase, and temperature. factor. Sometimes called relative HPLC packing. It has an amorphous
s2: See peak variance. retention. structure, is porous, and is composed of
Salting-out effect: Using a highcon- Selectivity coefficient (kA/B): In siloxane and silanol groups. It is used in
centration salt buffer in the mobile ionexchange chromatography, the equi- all modes of LC as a bare packing for
phase to cause a low-polarity analyte to librium coefficient obtained by applying adsorption, as the support for liquidliq-
have a decreased solubility in water and the law of mass action to an ion uid chromatography or for chemically
therefore precipitate or come out of exchanger and characterizing the ability bonded phases, and as an SEC packing
solution. Most often used for the of an ion exchanger to select two ions with various pore sizes. Microparticulate
hydrophobic interaction chromatogra- present in the same solution using elec- silicas of 3-, 5-, and 10-m average par-
phy of proteins when proteins are pre- troosmotic flow. For example, the ticle diameter are used in HPLC. Com-
cipitated first at high salt concentrations exchange of Na+ for H+ kNa/H = pared with irregular silicas, spherical
and then eluted by gradual dilution ([Na]S[H]M)/([Na]M[H]S). silicas are preferred in modern analytical
using reversed-gradient elution. HPLC columns because of their packing
reproducibility and lower pressure drops.
Semipreparative chromatography:
Sample capacity: Refers to the Refers to preparative LC performed on
amount of sample that can be injected analytical (4 5 mm i.d.) or slightly Sometimes called silica.
onto an LC column without overloading. larger (610 mm i.d.) columns. Normal Siloxane: The SiOSi bond. A prin-
Often expressed as grams of sample injection size would be milligram- to cipal bond found in silica gel or a silylat-
per gram of packing. Overloading is low-gram-size samples. edcompound or bonded phase. Stable,
defined as the sample mass injected Separation factor (a): A thermody- except at high pH values. Has little
when the column efficiency decreases by namic factor that is a measure of relative effect on the HPLC separation.
10% from its normal value; sometimes retention of two substances. Formerly Silylation: The reaction process of an
called sample loading. called selectivity or selectivity factor. The organochloro- or organoalkoxysilane
Sampling Rate: See Data Acquisition relative retention; a 5 tR29/tR19 5 with a compound that contains an reac-
Rate k2/k1, where tR29 and tR19 are the tive group. In LC, it refers to the process
Saturator column: See precolumn. adjusted retention times of peaks 2 and of derivatizing the solute before chro-
1, respectively, and k2 and k1 are the matography to make it detectable or to
Scaleability: In going from analytical
corresponding retention factors. prevent unwanted stationary-phase inter-
to preparative chromatography, refers to
Separation impedance (E): A figure actions. It also can refer to the process
of adding a chemically bonded phase sample for injection into an HPLC col- as the BET method using nitrogen
to a solid support or deactivating the umn or CE capillary. Sometimes refers adsorption.
packing to reduce surface activity. to the mobile phase used. See also Spherical packing: Refers to spheri-
Simulated moving bed: A chromato- eluent. cal, solid packing materials. In analytical
graphic system involving a series of Solvent demixing: Occurs when two HPLC, spherical packings generally are
columns and valves set up to simulate solvents with very different strengths preferred over irregular particles, but
the countercurrent movement of the A is the weak solvent, and B is the irregular particles often are used in
mobile and stationary phases and enable strong solvent are used with unmodi- preparative work because of their
the continuous removal of product and fied silica or alumina. The strong solvent lower cost.
reapplication of sample. A complex (B) will be adsorbed preferentially by the Standards: A sample which contains
form of recycle chromatography used in active surface of the stationary phase known quantities of the compounds of
preparative-scale chromatography. until it is saturated; until this occurs, the interest. Standards are used to help iden-
weak solvent (A) will be enriched or tify sample peaks by comparing the time
demixed as it travels down the column.
Size-exclusion chromatography
(SEC): Same as steric exclusion in which they elute to the retention
Eventually, when the entire column is times obtained through the injection of
saturated with solvent B, this solvent will
chromatography.
Slurry packing: The technique most the sample under the same conditions.
often used to pack HPLC columns with be eluted, mixed with solvent A at the For quantitation, external standards are
microparticles. The packing is sus- initial strength, and sample components compounds that are used to construct
pended in a slurry of approximately will be eluted with the sudden change in calibration curves of detector output
10% (w/v) and rapidly pumped into solvent strength. (peak area or peak height) vs. concentra-
the empty column using special high- Solvent selectivity: Ability of a sol- tion; the concentration of unknowns are
pressure pumps. ventto influence selectivity. For example, determined by fitting the detector out-
Snyder o: Solvent-strength parame- a change in solvent strength from 5% to put to the calibration curve. Internal
terin adsorption chromatography. The 10% solvent B or a change from standards are compounds of known
energy of solvent adsorption per unit methanol to acetonitrile as the reversed- concentration with different retention
surface area occupied by the solvent. phase organic modifier will affect band times that are added to the sample and
Soap chromatography: The earlier spacing. relative detector responses between the
name for ion-pair chromatography. Solvent-selectivity triangle: A useful internal standard and the unknown are
Long-chain soaps or detergents were guide for choosing among different sol- compared in order to quantitatively
used as the mobile-phase additives. vents for changing band spacing. Solvent measure unknown compounds.
Sol gel: Silica gel formed by the ag- selectivity is dependent on dipole Stagnant mobile phase: The fraction
gregation of silica sol. Generates Type B moment, acidity, and basicity of the of the mobile phase contained within
silica gel with lower surface acidity, lower solvent molecule. See reference 4 for the pores of the particle.
trace metal, lower surface area and details. Stationary phase: The chromato-
porosity, and greater high-pH stability Solvent strength: Refers to the ability graphically retentive immobile phase
than older Type A silica gels. of a solvent to elute a particular solute involved in the chromatographic
Solid-phase extraction (SPE): A tech- or compound from a column. Snyder process. The stationary phase in LC can
nique for sample preparation using a described this quality for linear elution be a solid, a bonded, an immobilized or
2040 m dp solid-phase packing con- adsorption chromatography (liquidsolid a coated phase on a solid support or a
tained in a small plastic cartridge, disk, chromatography) on alumina and quan- wall-coated phase. The stationary phase
or in the wells of a 96-well flowthrough titatively rated solvents in an eluotropic often characterizes the LC mode. For
plate. The solid stationary phases used series. Less-extensive data are available example, silica gel is used in adsorption
are identical to HPLC packings. for silica and carbon adsorbents. See chromatography and octadecylsilane
Although related to chromatography, the also Snyder o. bonded phase is used in reversed-phase
principle of SPE is different and is Sorb: The process of being retained chromatography.
sometimes called digital chromatogra- by a stationary phase when the retention Stationary zone: To be distinguished
phy. The process as most often practiced mechanism adsorption, absorption, from the stationary phase. The station-
requires four steps: conditioning the sor- or partitioning is unclear. ary zone includes the stagnant mobile
bent, adding the sample, washing away Sorbent: Refers to a packing used in phase and the chromatographically
the impurities, and eluting the sample in LC. Common sorbents are polymers, sil- active stationary phase.
as small a volume as possible with a ica gel, alumina, titania, zirconia, and Stepwise elution: Using eluents of
strong solvent. chemically modified materials. different compositions during a chro-
Solid support: Same as support. SPE: See solid-phase extraction. matographic run. These eluents are
Solute: See also analyte. Specific surface area: The surface added in a stepwise manner with a
area of an LC packing based on meas- pump or a selector valve. Gradient elu-
Solvent: The liquid used to dissolve a
urement by an accepted technique such tion is the continuous version of chang-
ing solvent composition. (SFC): A technique that uses a supercriti- linking resins will swell and shrink more
Steric exclusion chromatography: A cal fluid as the mobile phase. The tech- than highly cross-linked resins. If
major mode of LC in which samples are nique has been applied to the separation swelling occurs in a packed column
separated by virtue of their size in of substances that cannot be handled blockage, increased back pressure can
solution. Also known as size-exclusion effectively by LC (because of detection occur, and column efficiency can be
problems) or GC (because of the lack of affected.
volatility). Examples include separations
chromatography, gel-permeation
of triglycerides, hydrocarbons, and fatty IIIIIIIIIIIIIIII

chromatography, gel-ltration
chromatography, and gel chromatogra- T
phy. Steric exclusion chromatography is acids. GC detectors and HPLC pumps Tailing: The phenomenon in which a
used most often for polymer separation have been used together in SFC. normal Gaussian peak has an asymmetry
and characterization. Superficial velocity (us): The hypo- factor greater than 1. The peak will have
thetical velocity that a mobile phase an extended trailing edge. Tailing is
would have if the same column were caused by packing sites that have both a
Sterically protected bonded phase:
Bonded phase that has sterically protect-
ing bulky functional groups such as operated unpacked but with the same stronger-than-normal retention for the
isopropyl and isobutyl surrounding a flow rate; us = F/Ac, where F is the flow solute and slower desorption kinetics. A
siloxane covalent surface bond. Prevents rate and Ac is the cross-sectional area of typical example of a tailing phenome-
attacks on siloxane bond, catalyzed the tube. non would be the strong adsorption of
hydrolysis, and loss of bonded phase Superficially porous packing: Same amines on the residual silanol groups of
at pH levels less than 3. as porous-layer bead. a low-coverage reversed-phase packing
at intermediate pH values. Tailing also
Support: Refers to solid particles. A
can result from injecting an excessive
Straight-phase chromatography:
Same as normal-phase chromatogra- support can be naked, coated, or have a
mass or sample, badly packed columns,
chemically bonded phase in HPLC.
excessive extracolumn volume, poor fit-
phy.
Strong anion exchanger: Anionex- Normally the solid support doesnt con-
tings, excessive detector volume, and
change packing with strongly basic iono- tribute to the chromatographic process.
slow detector response. See Figure 1.
genic groups such as tetraalkylam- Suppressor column: Refers to the col-
monium groups. umn placed after the ion-exchange col-
Tailing factor: U.S. Pharmacopeia
measure of peak asymmetry defined as
Strong cation exchanger: Cationex- umn. Its purpose is to remove or
the ratio of the peak width at 5% of the
change packing with strongly acidic suppress the ionization of buffer ions so
apex to twofold the distance from the
ionogenic groups such as sulfonate that sample ions can be observed in a
apex to the 5% height on the short time
groups. weakly conducting background with a
side of the peak. Greater than unity for
Strong solvent: In general, refers to a conductivity detector. Sometimes mem-
tailed peaks. See also asymmetry factor.
solvent which is a good solvent for a brane suppressors are used rather than a
column. Temperature programming: Chang-
chemical compound; in chromatography,
ing column temperature as a function of
refers to the mobile phase constituent Surface area: Refers to the total area
time during the separation. Rarely used
that provides a higher solvent strength of the solid surface in an adsorbent as
in HPLC; if so, usually in a stepwise
that causes an analyte to elute more determined by an accepted measurement
manner.
quickly from the column; in a water- technique such as the BET method,
acetonitrile binary solvent system for which uses nitrogen adsorption. The Ternary mobile phase: Mobile phase
reversed-phase LC, acetonitrile would surface area of a typical porous adsor- that is a mixture of three solvents or
be considered to be the strong bent such as silica gel can vary from less buffers.
solvent. than 100 to 600 m2/g. Theoretical plate (N): A concept
Sub-2-m: A term that refers to the Surface coverage: Usually refers to described by Martin and Synge. Relates
use of porous packings below 2-m the mass of stationary phase per unit chromatographic separation to the the-
average particle diameter; current prod- area bonded to an LC support. Often ory of distillation. Length of column
ucts vary from 1.5- to 2.0-m expressed in micromoles per square relating to this concept is called height
meter of surface. Sometimes the per- equivalent to a theoretical plate. See also
Sulfonyl cation exchanger: A strong
centage of carbon is given as an indica- HETP. Plates are calculated as N =
cation-exchange functionality found in
tor of surface coverage. 16(VR/wb)2 = 16 (tR/wb)2, where VR
resin-based packings, usually propyl-
is the retention volume, wb is the width
SO3H. May come in cationic forms Swellingshrinking: Process in which
at the peak base, and tR is the retention
such as sodium, ammonium, silver, and resins and gels increase or decrease their
time. See also N.
calcium. volume because of their solvent envi-
ronment. Swelling is dependent upon Thermally tuned tandem column
the degree of cross-linking; low-cross- chromatography: A form of LC in
Supercritical fluid chromatography
which two columns with distinctly dif- oftrace amounts of hydrophobic com- matography followed by SEC. See also
ferent selectivities are placed in tandem pounds such as polynuclear aromatic multidimensional chromatography.
and operated at two temperatures to hydrocarbons from water using a Type A silica: Silica gel formed by
optimize the resolution or analysis reversed- phase packing. A strong gelling soluble silicates. Generally has
speed. Both columns use a common elu- solvent such as acetonitrile will elute higher acidity, higher surface area and
ent, and the entire sample passes the enriched compounds. porosity, more trace metals, and poorer
through both columns and is detected Triethylamine: A very common addi- high-pH stability than Type B silicas.
with a single detector. It is not a two-di- tive used to block silanol groups in Type B silica: See sol gel.
mensional technique because each sam- reversed-phase chromatography when t0: See void time.
ple component provides only one peak. separating basic analytes.
Titania: An uncommon adsorbent Trifluoroacetic acid: A very common
used in adsorption chromatography. additive in reversed-phase chromatogra- U IIIIIIIIIIIIIIII
tm: See migration time. phy for peptides and proteins. u: See linear velocity and velocity.
tM: Holdup time. Tryptic digestion: A method for se- ue: See interstitial velocity.
Tortuosity or tortuosity factor: A lectively and reproducibly dissecting
uM: See mobile-phase velocity.
packed-column property that controls peptide chains of proteins to yield a
characteristic pattern of smaller units us: See supercial velocity.
the inhibition of longitudinal diffusion
of the solute as it diffuses along the col- that enables analysis of the parent uz: See zone velocity.
umn axis. The B term in the van protein by gradient elution reversed- UHPLC: Refers to Ultra High Pressure
Deemter equation is proportional to the phase LC. Liquid Chromatography; often loosely
tortuosity. See also B term, g, and Turbulence: The state in which fluid used for any separation performed over
velocity fluctuates randomly at a point. the pressures of conventional pumps
(400 bar); original meaning was for sepa-
molecular diusion term.
See also Reynolds number and turbu-
rations in the 20,000 psi+ range.
Total mobile-phase volume (Vt):
The total volume of mobile phase in an lent ow.
SEC column. Also known as totally in- Turbulent flow: A form of fluid
cluded volume. Same as VM. motion in which the flow ceases to be
Total permeation volume (Vp): The smooth and steady and becomes chaotic V IIIIIIIIIIIIIIII
retention volume of an SEC packing in and fluctuates with time. It is character- Vacancy chromatography: Technique
which all molecules smaller than the ized by a pressure drop significantly in which a mobile-phase additive causes
smallest pore will be eluted. In other higher than what would be extrapolated a positive detector signal output. When
words, all molecules totally permeate all from the laminar region to achieve the a solute is eluted from the column, it
of the pores at Vp and are eluted as a same volumetric flow rate. dilutes the signal and generates a nega-
single peak. Same as VM. tive peak or vacancy. The technique has
been applied primarily to single-column
Turbulent flow chromatography:
Total porosity (T): Ratio of the total Chromatography performed at very high
linear velocities with large particles ion chromatography in which mobile
volume of mobile phase in the column
under conditions using high Reynolds phases such as citrate and phthalate
to the total column volume; 5 VM/Vc
numbers. At these conditions, the H ver- buffers absorb in the UV region. When
5 e 1 i(1 2 e); where VM is the
sus n curves show a decrease in H as n a nonabsorbing anion is eluted, it dilutes
mobile-phase volume, Vc is the column
increases. See Figure 2. the UV-absorbing background and
volume, e is the interstitial porosity,
causes a negative peak; the detector out-
and i is the intraparticle porosity. tw: See bandwidth.
put leads usually are reversed so that the
Totally porous packing: The station- Two-dimensional chromatography: chromatogram looks normal. It also has
ary phase is a porous matrix, and solutes A procedure in which part or all of the been used in CE for detection.
penetrate the porous matrix to interact separated sample components are sub-
van Deemter equation: An equation
with the stationary phase. jected to additional separation steps. It
used to explain band broadening in
tR: See retention time. can be performed by conducting a par-
chromatography. The equation repre-
tR9: See adjusted retention time and ticular fraction eluted from the first col-
sents the height of a theoretical plate
umn into a second column or system
(HETP) and has three terms. The A
that has a different separation character-
retention time.
Trace enrichment: Technique in term describes eddy dispersion or diffu-
istic. It includes techniques such as
which trace amounts of compounds are sion that results from axial velocity het-
twodimensional TLC using two eluent
retained on an HPLC or precolumn erogeneity. The B term is for the
systems in which the second eluent is
packing out of a weak mobile phase or contribution of molecular diffusion or
applied after rotating the plate through
solution and then are eluted by adding a longitudinal diffusion of the solute
90. It also includes LC followed by GC
stronger mobile phase in a concentrated while passing through the column. The
and one LC mode followed by a differ-
form. The technique has been applied C term is the contribution from inter-
ent mode such as reversed-phase chro-
most successfully in the concentration phase mass transfer, which allows for
OCTOBER 2008 GLOSSARY HPLC 41
the finite rate of transfer of the solute excluded volume (Ve) in SEC. surface area, high-porosity, rigid particle.
between the stationary phase and mobile Vp: See total permeation volume.
phase. In its simplest representation, h 5 VR: See retention volume and elution
A 1 B/n 1 Cn. See also reduced plate Z IIIIIIIIIIIIIIII
height and reduced velocity. Zero dead volume: Any fitting or
volume.
VR9: See adjusted retention volume.
Vc: See column volume. component that has no volume that is
Vt: See total mobile-phase volume.
Vd: See dead volume. unswept by the eluent.
Vo: See exclusion volume.
Ve: See interstitial volume. Zirconia: Porous zirconium oxide.
Velocity (u): Same as linear velocity. Used as a chromatographic sorbent,
usually coated or bonded with polymeric
veo: See electroosmotic ow. W IIIIIIIIIIIIIIII organic phase.
Vi: See intraparticle volume. Wall effect: The consequence of a
Zone: See band.
Viscosity (h): Also called mobile- looser packing density near the walls of
a rigid HPLC column. The mobile phase Zone velocity (uz): The velocity at
phase viscosity. The viscosity of the mo-
has a tendency to flow slightly faster which the solute zone travels; uz =
bile phase varies with the temperature
near the wall because of the increased uM/(1 + k) = L/tR, where uM is the
of the column. Low-viscosity mobile
local permeability. The solute molecules mobile-phase velocity, k is the retention
phases generally provide better effi-
near the wall are carried along faster factor, L is the column length, and tR is
ciency than less-viscous ones because
than the average of the solute band, the retention time.
diffusion coefficients are inversely re-
lated to solvent viscosity. For example, and,consequently, band spreading results
column efficiency is higher in reversed- and the column loses efficiency. Zwitterions: Compounds that carry
phase chromatography with acetonitrile wb: See peak width. both positive and negative charges in
as an organic modifier than with iso- solution.
Weak anion exchanger: Anionex-
propanol, which is more viscous. Col- change packing with weakly basic iono-
umn back pressure is directly genic groups such as amino
References
proportional to solvent viscosity. (1) R.E. Majors and P.W. Carr, LCGC
diethylamino ethyl groups.
19(2) 124-162 (2001).
VM: See holdup volume. Also mobile- Weak cation exchanger: Cation (2) Nomenclature for Chromatogra-
phase volume. exchange packing with weakly acidic phy In Pure and Appl. Chem. 65
Void: The formation of a space or ionogenic groups such as carboxyl (4), 819-872 (1993).
gap, usually at the head of the column, groups.
caused by a settling or dissolution of the Weak solvent: In general, refers to a
column packing. A void in the column solvent which is a poor solvent for a
Peter W. Carr
leads to decreased efficiency and loss of chemical compound; in chromatography,

Peter W. Carr is a professor of chemistry in
resolution. Even a small void can be dis- refers to the mobile phase constituent
the Department of Chemistry, University
astrous for small-particle microparticu- that provides a low solvent strength that
of Minnesota, 207 Pleasant Street SE,
late columns. The void sometimes can causes an analyte to elute more slowly
Minneapolis, MN 55455-0431, and is a
be filled with glass beads or the same from the column in a water-acetonitrile
member of LCGCs editorial advisory board.
porous packing used in a column. binary solvent system for reversed-phase

Void time (t0): The elution time of an LC, water would be considered to be the
Ronald E. Majors
unretained peak; also called the dead weak solvent.

Ronald E. Majors, Column Watch and
time and the holdup time (tM). The WilkeChang equation: A semiem-
Sample Prep Perspectives Editor
void volume is determined by multiply- pirical equation used to estimate diffu-

Ronald E. Majors is Senior Chemist, Columns
ing the void time and the flow rate. sion coefficients in liquids as a function
and Supplies Division, Agilent Technologies,
Void volume (VM): The total volume of solute molecular size and solvent
Life Sciences Chemical Analysis, Wilmington,
of mobile phase in the column; the viscosity.

Delaware, and is
remainder of the column is taken up by

also a member of
packing material. This volume can be

LCGCs editorial
determined by injecting an unretained IIIIIIIIIIIIIIII

advisory board.
X
substance. Also called dead volume. The Xerogels: Gels used in SEC that swell
symbol V0 is often used to denote the and shrink in different solvents. Also
void volume. This is valid only for a col- refers to silica-based packings that are
umn packed with nonporous particles. prepared from acidification of soluble
V0 is valid when used to denote the silicates to generate an amorphous, high-
42 LC EDUCATION SERIES OCTOBER 2008
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HPLC Application Guide and Glossary of Terms

HPLC APPLICATION AND

products | applications | software | services

Our new LC solutions revolve around your needs.

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LC SOLUTIONS THAT REVOLVE AROUND YOUR NEEDS

HPLC APPLICATION AND EDUCATION NOTEBOOK

HPLC Applications That Revolve Around Your Needs

Page 5 RESOLUTION Agilent 1200 Series Rapid Resolution LC system

Page 10 SPEED Improving the Effectivenss of Method Translation for

HPLC Industry Applications

Page 18 Examples for Food, Environmental, Forensics, Pharmaceutical

Glossary of Liquid-Phase Separation Terms

Page 19 Updated HPLC Terminology and definitions.

Page 42 Complimentary Secrets of Agilent ChemStation' eSeminar

Page 43 Complimentary 'Optimize your LC regardless of manufacturer'

RESOLUTION Agilent 1200 Series Rapid Resolution

Fast and unambiguous determination of purity and

I n the quest to achieve highest throughput in LC/MS analyses,

Figure 1: Agilent 1200 Series Rapid Resolution LC system with Agi-

Results and Discussion

connecting the capillary between the UV detector and ESI inter-

ditions. Figure 6 shows the achieved mass accuracy errors of the

Figure 6: Mass accuracy errors of the analyses of a set of chemical

DAD (80 Hz) TOF (100 1000 Da) Cycletime Throughput

2. Udo Huber, High throughput HPLC Alternating

Figure 7: Histogram of the mass accuracy errors of the analyses

SPEED Improving the Effectivenss of Method

M ethods developed on older columns packed with large 5-

SELECTIVITY Unique Selectivity and High-Throughput

C urrently, much HPLC method development is being driven

Large changes in N result in small changes in chromatographic

Figure 3, these compounds can be readily separated at pH 2.5 in

Figure 5: Comparison of acetonitrile and methanol solvent

Figure 3: Determining optimum flow rate for RRHT columns for

Figure 6: Steroid separation on fast and selective SB RRHT

Figure 7: SB-Phenyl with methanol shows substantial differ-

brate, allowing selectivity to be quickly evaluated using many dif-

Food and Flavor Analysis Pharmaceutical Impurity Profiling

Rapid Separation and Identification of Carbonyl

Access the complete applications online by visiting

versible and usually occur on packings

signal output or sample concentration

versus time or elution volume during the

Open-column chromatography, HPLC, dimethyloctylamine at 1050 mM con- divinylbenzene is a typical cross-linking

M IIIIIIIIIIIIIIII MEKC: See micellar electrokinetic smaller are considered micro-LC

of triglycerides, hydrocarbons, and fatty IIIIIIIIIIIIIIII

leads to decreased efficiency and loss of chemical compound; in chromatography,

porous packing used in a column. binary solvent system for reversed-phase

unretained peak; also called the dead weak solvent.

void volume is determined by multiply- pirical equation used to estimate diffu-

of mobile phase in the column; the viscosity.

remainder of the column is taken up by

packing material. This volume can be

determined by injecting an unretained IIIIIIIIIIIIIIII