SUPPLEMENT TO

Consistent Results
The full MS portfolio from Agilent

With a full portfolio of leading Mass

Spectrometry products, Agilent helps
businesses worldwide optimize their
assets and uptime. Just what you’d
expect from a company with over 35
5973 inert GC/MSD years of analytical testing expertise.

Our entire line of MS systems offer newly

developed technologies – they are more
7500 Series ICP-MS reliable, efficient, and produce higher
quality data than ever before. Each is
developed and tested to meet the individ-
ual needs of labs worldwide. Plus, our
global services and support network will
1100 Series LC/MS Quad,
Ion Trap, and TOF systems help you maximize the value, longevity,
and performance of your systems.

u.s. and canada 1-800-227-9770 Contact your Agilent representative or

www.agilent.com/chem/ms visit our web site for more information.

©Agilent Technologies. 2005 MC5307C


Fast, Faster, Ultrafast: Optimization of
Cycle Times in Liquid Chromatography
for Optimum Speed and Resolution
T
he demand for greater sample
throughput and faster results in
urgent situations has driven the
creation of fast or ultrafast analytical liquid
chromatography (LC) methods. Today,
new and enhanced LC column and instru-
ment technologies are available to maxi-
mize sample throughput and provide sub-
stantial productivity improvements in the
analytical laboratory.
These new technologies are used to cre-
ate solutions to reduce cycle time (the time
between injections) and can be adapted to
any specific application needs. These range
from high-throughput screening using
ultrafast LC–mass spectrometry (MS)
methods to any other applications where
high resolution and shortened analysis
times are desired. In this primer, we will
present and discuss different approaches to
reducing cycle time for a variety of differ-
ent application needs. We will consider
both improvements in column technology
and LC instrument design.
Several practical examples are presented,
which include recommendations on how
to optimize standard LC equipment for
fast and ultrafast applications by utilizing
current available replacement parts for
better performance.
These examples demonstrate that:
● New column technology enables ultra-
fast analysis with runtimes
40 s.
● New column technology enables fast
and ultrafast LC without loss in resolu-
FEBRUARY 2005 FAST, FASTER, ULTRAFAST 3
tion but up to 20 higher throughput.
● The combination with high speed LC
equipment enables cycle times
60 s.
● When combined with automated and
sophisticated data evaluation, rapid
data review of huge numbers of chro-
matograms and results is possible with-
out compromising data quality.
● Furthermore, the needs regarding data
acquisition, chromatogram integration,
reporting, data storage, and data trans-
fer to a laboratory information manage-
ment system (LIMS) are discussed.
Introduction
The demands of high sample throughput
in condensed timeframes have given rise to
high efficiency, fast LC. From the process-
ing of hundreds of samples in overnight
runs, to efficient and timely screening of
metabolic studies, to rapid method devel-
opment, and to reducing solvent disposal
costs, fast chromatography has become a
necessity in the chromatography lab.
Using this new methodology, results can
be reported in a few hours, rather than a
day or even more. Complete validated
results in such a short time mean that
manufactured goods can be released the
same day that they are produced. The end
result is greater productivity for customers
and greater cost efficiency
What Is Fast?
Fast is a relative term that is dependent
4 FAST, FASTER, ULTRAFAST FEBRUARY 2005

Eclipse XDB-C8 Rapid Resolution Eclipse XDB-C8

150 mm x 4.6 mm, 5 m 75 mm x 4.6 mm, 3.5 m
N = 12,000 N = 10,500

1 Analysis Solvent 1 Analysis Solvent

time waste 2 time waste
2 3 5
3 5 27 min 27 mL 14 min 14 mL
4 4
6 6
7 7

0 5 10 15 20 25 30 0 5 10 15
Time (min) Time (min)

Figure 1: Mobile phase: 35% 20 mM Na2HPO4, ph 7: 65% Methanol Temperature: 35 C.

Sample: 1. Uracil, 2. Butyl Paraben, 3. Propranolol, 4. Napthalene, 5. Dipropyl Phthalate,
6. Acenaphthene, 7. Amitriptyline.

upon each user’s requirement for speed and To provide a simple structure for this
resolution. If a 20-min separation can be discussion, we have categorized fast LC
completed in less than 5 min without res- into the following areas:
olution loss, the analyst clearly will tell you ● Ultrafast LC means cycle times
that is fast LC.
1 min.
An analyst with a 5-min run per sample ● Fast LC means cycle times
5 min.
would tell you that fast LC should be 1 ● Conventional LC means cycle times
min or less. Therefore, “fast” LC is a rela-  5 min.
tive term and is defined by the user as the We will examine applications in these
reduction in total analysis time or cycle areas and determine the columns and
time while maintaining the needed instrumentation needed to achieve these
resolution. cycle times.

A:B 70:30 2 80:20 1 2 90:10

3
1 3
5 6 Equilibration volume: 7.5 mL
4
4 5
6

0 1 2 3 0 1 2 3 0 1 2 3 4 5 6 7 8
Time (min) Time (min) Time (min)

Figure 2: Mobile Phase: A: 50 mM Na2HPO4/ 10 mM TBA (pH 7) B: Acetonitrile / 10 mM TBA

Flow Rate: 1.0 mL/min Temperature: 35 °C, Detection: UV 254 nm, Sample: 1. NAD, 2. GMP,
3. AMP, 4. NADH, 5. ADP, 6. ATP.
 FEBRUARY 2005 FAST, FASTER, ULTRAFAST 5

● short column of 100 mm or less

● Columns packed with stationary phases
N = 20,441
3
250 mm x 4.6 mm, 5 m
of small particles of 3–3.5 m or
1
2 N = 20,078
4

2 m.
0 5 10 15 20 25 30
3
N = 12,133 150 mm x 4.6 mm, 5 m
1
2 N = 12,351
Columns Packed with Polymeric
4

0 5 10 15 20 25 30
or Silica-Based Monolithic
3 N = 11,468 Stationary Phases
N = 11,615 100 mm x 4.6 mm, 3.5 m
1
2
4 The second step is to select the appropriate
0
3
5 10 15 20 25 30 instrumentation. Recently, for fast and
N = 10,258 75 mm x 4.6 mm, 3.5 m
1 N = 10,148
ultrafast LC, three approaches have been
2
4
used:
0 5 10 15 20 25 30
3
N = 6037 ● High-speed, high-capacity LC systems
50 mm x 4.6 mm, 3.5 m
N = 6532
12
4 (commercially available instrumenta-
0 5 10 15 20 25 30
tion)
Time (min)
● High-speed, high-pressure LC systems
Figure 3: Influence of column length on run (research instrumentation like that pro-
time and plate numbers. Columns: ZORBAX posed in the literature [1])
SB-C18, Mobile Phase: A: 50% 20 mM
NaH2PO4, pH 2.8, B: 50% ACN, Flow Rate: ● High-speed, high-temperature LC sys-
1 mL/min, Temperature: 25 °C, Sample: tems (modified standard LC instru-
1. Estradiol, 2. Ethynylestradiol, 3. Dienestrol, mentation like that proposed in the lit-
4. Norethindrone. erature [2,3])
What Makes HPLC Fast? Data evaluation is the third important
To reduce analysis times in chromatogra- aspect that needs to be optimized. The
phy, a user first must select an appropriate needs here are:
column configuration. Several excellent ● Reliable and correct automated
column choices currently are available: integration

Time
36 s for drawing sample (26 s), 42 s for injection and analysis Equilibration time 24 s
Needle wash (10 s) for first run from second run on

Figure 4: Fast analysis of drugs with cycle time of 1 min 6 s with overlapped injection
(31 runs/h), without overlapped injection cycle time of 1 min 54 s (54 runs/h) are possible. Short
column, 4.6  30 mm Zorbax SB C-18, High flow rate, 3 ml/min, Gradient: from 40 to 55%
organic phase in 0.3 min, Detector sampling rate, peak width  0.1 min, Injection volume: 1 l
in overlapped injection mode. Compounds: Hydrocortisone, Beclomethasone, Hydrocortisone
acetate.
6 FAST, FASTER, ULTRAFAST FEBRUARY 2005
Wellplate Automation 1100 HT
handler interface system
Figure 5: Agilent 1100 Sample Capacity
Extension.
● Automated and fast customized report
generation
● Automated data storage and archiving
of raw data and results
● Automated transfer of data to other
data-handling or evaluation devices.
The objective of this primer is to use
examples with complete instrument and
column recommendations that will permit
users to perform conventional methods
faster or to perform fast and ultrafast appli-
cations successfully. This will be done with
examples that:
● Present the latest developments in
packed column technology for fast
analysis (for example, 1.8-m particle
size columns).
● Provide recommendations for optimiz-
ing instrumentation for fast and ultra-
fast analysis (for example, achieving the
lowest delay volume).
● Provide recommendations for improved
data evaluation software including
automated report generation with rele-
vant results.
HPLC Column and Instrument
Technology Advancements
Recent developments in column
technology: The column is the heart of
chromatography; everything else is built
around it. To improve the speed of analy-
sis, the column length can be reduced or
the flow rate can be increased. But without
changing any other parameters, these
approaches can compromise results and
increase analysis costs. Thus, the ideal
approach improves analysis speed without
compromising the quality of results. Let’s
first review some advancements in column
technology, which are designed to improve
sample throughput.
Recently, new column-packing materials
have been developed, which can be utilized
for fast and ultrafast applications. In addi-
tion, these materials offer enhanced effi-
ciencies and can be used for conventional,
fast, and ultrafast applications.
We have categorized fast LC columns
into two technology areas.
50 mm x 4.6 mm, 3.5 m
50 mm x 4.6 mm, 1.8 m
2
Plates (N)
1. 3476
with twice the efficiency
2. 4585
3. 5673
4. 6180
2 Plates (N)
1. 6560
3
4
3
2. 8958
3. 11,508
4. 12,266
1
4
1
0.0 1.0 0.0 0.5 1.0
Time (min) Time (min)
Figure 6: Influence of particle size on reso-
lution. Columns: ZORBAX SB-C18, 50  4.6
mm, Mobile Phase: 25% Water: 75% MeOH,
Flow Rate: 1.5 mL/min, Temperature: RT,
Detection: UV 254 nm, Sample: QC: 1. Uracil,
2. Phenol, 3. 4-Cl-Nitrobenzene, 4. Toluene.
 FEBRUARY 2005 FAST, FASTER, ULTRAFAST 7

1
RS(1,2) = 4.8 2 250 mm x 4.6 mm, 5 m
3 29.65
N = 21,848 4 N = 22,680

1 2
3
RS(1,2) = 3.5 12.71 100 mm x 4.6 mm, 3.5 m
4 N = 11,691

1 2
N = 6568
RS(1,2) = 2.9 30 mm x 4.6 mm, 1.8 m
3 4.15
4 N = 6104 1 mL/min

N = 6463
RS(1,2) = 2.9 30 mm x 4.6 mm, 1.8 m
2.09
2 mL/min
N = 6460

0 5 10 15 20 25 30
Time (min)

Figure 7: Columns: 50  4.4 mm ZORBAX SB-C18, 1.8 m, Mobile Phase: 50% 20 mM
NaH2PO4, pH 2.8: 50% ACN, Flow Rate: 1 mL/min, Temperature: RT, Detection: UV 230 nm.
Sample: 1. Estradiol, 2. Ethinylestradiol, 3. Dienestrol, 4. Norethindrone.

Columns packed with silica-based Columns packed with polymeric or

particles smaller than 2 m:
Advantages
● Columns of different length and inter-
nal diameter are available.
● Short columns packed with 1.8-m
particles show excellent resolution and
high plate numbers even for ultrafast
separations.
● A wide variety of available bonded
phases to meet a range of application
needs.
● Available column internal diameters
and chemistries make them compatibile
with LC–MS.
Disadvantages
● Higher back pressure at increased flow
rates. An appropriate particle size distri-
bution can contribute to reducing this
effect.
silica-based monolithic stationary
phases:
Advantages
● Fast and ultrafast LC at high flow rates
and low back pressures.
● Long columns ( 100 mm) can be
used due to lower back pressure.
● Comparable efficiency, resolution, and
precision are achieved, as with conven-
tional columns packed with 3-m par-
ticles or greater (4–7).
Disadvantages
● Conventional LC equipment often is
not designed for optimal performance
at flow rates greater than 3 mL/min.
For example, this can be due to flow
cell design, low detector response times,
and scan rates of the mass spectrometer.
● If a mass spectrometer is used, a splitter
8 FAST, FASTER, ULTRAFAST FEBRUARY 2005

might be required in front of the MS standard methods as well as new optimized

system to prevent sensitivity loss for ultrafast separations.
flow rates greater than 1 mL/min. For fast and ultrafast high-resolution
● Very high flow rates increase solvent HPLC, three instrument approaches are
costs and disposal costs significantly. currently in use:
● Limited choice of stationary phases and ● State-of-the-art HPLC equipment
suppliers. designed for reliability, high speed, and
Monolithic columns are a promising high capacity.
alternative to packed columns in the ● High-temperature instrumentation
future. Due to the extensive use of packed designed for temperatures above
columns, we will focus in this primer on 100 °C.
packed columns with smaller particle sizes ● High-pressure instrumentation
(1.8 m and 3–3.5 m) for conventional designed for back pressure  1000 bar.
analyses with shorter analysis times as well
as fast and ultrafast HPLC. High Speed, High Capacity HPLC
Recent developments in instrument Instruments
technology: The major factors qualifying Conventional HPLC instruments can be
an HPLC instrument for fast or ultrafast configured for use with almost every typi-
HPLC are a low delay volume minimizing cal column type with good results. Many
extracolumn effects and the availability of conventional methods with reduced analy-
an extended temperature or pressure range sis times also can be done successfully with
are. Some state-of-the-art LC equipment these instruments. However, as columns
already has been developed for high speed get smaller in length, internal diameter,
and throughput and can be tuned for opti- and particle size, high-speed, high-capacity
mum results. These current choices pro- instrumentation is needed to take full
vide the flexibility to be adapted for a wide advantage of these new columns (8). Extra
range of applications — well established delay volume for best mixing performance

Set PW <0.01 min

1.086
Flow rate: 3.5 mL/min
Set PW = 0.1 min
Absorbance (mAU)

200 DAD cell = 1.7 L 0.762 200

150 150
0.306 0.944
100 100
0.614 0.761 1.086
0.305
50 50 0.612 0.943
0 0
0.0 0.2 0.4 0.6 0.8 1.0 1.2 0.0 0.2 0.4 0.6 0.8 1.0 1.2

Figure 8: Influence of data rate settings. Column: 30  4.6 mm Zorbax, Mixer: Upchurch
mixer (Oak Harbor, Washington), Mobile phase: Water/ ACN  90/10, 3.5 ml/min, Gradient: at
0.3 min 10% ACN, at 1 min 95%ACN, at 1.18 min 95% ACN, at 1.19 min; 10% ACN, Injection
volume: 1 l with overlapped injection, Column Temperature: 60 C, DAD: 1.7 l cell,
wl 210/8 nm, ref wl 360/100nm, PW
0.01 or PW  0.1, slit width 4 nm.
 FEBRUARY 2005 FAST, FASTER, ULTRAFAST 9

USP L1 columm: ZORBAX Eclipse XDB-C18, 250 mm x 4.6 mm, 5 m Peak tR N RS 2

Injection: 8 L 1
1 6.63 12,737 0
2 11.19 18,552 15.8

0 2 4 6 8 10 12
Time (min)
2
1 Rapid Resolution HT column (L1) Peak tR N RS
ZORBAX Eclipse XDB-C18, 50 mm x 4.6 mm, 1.8 m
Injection: 2 L 1 1.40 11,421 0
2 2.33 12,909 12.3

0 1 2 3
Time (min)

1 2 Rapid Resolution HT column (L1) Peak tR N RS

ZORBAX Eclipse XDB-C18, 30 mm x 4.6 mm, 1.8 m
Injection: 2 L 1 0.85 5855 0
2 1.43 7300 8.6

0 1 2 Minimum resolution exceeded with the shortest column

Time (min)

Figure 9: Short column length choices to improve throughput - USP analysis of guaifenesin.
Sample: 1. Guaifenesin: 0.04 mg/mL, 2. Benzoic Acid: 0.10 mg/mL; Minimum Resolution
Required  3.0 Eluent: 40% Methanol: 60% Water:1.5% Glacial Acetic Acid, Flow: 1.0 mL/min
Temp: 25 C.

or a complex autosampler design can cause
a longer run time. The dwell volume (the
volume between gradient mixing point
and head of the column) and system delay
volume (extracolumn volume from the
pump to the detector) are optimized to
minimize instrument contributions to
peak dispersion.
During the past few years, high-speed
autosamplers have been introduced that
are able to inject a sample within 15 to
30 s. This enhanced speed of injection now
is ideal for use in gradient runs that take
less than a few minutes to complete. At the
same time, sample capacity was increased
to permit several hundred or thousands of
samples, which allowed 24  7 operation
and high sample throughput with short
cycle times. Valve systems were developed
to increase sample throughput doing sam-
ple wash steps and column regeneration in
parallel to the separation. Special configu-
rations with solvent- and column-switch-
ing valves decrease the time for method
development or enable more rapid and
automated sample preparation. With these
options available, a high-speed, high-
capacity LC system will enable fast results
and high-throughput without compromis-
ing resolution.
High-temperature instrumentation:
Originally, column thermostatting was
introduced to ensure optimum repro-
ducibility of retention times. The tech-
nique of increasing temperature to
decrease analysis time currently is used at
temperatures as high as about 80 °C with
state-of-the-art LC instruments. This
approach has been expanded to increase
the column temperature above 100 °C to
further increase the number of theoretical
plates for the same column length. This
has allowed run times to be decreased to
less than 1 min. Columns with internal
diameters of 2.1 or 4.6 mm with particle
sizes of about 3 m were used. Impressive
results were obtained (3). Standard instru-
mentation was used with modifications to
10 FAST, FASTER, ULTRAFAST FEBRUARY 2005

heat the LC modules in the sample flow as high as 7000 bar. The speed and effi-
path, which must be kept at elevated tem- ciency of this system have been demon-
peratures. Special column ovens are avail- strated in several publications (1).
able commercially to test this approach. Unfortunately, there is no commercial
Recommendations in the literature are LC system on the market that would allow
given on how to modify a state-of-the-art pumping pressures of 7000 bar. These
LC system accordingly. high pressures would compromise current
However, some stationary phases can technology used in commercial units to
show degradation at elevated temperature, shorten equipment life significantly.
either with respect to retention of com- Some systems and components are avail-
pounds or separation efficiency. The ana- able to allow pressures as high as 1000 bar.
lytes themselves can be unstable at these These systems have limited flow rates and
elevated temperatures. This has been therefore do not permit use with all com-
shown with recent investigations (9). monly used column internal diameters,
High-pressure instrumentation: The especially more desirable and commonly
group of J.W. Jorgenson demonstrated that used 4.6-mm i.d. columns.
small particles have a tremendous positive We will focus on examples that use state-
effect on column efficiency. Excellent reso- of-the-art instrumentation because this is
lution for tryptic digests and for other either available in most labs or can be
applications have been achieved, and the updated inexpensively (compared with the
lowest run times were obtained using cost of purchasing a complete new system)
columns with particle sizes of about 1 m. for fast and ultrafast analytical LC meth-
Jorgensen and his team have developed ods. If required, this instrumentation still
instruments that allow pumping pressures can be used for validated conventional

4
600 3
2
Flow cell with 6 8
Absorbance (mAU)

500 5 7 9
400 500-nL cell volume
300 1
200
100
0
0 0.2 0.4 0.6 0.8 1.0 1.2

4
500 2 3
8
6
Standard flow cell with
Absorbance (mAU)

5 7
400 9
300
13-L cell volume 1
200
100
0
0 0.2 0.4 0.6 0.8 1.0 1.2

Time (min)

Figure 10: Influence of additional dispersion volume on run time and resolution, conditions
see Figure 15, except flow rate  2.3 ml/min and column temperature  32 ºC.
 FEBRUARY 2005 FAST, FASTER, ULTRAFAST 11

High-pressure Standard assembly without standard mixer

gradient pump and 80-L filter

400 mm x 0.17 mm capillary

High-speed well
plate sampler

200 mm x 0.17 mm capillary

3-L Thermostated
heat exchanger column
compartment
105 mm x 0.17 mm capillary
Rapid resolution
HT column
105 mm x 0.17 mm capillary
Union

220 mm x 0.125 mm capillary

Diode-array
DAD equipped with a 50-nL flow cell
detector

80 mm x 0.125 mm capillary

Waste

Figure 11: Agilent 1100 System configuration for ultrafast LC without automated alternat-
ing column regeneration.

methods. High-temperature and high-
pressure instrumentation can require a
higher degree of maintenance as operating
conditions add more stress.
We will focus on examples that use high
performance standard instrumentation
because this is readily available in most labs
and can be inexpensively updated (in com-
parison to purchasing a complete new sys-
tem) for high-throughput analytical LC
methods. As we review these examples, we
will provide additional detail on all of the
components of the LC system, including
data handling and data storage.
Practical Examples of Faster
Analysis: Column and Instrument
Recommendations
This chapter provides examples that illus-
trate the practical use of faster conven-
tional analyses as well as fast and ultrafast
HPLC. Column parameters as well as
instrument settings must be reviewed and
optimized carefully, and recommendations
are provided for each type of analysis: con-
ventional, fast, and ultrafast HPLC.
Faster conventional analyses — switch
to smaller particle size and shorter column:
A typical analytical LC method might be a
simple isocratic separation of as many as
10 components. This type of separation on
conventional 150 or 250 mm  4.6 mm,
5-m dp columns often will take 30 min
or more. The first step in reducing the
cycle time for a method such as this is to
replace a traditional column with one that
provides the same efficiency in a shorter
length. This can be done with a column
packed with 3–3.5 m particles.
Figure 1 shows an example where analy-
12 FAST, FASTER, ULTRAFAST FEBRUARY 2005

sis time was reduced by 50% for an iso-
cratic separation of seven components.
The analysis on the 150-mm long, 5-m
column took 27 min. When the column
length was cut in half and the particle size
reduced to 3.5 m, the analysis time was
reduced by 50% with no loss in resolution.
Using the long column, only 18 runs can
be performed within an 8-h day, while 34
runs can be completed in the same time
using the short column. To do this analysis
required conventional LC instrumentation
with no changes and is the simplest way to
decrease analysis time.
The same approach can be used during
method development, as in Figure 2. The
shorter column with the 3.5-m particle
size was used for method development.
The demands of the method were for base-
line separation for all six peaks. The iso-
cratic conditions were changed after each
run using a sequence. The method devel-
opment was performed within half an
hour.
In the previous two examples, we saw
column length reduced to speed up analy-
sis. This is the first key step to decreasing
run time. In Figure 3, an example is given
showing the influence of column length on
run time, and includes short columns (10).
Run time has been shortened from
about 30 min to 7 min; a 77% reduction
in analysis time. All peaks remain baseline
separated, and proper quantitation is
possible.
These methods improved dramatically
on analysis time but are still considered
conventional LC methods with cycle times
of approximately 5 min or more.
Recommended instrumentation for
faster conventional analyses: Standard
HPLC equipment can be used for these
faster conventional methods with excellent
results.
Recommended column choices for
faster conventional analyses: Standard
4.6- or 2.1-mm i.d. columns with lengths
of 75–150 mm and 3.5-m particles are a

1.08
1 2 Pressure
Ambient 245 bar
temperature T = 50 °C
Flow rate = 1.0 mL/min

1 2 0.93
T = 50 °C
35 °C 225 bar
Flow rate = 1.5 mL/min

1 2 0.78
50 °C 200 bar
Can increase flow rate to decrease
analysis time even further
Increasing temperature reduces analysis
2 0.65 time by 40% or more and pressure by 25%
1
60 °C 189 bar

0.0 0.5 1.0 1.5

Time (min)

Figure 12: Influence of column temperature on run time. Column: rapid resolution HT SB-C18
30  4.6 mm, 1.8 mm, Mobile Phase: 40% water: 60% methanol, Flow Rate: 1 mL/min, Detec-
tion: UV 254 nm, Temperature: as noted, Sample: 1. Triamcinolone , 2. Hydrocortisone.
 FEBRUARY 2005 FAST, FASTER, ULTRAFAST 13

1100 HT
4
Flow rate 2.6 mL/min
2 3
8 Temperature 32 °C
5 6 7 Pressure 346 bar
9
Absorbance (mAU)

400 1
Cycle time 1.5 min
Run time 1.2 min
200
Analysis 0.912 min
time
0 RS (4,5) 2.76

0 0.2 0.4 0.6 0.8 1.0 RT% RSD <0.2%

Time (min)

Figure 13: Ultrafast gradient analysis of nine Alkylphenones. System configuration without
automated alternating column regeneration. Chromatographic conditions: Column: 50 
4.6 mm Zorbax StableBond-C18, 1.8 m, Injection: 1 L, Separation: Mobile phase: A: water
 0.1% HCOOH; B: acetonitrile  0.1% HCOOH. Gradient: from 50% B to 100% B in 0.65 min,
hold over 0.2 min. Stop time  1.2 min. Sample: alkylphenones and acetanilide (100 ng/L
each) consisting of: 1. acetanilide, 2. acetophenone, 3. propiophenone, 4. butyrophenone,
5. benzophenone, 6. valerophenone, 7. hexanophenone, 8. heptanophenone, 9. octanophe-
none Temperature: 32 °C, DAD detection: UV signal  245 nm, 10 nm Reference  360 nm, 80
nm Slit: 8 nm, Peak width (response time)  0.01 min (0.1 s), that is, 20 Hz data acquisition rate.

great choice for faster analyses. They pro- time (the time between two injections)
vide improved efficiency in shorter column even more.
lengths with shorter analysis times, while To improve the speed of an analysis, the
delivering excellent quantitative results. cycle time must be minimized. Typically,
cycle time is influenced by the run time,
Fast Analysis (
5 min cycle time) the equilibration time of the column, time
The previous examples demonstrated needed for the detector to balance or equil-
improved speed and greater throughput ibrate, the time needed for the injection,
but still utilized conventional analysis and the overall response time of both the
times of greater than 5 min. The next goal data acquisition and the data evaluation
is to achieve fast analysis with run times of work station.
less than 5 min. This time is necessary Fast analysis with short 4.6-mm i.d.
when there is a very high number of sam- columns with 3.5-m particles: This
ples or when demand for results is required example (Figure 4) is a gradient example
quickly. This can be a typical need in and uses a very short column (30-mm
process control or drug-screening long), a high-pressure pump, and high-
applications. speed autosampler technology to achieve
Therefore, the next step is to choose an very short cycle times.
even shorter column and use higher flow Influence of high-pressure mixing pumps:
rates to reduce analysis time further. If the The influence of a pump on speed for an
goal is to achieve the highest throughput isocratic analysis is relatively low. For gra-
on the shortest columns, then we must also dient analysis, the importance of low dwell
take advantage of the improvements in volume is significant. The faster gradient
instrument technology to reduce the cycle changes can reach the top of the column,
14 FAST, FASTER, ULTRAFAST FEBRUARY 2005

the faster the peaks can be eluted and
shorter run times will result. High-pressure
mixing gradient pumps typically have
lower dwell volumes than low-pressure gra-
dient pumps and are the best choice for
shortening run times and equilibration
times. However, some solvents might
require a minimum mixing volume to
ensure a stable detector baseline.
Influence of autosampler speed and sample
capacity: Some high-speed autosamplers
can inject 1 L within 15 to 30 s. This is a
substantial improvement over previous sys-
tems with injection times of 1 min or
more. Some systems permit overlapped
injections in which the next sample can be
drawn up during the runtime of the cur-
rent sample and kept in the sample loop
until the current run has ended. As soon as
the LC system is ready, the “parked” sam-
ple is injected immediately. Ideally,
autosampler wash steps occur while the
sample is analyzed already and no extra
time is needed. In Figure 4, we illustrate
the separation of drugs using an over-
lapped injection mode and fast gradient
application on the 30-mm-long column
(11).
This graph shows that cycle times can be
reduced significantly using overlapped
injections, and sample throughput can be
increased from 31 samples/h to 54 sam-
ples/h, an increase in speed of 57%.
Autosampler sample capacity is another
very important issue in fast and ultrafast
LC. An autosampler that is fast, but which
can handle only 100 samples is not a suffi-
cient high-throughput solution for large
sample loads or 24  7 h operation in a
service lab.
A typical autosampler that can have
extended capacity if needed is recom-
mended for high sample throughput. This
type of system is shown in Figure 5, with
an additional module that enhances the
basic system. The extra module enables the

Gradient Equilibration
Standard assembly without standard mixer High-pressure High-pressure Standard assembly
and with 80-L filter gradient pump 1 gradient pump 2 without standard mixer

400 mm x 0.17 mm capillary

High-speed well
plate sampler 0.17-mm i.d. capillary

208 mm x 0.17 mm capillary

Thermostated
2PS/10PT valve
column compartment
105 mm x 0.17 mm capillary
220 x 0.125 mm

105 mm x 0.17 mm capillary

Column 1 Column 2

105 mm x 0.17 mm capillary 105 mm x 0.17 mm capillary

DAD equipped with a 500-nL flow cell Diode-array

detector 0.508-mm i.d capillary
80 mm x 0.125 mm capillary

Waste

Figure 14: Agilent 1100 System configuration for ultra-fast LC with automated alternating
column regeneration.
No false starts
with the power of a single solution
ZORBAX Eclipse XDB HPLC columns provide
the power to eliminate false starts. From
Eclipse XDB-C18 the first injection resolve your most diffi-
Nearest Competitor A cult separations through Eclipse XDB
Nearest Competitor B columns’ wide selection of bonded phases
and dimensions.

Eclipse columns come in four phases to

meet any application need: Cyano, C18, C8
and Phenyl. Unique eXtra Densely Bonded
Column: Eclipse XDB-C18
phases provide stable separations in a pH
4.6 x 150 mm, 5µm range of 2.0 to 9.0. From 1.8µm to 7µm and
(p/n 993967-902) from fast LC to preparative separations,
Mobile phase: 90% 25mM Na2HP04 , pH 7.0:10% ACN Eclipse columns ensure productive results.
Flow rate: 1.5 mL/min
Temperature: 40 oC No other column affords you the power to
Sample: Procainamide
get the results you need. Eclipse XDB
columns provide a single solution approach
through excellent peak symmetry and wide
Visit our Web site to get your free pH compatibility. Our exclusive double end-
Eclipse technical brochure brief and/or capping provides extended column life and
Column Selection Guide. eliminates undesirable surface interactions.

Combine Eclipse XDB with the Agilent

u.s. and canada 1-800-227-9770 (Option 1) 1100 HPLC for the most powerful, single
www.agilent.com/chem/13eclipse solution to your separation needs.

© Agilent Technologies, Inc. 2005 MC10286

16 FAST, FASTER, ULTRAFAST FEBRUARY 2005

storage of as many as 80 shallow-well pounds on the same column type with

plates (14.4-mm height) for a total sample
capacity of more than 7800 samples. Sam-
ple tracking via barcode can be an appro-
priate complementary step to ensure
rugged operation.
Instrument recommendations for fast
analysis: Gradient pumps with high-pres-
sure mixing as well as high-speed autosam-
plers are a prerequisite to enable fast gradi-
ents with cycle times less than 5 min.
Sample capacity extensions with sample
tracking capabilities enable robust opera-
tion in a routine lab.
Column recommendations for fast
analysis: For a simple, fast analysis, a short
column (50 mm or shorter) with 3.5-m
particles can be used. These columns pro-
vide a fast analysis with good results.
High-efficiency, fast analysis with 4.6-
mm i.d. columns with 1.8-m particles:
The next change in the column technology
is to use even smaller particles than in the
previous examples (that is, 1.8-m parti-
cles). These particles provide extremely
high resolution with very short column
lengths (10). In Figure 6, the two chro-
matograms show the analysis of four com-
identical length and internal diameter. The
only difference is the particle size. The res-
olution for the 1.8-m particle size col-
umn is significantly better. This is due to
the high efficiency possible with these
small particles.
In Table I, a comparison is made
between column efficiency, expressed in
plate numbers, and particle sizes for differ-
ent column lengths.
For example, comparing a 150-mm-
long column with a particle size of 5 m to
a 50-mm-long column with a 1.8-m par-
ticle size, nearly the same efficiency can be
expected. For the 1.8-m particle size, a
reduction in run time of 67% can be
achieved with no observable loss in effi-
ciency and resolution.
Figure 7 shows the capability of short
columns with 1.8-m particles to main-
tain resolution with very short analysis
times. This is the same separation of estro-
gen-related compounds shown on
columns with 3.5-m particles, now sepa-
rated on columns with 1.8-m particles.
The bonded phase remains the same, only
the particle size changes to the smaller

1100 HT
Flow rate 2.6 mL/min
Temperature 32 °C
3 4 Pressure 350 bar
600 2
6 8
Absorbance (mAU)

500 7 Cycle time 1.3 min

5 9
400 1 Run time 1.2 min
300 Analysis time 0.92 min
200 RT 0.66
100 Avg4_PW 0.79 s
0
RS (4,5) 2.62
0 0.2 0.4 0.6 0.8 1.0 1.2 % RSD n.d.

Time (min)

Figure 15: Ultrafast gradient analysis of nine alkylphenones. System configuration with auto-
mated alternating column regeneration. Conditions, see Figure 13.
From workhorse to racehorse
A scaleable HPLC for ultimate reliability at every turn
Pumping systems Injection systems
Detectors Fraction collectors
Agilent 1100 Series LC Systems—50,000 systems installed
• More than 50 modules to build your best LC
• From isocratic pumps to TOF-MS detectors
• From precise shallow gradients to 30 sec run time
• Perfect fit for peak widths shorter than 1 sec
• Stable flows from 100 ml/min down to 10 nl/min
• From walk-up to automated 24/7 operation
• Capacity from 100 to 30,000 samples
u.s. and canada 1-800-227-9770
www.agilent.com/chem/1100
©Agilent Technologies, Inc. 2005 MC8408B
Agilent Technologies has been setting the
pace in LC technology for over 30 years.
It’s why 50,000 Agilent systems have been
installed worldwide. And why Agilent’s
1100 Series LC and LC/MS Systems are
considered such workhorses. The open
architecture gives seamless integration
and unprecedented scalability to more
than 50 modules. You can mix and match
modules to adapt to the ever-changing
demands of your lab, providing the best
set of features for every application.
The 1100 Series LC Systems are also the
ultimate racehorses. State of the art
instrumentation has been combined with
®
the latest ZORBAX column technology to
give optimum speed, resolution and sensi-
tivity. Save up to 95% in analysis time,
double your efficiency and gain orders of
magnitude in sensitivity. Agilent nanoflow,
capillary and standard LC systems will
exceed your expectations at every turn.
For the best in reliability, scalability and
record-breaking performance, contact Agilent.
18 FAST, FASTER, ULTRAFAST FEBRUARY 2005

(a) (b) 0.75 mL/min

0.25 mL/min

-
-
0 1 2 3 4 5 6 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.3

Time (min) Time (min)

Figure 16: Influence of flow rate on run time Columns: ZORBAX Rapid Resolution HT SB-C18,
30  1.6 mm, 1.8 mm, Mobile Phase: A: 20 mM Na2HPO4 pH 2.8, B: ACN, Gradient: A: 30–70%B
in 5 min, hold for 1min. B: 30–80% B in 1.1 min, hold for 0.3 min, Flow Rate: see below, Tem-
perature: Ambient, Autosampler: 1100, Bypass Mode, Detection: UV 230 nm, Sample: 1. Estriol,
2. Estradiol, 3. Ethynylestradiol, 4. Dienestrol, 5. Norethindrone.

1.8-m particles. This results in easy and
fast scaling of method parameters to obtain
these high resolution, very fast results.
Detector response time (data rate) and
flow-cell considerations: Fast and ultrafast
peaks like those in Figure 7 require an
appropriate data rate to obtain reliable
results. A high enough data rate enables
accurate detection of the faster eluted
peaks. To achieve accurate results, enough
data points must be collected to define the
height and shape of the peak correctly; this
is possible only with a fast data rate. The
example in Figure 8 shows the improved
peak shape and width when a correct data
rate setting is used with a UV detector.
State-of-the-art UV detectors can be
used even at flow rates above 3 mL/min
without loss of information. A detector cell
with an efficient elution behavior and a cell
volume that is in line with the peak vol-
ume plus correct data rate settings ensure
reproducible results and lower limits of
detection.
Assays for active pharmaceutical ingredi-
ents are one example in which very short
1.8-m columns can be used to speed up
assays. By substituting these columns for
longer columns with larger particle size
packings, it is possible to make dramatic
gains in productivity. Figure 9 shows
results for the USP assay of guaifenesin
with benzoic acid as the internal standard
(12).
Both 50 mm  4.6 mm and 30 mm 
4.6 mm, 1.8-m columns were substi-
tuted to evaluate different choices in col-
umn lengths for high throughput applica-
tions. The 50-mm column length
improves throughput 5, while maintain-
ing 70% of the efficiency and 78% of the
resolution of the 250-mm, 5-m column.
The 30-mm, 1.8-m provides 8 the
sample throughput and resolution Rs of
8.6, still nearly 3 the method require-
ments for resolution. These comparisons
are all done using the same bonded phase,
so the results are due to the changes in col-
umn length and particle size.
Standard configurations provide very
good performance for chromatography
with 4–4.6 mm i.d. columns and small,
3.5-m particle packings as long as run
times are above or close to 5 min and flow
rates are between 1 and 3 mL/min, as we
saw in the earlier examples. But, for special
Charging forward
HPLC technology that’s been leading for more than 30 years
Agilent 1100 Series LC Systems—50,000 systems installed
• Patented variable stroke high-pressure gradient mixing
• Flow-through injector design
• Ultrafast column technology
• Active electronic flow control
• Wireless column identification module
• Patented online and offline diagnostics
• Predictive early maintenance feedback (EMF)
u.s. and canada 1-800-227-9770
www.agilent.com/chem/1100
©Agilent Technologies, Inc. 2005 MC8409B
Agilent Technologies has always led the
pack in HPLC innovation. That’s why over
50,000 1100 Series LC and LC/MS Systems
have been installed worldwide—a total of
300,000 modules performing with expert
robustness day in and day out. From routine
testing to leading edge proteomics, you can
charge forward with powerful performance.
These innovations include a patented vari-
able stroke for optimum retention time
precision and minimum composition ripple;
a flow-through injector design for superior
®
precision; 1.8 µm ZORBAX column tech-
nology for ultimate speed and efficiency;
and a Peltier-controlled column compart-
ment for retention time stability. The
innovation continues, including purification
solutions, active flow-controlled capillary
and nano LC, and level-4 instrument control.
To lead the field in your research, install
the worldwide leader in HPLC systems. If
you’re using anything less, it’s time you
left it behind.
20 FAST, FASTER, ULTRAFAST FEBRUARY 2005
150 mm x 2.1 mm, 5 m
70-min gradient
0.2 mL/min
15 20 25
50 mm x 2.1 mm, 1.8 m
10-min gradient
0.5 mL/min
3 4
50 mm x 2.1 mm, 1.8 m
30-min gradient
0.5 mL/min
5 10
Figure 17: Conditions: Mobile Phase A: Water w/ 0.1% TFA, B: ACN w/0.1% TFA, Gradient
2%B to 50%B, Temperature: 50 C, Detection: UV 214 nm Sample: HSA Tryptic Digest, injection
8 l (120 pmol total on column).
demands, like fast or ultrafast LC with
optimum resolution, the standard instru-
ment configuration should be modified as
discussed here. Capillaries or tubings in the
flow path must be exchanged according to
the vendor’s recommendation. An appro-
priate detector cell should be selected, and
the mixing device should also be of the
lowest possible volume for gradient separa-
tions. These options are readily available,
and these changes can be made in a mini-
mal amount of time.
At higher flow rates, the influence of the
correct instrument setup is also significant.
In Figure 10, two instrument setups are
compared. The comparison of peak width
for both chromatograms shows that mini-
mizing extra peak dispersion (as the result
of extra column volume) with the properly
configured instrument enables faster run
times without loss in resolution (13).
Figure 11 shows the modifications
needed for a typical LC system. A mixer
with an 80-L volume replaced the mixing
120 peaks
60 min
30 35 40 45 50 55
125 peaks
10 min
5 6 7 8 9 10
156 peaks
25 min
15 20 25
Time (min)
device with a volume of 320 L, the detec-
tor cell was minimized to a volume of
500 nL, and the detector capillaries were
changed.
Two additional chromatographic
parameters also can be optimized to reduce
analysis time and achieve fast analysis.
They are flow rate and temperature. Many
people increase flow rate to decrease analy-
sis time. It also is possible to adjust both
parameters at one time. Figure 12 shows an
example in which temperature was raised
to decrease run times. At the same time,
due to the decreased viscosity and reduced
back pressure of the mobile phases at ele-
vated temperatures, higher flow rates can
be used.
Instrument recommendations for high
efficiency, fast analysis: In addition to the
instrument recommendations for fast
analysis with high-pressure mixing —
binary pumps and high capacity, high-
speed autosamplers — it is critical to
choose a detector setup with a fast data rate

to accurately define the narrow peaks in
high-efficiency chromatography. These
will allow accurate detection of the peak
and permit good, quantitative results.
Column recommendations for high-
efficiency fast analysis: The isocratic
examples mentioned earlier demonstrate
the high resolution achievable for fast and
ultrafast LC by simply scaling down with
4.6-mm i.d. columns with high resolution
1.8-m particles. This small particle size
provides extremely high efficiency, while
the4.6-mm internal diameter permits stan-
dard injection and sample sizes and easy
scaling from conventional 4.6-mm i.d.,
3-m or greater columns with predictable,
reliable, and robust results.
Ultrafast Gradient Analysis

1-min Cycle Time Using 1.8-m
Particle Size Columns
Gradient elution is used widely for fast
separations of complex samples. Ultrafast
600
Absorbance (mAU)
500
400
300
200
100
0
0.25 0.50
Absorbance (mAU)
800
600
400
200
0.25 0.50
Figure 18: Influence of instrument setup on performance. Column: 50  2.1 mm Zorbax
StableBond-C18, 1.8 m 50–95%B in 0.95 min, hold over 0.55 min, 0.70 mL/min, 350 bar,
Room Temp.
FEBRUARY 2005 FAST, FASTER, ULTRAFAST 21
gradient separations can also be done effec-
tively with 1.8-m particle size columns,
with both 4.6-mm and 2.1-mm internal
diameters. The combination of the high
resolution of the 1.8-m particle size and
steep gradient analysis results in extremely
high peak capacity and the ability to sepa-
rate complex samples in a very short time.
Sequential operation mode: In the
next example, the ultrafast analysis of eight
alkylphenones and acetanilide is presented
(13). The Agilent standard 1100 LC sys-
tem was modified to meet the demands of
extremely short cycle times, as shown
before (Figure 11).
This system and the principle of “auto-
matic delay volume reduction,” in which
the autosampler delay volume is excluded
automatically from the flow path once the
sample has cleared the sample loop,
resulted in cycle times of 1.5 min with
baseline separation for all peaks (Figure
13). The flow rate was set to 2.6 mL/min
Poor instrument setup
W4 = 2.26 s
R (4,5) = 1.83
0.75 1.00 1.25 1.50 1.75 2.00
Optimized setup
W4 = 1.54 s
R (4,5) = 2.27
0.75 1.00 1.25 1.50 1.75 2.00
Time (min)
22 FAST, FASTER, ULTRAFAST FEBRUARY 2005

and the back pressure was kept around 346
bar, which is below the 400 bar limit on
some liquid chromatographs. The modifi-
cations involved replacement of the stan-
dard mixer in the pump with a low volume
mixer. A 500-nL UV cell replaced the stan-
dard diode-array detector cell, and wider
capillaries were used for the 500-nL cell to
avoid higher back pressure. Overlapped
injections also are used in this example to
provide faster sample throughput, as
described earlier. More samples are ana-
lyzed during a given period of time when
both automatic delay volume reduction
and overlapped injections are used.
Operation with parallel column
regeneration: Because gradient elution
requires the column to regenerate before
subsequent runs, an automated column
regeneration system reduces cycle time
dramatically, especially with gradients that
take less than a minute. A column regener-
ation valve (two-position/10-port valve)
allows simultaneous analysis on one col-
umn while a second, identical column is
flushed and equilibrated by an additional
regeneration pump (Figure 14). Depend-
ing upon whether the regeneration pump
is an isocratic or gradient pump, column
equilibration or column wash and equili-
bration can be performed while the next
analysis already is in progress. The two-
position/10-port valve can be integrated
into the thermostated column compart-
ment for full temperature control.
For this sample, using a system with a
two-position, 10-port valve for alternating
column regeneration, the cycle time was
reduced to 1.3 min, using the same chro-
matographic conditions (Figure 15). All
peaks were still baseline separated and 13%
cycle time is saved.
Instrument recommendations for
ultrafast analysis: These examples
demonstrate that with only a few modifi-
cations, a currently available HPLC system
is suited perfectly for fast and ultrafast gra-
dient analysis, even of complex mixtures
that require high resolution. With the
smallest particle size columns, 1.8 m, the
only additional instrument changes
required are to optimize the data rate for

Table I: Efficiency versus column length with different particle sizes

Column Length Particle Size Efficiency Typical Pressure* Analysis Time
Reduction (%)

150 mm 5 mm 12,500 86 bar -

3.5 mm 21,000 157bar -
1.8 mm N.A. - -
50 mm 5 mm 4200 44 bar 67%
3.5 mm 7000 62 bar 67%
1.8 mm 12,000 210 bar 67%
30 mm 5 mm N.A. 20 bar -
3.5 mm 4200 37 bar 80%
1.8 mm 6500 126 bar 80&
15 mm 5 mm N.A. - -
3.5 mm 2100 17 bar 90%
1.8 mm 2600 55 bar 90%
Chromatographic conditions:
Pressure determined with 60:40 MeOH: water, 1 mL/min. 4.6 mm id.
Room Temperature
Column only – optimized HPLC system adds approximately 60 bar to each pressure
 FEBRUARY 2005 FAST, FASTER, ULTRAFAST 23

accurate detection of these low-volume,
high-efficiency peaks.
Column recommendations for ultrafast
analysis: The high resolution shown here
is possible using high-throughput columns
with small, 1.8-m particle sizes. The
superior efficiency provides the resolution
of much longer columns, but the short col-
umn lengths, 50 or 30 mm, permit fast
analysis. The 4.6-mm i.d. columns are easy
to use and allow scaling down of current
conventional methods. With many
bonded phases available, methods easily
are matched or adapted as needed. Solvent
cost is saved due to the very short run
times. While 4.6-mm i.d. columns provide
ease of use, narrow-bore 2.1-mm
i.d.columns offer substantial advantages in
solvent costs and sensitivity. They are oper-
ated at lower flow rates and thus are com-
patible with many different detector
options, including UV detectors and mass
spectrometers. These columns with smaller
internal diameters also can be used for fast
and ultrafast analysis with optimized LC
systems.
UV detection: In Figure 16, a gradient
method with a conventional analysis time

High-pressure Standard assembly

gradient pump without standard mixer

400 mm x 0.12 mm capillary

High-speed well
plate sampler

200 mm x 0.12 mm capillary

Column oven
3-L
heat exchanger

70 mm x 0.12 mm

Column

300 mm x 0.1 mm capillary

Diode-array DAD equipped with a 500-nL flow cell

detector

80 mm x 0.125 mm capillary

600 mm x 0.13 mm PEEK

Mass
spectrometer

Figure 19: Agilent 1100 instrument configuration for 2.1 mm, 1,8 m column with DAD and
MS.
24 FAST, FASTER, ULTRAFAST FEBRUARY 2005

( 5 min) on a 2.1-mm i.d. short column
is shown with UV detection. This method
was converted easily to a very fast LC
method by increasing the flow rate. With
such a short column length, the flow rate
can be increased without exceeding the
pressure limit of the column or the instru-
mentation. The flow rate was increased by
a factor of 3. This resulted in a decrease in
run time by a factor of 4.5. Resolution still
is very good, and area and retention time
precision are comparable for both
applications.
Tryptic protein digests typically are pre-
cious and complex samples that are prefer-
ably separated on a small i.d. column for
higher sensitivity with the limited sample
size (14). Resolution of these samples and
other complex biological fluids can be
improved significantly using columns with
particle sizes less than 2 m. This is
demonstrated by a comparison of two
columns, differing in length and particle
size, on the analysis of a tryptic digest of
human serum albumin. The chro-
matograms in Figure 17 illustrate the
difference.
The first trace shows the results
obtained using a 150 mm  2.1 mm,
5-m column and gradient conditions.
Using a shorter 50-mm column with the
same stationary phase but with smaller
particles (1.8 m), higher resolution is
obtained in one-sixth the time, up to
500% faster, due to the combined effects
of using smaller particles and reducing the
column length by a third, increasing the
flow rate by a factor of 2.5 and reducing
the gradient time from 70 min to 10 min.
The resolution can be increased further by
increasing the gradient time from 10 to 30
min, while the analysis time still is less than
half that of the original method.
For flow rates below 1 mL/min and
2.1-mm i.d. columns, the optimum setup
is a little different. To optimize the stan-

1
7.0

6.0
TIC (x 106)

5.0

4.0 2

3.0

2.0

0.5 1.0 1.5 2.0 2.5

Time (min)

Figure 20: High-throughput LC–MS with rapid resolution HT columns. APCI analysis of Clin-
damycin and Lincomycin Column: ZORBAX Rapid Resolution HT SB-C18 30  2.1 mm, 1.8 m
Mobile Phase: Gradient: 15–50% B in 1 min, hold for 1.5 min, A: 0.2% formic acid pH, 2.8 B:
ACN  0.2% formic acid, Post time: 1.5 min, Flow Rate: 0.5 mL/min, Injection Volume: 1 L,
Temperature: Ambient, HPLC: Agilent 1100 with WPS and ADVR on, Detection: APCI, Positive
ion MS Conditions: Peak width: 0.10 min Scan: 150–600 Da, step 0.1 Fragmentor: 70, Gas
Temp: 350 ºC Vaporizer: 350 ºC Drying gas: 12 L/min, Nebulizer pres. 50 psi, Vcap 3000V,
Corona: 4.0 mA, Sample: 1. Lincomycin 2. Clindamycin.
 FEBRUARY 2005 FAST, FASTER, ULTRAFAST 25

dard LC configuration, short 0.12-mm i.d. accurate identification of close eluted com-
capillaries replaced the long 0.17-mm i.d. ponents possible, this combination is ideal
flow capillaries. As a mixing device a short to analyze unknowns.
0.12-mm i.d. capillary was used. The The analysis of antibiotics with MS
500-nL UV detector cell with appropriate detection using narrow-bore columns
inlet and outlet capillaries ensures opti- packed with 1.8-m packing material is
mum peak shape. shown in Figure 20 (15).
Figure 18 dramatically shows the influ- The separation of clindamycin and its
ence of the correct configuration on peak precursor product lincomycin was done
width and consequently on resolution with within 1.5 min, and the cycle time was
narrow bore, 2.1-mm i.d. columns. The 3 min. The mobile phase selected and the
required instrument modifications are flow rates of 0.5 mL/min are compatible
shown in Figure 19. with most LC–MS ionization techniques.
LC–MS detection: Narrow-bore Atmospheric pressure chemical ionization
columns are the ideal choice for LC–MS was chosen and provided good sensitivity
work. They provide excellent sensitivity at for these compounds.
lower flow rates. While the mass spectrom- The spectra of the two components
eter is chosen for quick and accurate iden- show predominantly the expected [M+H]+
tification of compounds, the high-resolu- ions for both lincomycin and clindamycin
tion column packing is especially critical to phosphate (Figure 21).
avoid ion suppression. By resolving ana- In fast and ultrafast HPLC, mass spec-
lytes from early eluted matrixes or excipi- trometers with different ionization and
ent components and obtaining the most detection processes can be utilized. Typical

4.0 Lincomycin spectrum 407.1 [M + H]+ Max: 403,456

Abundance (x 106)

1.45 min
3.0

2.0

1.0
160.1 176.1 219.1 391.1 423.0 519.0
355.0 371.1 437.1 577.0
0.0
200 300 400 500 600
1.2
[M + H]+ 505.1 Max: 115,512
Clindamycin phosphate spectrum
1.0
1.84 min
Abundance (x 106)

0.8 507.0

0.6
160.1 506.0
0.4 219.1
407.1
176.1
0.2 156.0
391.0 445.2 521.0
197.0 220.1 279.1 355.0 371.1 425.2 469.1 577.0 583.0
0.0
200 300 400 500 600
m/z

Figure 21: Mass spectra of clindamycin and lincomycin. High-throughput LC–MS with rapid
resolution HT column lincomycin MW  406.5 spectrum 407.1  [M + H]+, clindamycin phos-
phate MW  505 spectrum 505-506 [M+H]+.
26 FAST, FASTER, ULTRAFAST FEBRUARY 2005

100 100 100

279.1 285.0 311.1
80 80 80

60 60 60

40 1,2 40 3 40 4

20 270.0 20 20
221.0
311.1
0 0 0
200 400 600 200 400 600 200 400 600
m/z m/z m/z

5–95% acetonitrile in 0.5 min

600 1,2 600 Zorbax 15 mm x 4.6 mm, 1.8 m
Absorbance (mAU)

1,2
Absorbance (mAU)

400 400 3
3 4

200 4 200

0 0
0 0.4 0 0.1 0.2 0.3 0.4 0.5 0.6
Time (min) Time (min)

Cycle time of 1 min achieved by

• Short small particles column: 15 mm x 4.6 mm, 1.8 m SB-C18
• High flow rate: 4 mL/min
• Steep gradient: 55–95% organic in 0.5 min
• Coelution acceptance: Selected ion monitoring
• Overlapped injection: Save sample-draw and wash time
• Column regeneration: Save column regeneration time

Figure 22: Ultrafast, high-throughput LC–MS analysis of sulfa drugs.

examples are: analysis.

Single Quadrupole: ● High calibration stability
● Sensitive and secure molecular weight Typically, these detectors cannot be used
information for rapid screening pur- at flow rates above 1.5 mL/min without a
poses. loss in sensitivity. Some mass spectrome-
● Fast scanning for scan rates up to ters can suffer even at lower flow rates. If
5250 m/z/s. higher flow rates are used, a splitter must
● For qualitative/quantitative analysis. be installed in front of the mass spectrom-
● Highest sensitivity and accuracy in eter. In Figure 22, an example is given for
selected ion mode. the ultrafast analysis of sulfonamides using
● Special versions allow fast pos/neg a quadrupole mass spectrometer or an ion-
switching. trap mass spectrometer system (16). High
Ion Trap: flow rates were applied and a column-
● Fully automated and manual MSn to switching valve was used to switch between
characterize compounds and elucidate two identical columns. Having finished
structures. the run on the first column, the injection
● Special version for highest sensitivity & valve and the column selection valve were
ultrahigh scan rates up to 26,000 m/z/s. switched such that the next sample was
Time of Flight: injected immediately onto the second col-
● Highest resolution and mass accuracy umn. In the meantime, a second pump,
(
3 ppm) for secure identification providing the starting concentration of the
and characterization. gradient, regenerated the first column.
● Extremely fast duty cycles for ultrafast Applying this setup, the cycle time was as
 FEBRUARY 2005 FAST, FASTER, ULTRAFAST 27

low as 1.1 min. accuracy, only the amount of noise can-

In Figure 14, the instrument set up for celling due to summing fewer data points
such fast cycle times is shown. (Figure 23). As a result, TOF is the pre-
Influence of MS data acquisition rate: ferred mass analyzer for high speed
Increasing the acquisition rate for MS chromatography.
detection may have an impact on other Instrument recommendations for fast
performance factors. For example, resolu- and ultrafast analysis with narrow-bore
tion is sacrificed on quadrupoles when columns and UV or MS detection: With an
increasing the scan speed to m/z 5000/s. In optimized flow path (for example, as
TOF detectors, the key performance factor shown in Figure 19 with 0.12-mm i.d.
is mass accuracy. TOF detectors typically capillaries) a state-of-the-art HPLC instru-
acquire one scan or “transient” in 70 – 100 ment can be updated for ultrafast HPLC
s and add as many as possible given the or HPLC–MS using narrow bore columns.
chromatographic peak width. As a result, The minimized system delay volume takes
faster acquisition rates do not impact mass gradient changes right to the column and

TIC: from 20_Hz_hi_lo.wiff

Max. 1.9e5 cps
0.49
1.6 0.37
Intensity (cps x 105)

1.2

0.33 0.41
0.8
0.27
0.4

0.0
0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50 0.55 0.60
Time (min) Max. 1053.1 counts
108.0450 156.0116
1000

Fragmentor 225 V
Intensity (counts)

800
285.0209
600 307.0026

400

308.9998
200 287.0194
328.9839

100 120 140 160 180 200 220 240 260 280 300 320 340 360 380
m/z (amu)

Figure 23: Analysis of four sulfa drugs with LC–ESI-TOF instrument at 40 Hz scan rate. A:
Water  0.1% Acetic Acid, B ACN  0.1% Acetic, 25%B to 60% @ 0.4 min, flow 3 ml/min
5 ng/ul each of sulfamethazine, sulfadimethoxine, sulfachloropyridazine and sulfamethizole
4.6  30  1.8 m Zorbax SB-C18 flow split to MS 2:1, Fragmentor 175 (175 & 225), drying gas
13 L/min @ 325 C, nebulizer 60 psi, Transient length 70 usec, scan range m/z 100–1000, 141 tran-
sients/scan, dual fragmenter data, 212 transients/scan.
28 FAST, FASTER, ULTRAFAST FEBRUARY 2005
Sequence 1
Run Run Run
Figure 24: Automation model with a "Hypersequence.”
speeds up the analysis. The autosampler
delay volume must be taken out of the flow
path automatically after the sample has
reached the top of the column. This was
discussed previously as “automatic delay
volume reduction.” This contributes to
reduced run times and reequilibration
times because of the lower volume. MS
detectors with sufficient scan rates ensure
sufficient data to characterize fast-eluted
compounds properly.
Column recommendations for fast
and ultrafast analysis with narrow-bore
columns and UV or MS detection: Nar-
row-bore, 2.1-mm i.d. columns provide
enhanced sensitivity with smaller sample
sizes. These columns also are operated at
lower flow rates and are compatible with
both UV and MS detectors. When com-
bined with 1.8-m particles, high effi-
ciency can be achieved for fast analysis
with complex samples. Therefore, high-
resolution, fast analysis of complex samples
is an achievable goal.
How do you select columns and set col-
umn parameters?: Fast and ultrafast LC
can be done with the highest resolution
and highest speed on columns packed with
1.8-m particles. Choose short columns
Hypersequence
Sequence 2 ... Sequence x
Run Run Run Run Run Run
packed with the appropriate bonded phase
material and with small particle size, either
3–3.5 m or for greater efficiency, 1.8 m
Choose flow rates as high as appropriate
for optimal resolution. Increase column
temperature to reduce analysis time and
back pressure.
How to select the optimum instrument
setup: State-of-the-art HPLC equipment
ideally is suited for fast and ultrafast LC
without compromising performance or
flexibility using standard and narrow-bore
columns, both with monolithic as well as
columns with particles smaller than 2 m.
High-pressure mixing gradient pumps, fast
autosamplers with capacity extension, and
fast-responding detectors are recom-
mended. For UV detection, low volume
flow cells are recommended. For gradient
operations, the dwell volume should be as
low as possible.
Using 2.1-mm i.d. columns such as
those often recommended for LC–MS
applications, short 0.12-mm capillaries
help further reduce the delay volume, yet
still are compatible with the flow rates used
with ultrafast HPLC.
Data System Considerations for

Fast and Ultrafast Analysis
Fast run times and fast cycle times are the
first step to increase sample throughput. A
second step, of equal importance, is to
ensure that setup of automatic unattended
analysis of samples (sequences) and evalua-
tion of results do not become the limiting
step. These hints are meant to provide
examples of how intelligent data acquisi-
tion and evaluation software can help to
increase sample throughput. The topics
discussed are related to sequencing, inte-
gration, customized reporting, data stor-
age, and archiving and data transfer for
further data evaluation.
Acquisition setup: The setup of
sequences of samples typically involves a
set of information like vial number,
method name, injection volume, sample
type, data file names, sample names, and
more, which are added into the sequence
lines. It is convenient for users if they can
use names as long as needed and configure
Baseline Example
Classical
Classical
No penetration
(Classical)
Advanced
Figure 25: Improved integration for automatic baseline correction.
FEBRUARY 2005 FAST, FASTER, ULTRAFAST 29
their own sequence table. It is also an
advantage if for further transfer of data to
a database, the appropriate study name can
be filled in.
Once a sequence has been set up and
started, it also is convenient to add more
sequences for more sample sets without the
need to wait for the end of the started
sequence. Using a so-called “hyper-
sequence” you can do this (17). A hyper-
sequence contains several sequences
aligned with their samples and also man-
ages the correct analysis of all samples. The
sequence itself contains all parameters that
are needed to perform the single runs. An
add-on of new samples and sequences
should be possible at any time. In Figure
24, the schema of such a hyper-sequence is
shown.
The use of integrated barcode readers
allows safe linking of sample and results
and automatic selection of sequences based
upon barcodes. It also is useful and saves
Description
1. Standard baseline tracking
2. Using timed events
1. Search for penetration
2. Move start–end of peak until
no penetration is left
1. Removal of baseline penetrations
2. Improve start–end of a peak
3. Reestablish the baseline for
a cluster of peaks
30 FAST, FASTER, ULTRAFAST FEBRUARY 2005

time if a hyper-sequence table can be
imported as a CSV file, for example, cre-
ated from Microsoft Excel.
Data evaluation — chromatogram
integration: In the past, most users looked
at the obtained chromatograms, at the
integration results, and took appropriate
action if the integration and baseline set-
ting were not appropriate. In the mean-
time, due to extreme workload and more
and more high throughput applications,
manually examining all chromatograms
and results has become a limiting factor for
fast applications. Therefore, it is necessary
for the integrator algorithm to be designed
such that even with a difficult baseline, the
automated integration is appropriate. In
Figure 25, a comparison is made between

Table II: Column and instrument recommendations for high-throughput analysis

Module Cycle time Cycle time Cycle time Cycle time
< 1min < 5min > 5min > 10m
Column 15, 30 or 50mm length, 50mm length, 75mm length, > 75mm length,
4.6mm id, 1,8µm 4.6 mm id, 4.6 mm id, 1,8 µm 4.6 mm id
particle size 1.8 µm particle size or 3.5µm 1.8, 3.5 or 5 µm
particle size particle size
Flow As short as possible As short as possible Standard length Standard length
capillaries with internal diameter with internal diameter with internal with internal
of ~0.17µm of ~0.17µm diameter of diameter of
~0.17 µm ~0.17 µm
Pump High pressure gradient High pressure gradient High pressure High pressure
pump with optimized pump with optimized gradient pump gradient pump
delay vol. for fast delay volume for with optimized or low pressure
gradient operation fast gradient operation delay volume for gradient pump
fast gradient
operation
Auto sampler High speed sampler High speed sampler High speed sampler High speed
(ALS) with flow through with flow through with flow through sampler or
design for fast design for fast design for fast vial standard
injection and injection and injection and auto sampler
cleaning cycles cleaning cycles cleaning cycles
ALS ALS should be ALS should be Extension is Extension is
capacity equipped with equipped with typically not a typically not a
extension capacity extension capacity extension must must
Column Thermostat with Thermostat with Thermostat with Thermostat with
compartment Peltier heating Peltier heating Peltier heating Peltier heating
and cooling for fast and cooling for fast and cooling for fast for optimum
method changes and method changes and method changes temperature
highest temperature highest temperature and highest stability
stability stability temperature stability
UV detector UV detectors with UV detectors with UV detectors with UV detectors
fast data rates fast data rates standard cells with standard
(min 20Hz) and (min 20Hz) and and long path flow cells
with low-volume with low-volume length for and long path
flow cells and flow cells and highest sensitivity length for
long path length long path length highest
for lowest peak for lowest peak sensitivity
dispersion and dispersion and
highest sensitivity highest sensitivity
Mass MS with fast scan rates MS with fast scan MS with moderate MS with
spectrometer and typically with flow rates and typically scan rates and moderate scan
splitter, if flow rate is with flow splitter, typically with flow rates and
>1ml/min if flow rate is splitter, if flow rate typically with
> 1ml/min is > 1ml/min flow splitter, if
flow rate is
> 1ml/min
Recommendations for 4.6mm id columns packed with stationary phases of particle size between 1.8 and 5µm. If 2.1 mm
id columns are used for cycle times less than 5 minutes the flow capillaries should be about 0.12mm id to minimize
peak dispersion.

different integration algorithms.
A chromatogram is shown in which
automated tracking of a baseline is not
easy. Baseline corrections are necessary
because baseline penetration occurs and
most peaks are on an upslope of the base-
line. Penetration is where the constructed
baseline crosses the real signal and gives
negative areas, particularly if the user has
set the “baseline at valley” timed event.
Classical integrators cannot handle these
Figure 26: Customized report.
FEBRUARY 2005 FAST, FASTER, ULTRAFAST 31
penetrations, and the user must spend
valuable time to set more integration
events to ensure proper baseline tracking.
Modern integrators have new functions
or options in which first a standard base-
line is found and at the same time, baseline
penetrations are detected and advanced
baseline tracking occurs.
Most recently, the user can determine
whether or not these new options should
be set. When this option is set, the peak
32 FAST, FASTER, ULTRAFAST FEBRUARY 2005
cluster will be searched for baseline pene-
trations. The start and end of the peak are
shifted toward the top of the peak until
there is no penetration left.
Furthermore, the integrator will try to
improve the start and end location of a
peak and reestablish the baseline for a clus-
ter. In many situations, this will give a bet-
ter and more stable (less dependent upon
the slope sensitivity setting) baseline. The
better the automated integration of chro-
matograms is, the less interaction of the
user is needed. One can have more trust in
the correctness of automated created base-
line, and there is no need to look at any
chromatogram.
Data reporting generation, archiving,
and transfer to a LIMS: For high through-
put analysis, hundreds and hundreds of
reports must be created. The need here is
to get only the amount of information that
is important for judging the results and for
making the right decisions. In most cases,
report generation must be customized
according to the needed report content
Figure 27: Tool bar of the LimsLink CDS worksheet, showing that all six samples have been
retrieved.
and format.
State-of-the-art data-evaluation software
offers both standard reports and also the
possibility to generate customized report
templates, which are then used for all fol-
lowing applications.
In Figure 26, an example of a cus-
tomized report, based on an Agilent
(Wilmington, Delaware) ChemStation
Plus report generation tool is given.
Results of all data files of a sequence are
combined. The report includes statistical
and quantitative data for the calibrated
peaks, names of used study and applied
method and data, as well as the chro-
matograms of the single runs (18). To
make data management easier, the
acquired data are transferred to a database
and are stored in studies or other data-
management tools. This allows tracking of
different sequences with, for example, dif-
ferent applications on different working
days.
These data-management tools allow a
central and secure repository for organiza-

tion, review, approval, and storage of chro-
matographic data. Safe archiving of raw
data, chromatograms, and results is pro-
vided using these enhanced software tools.
Furthermore, laboratories also are faced
with the requirement to accurately and
efficiently transfer data from the chro-
matography data system (CDS) to a LIMS.
An example of such a data-handling sys-
tem is the Agilent Enterprise Content
Management (ECM) tool. The Agilent
ECM is the umbrella system that encom-
passes all of Agilent’s application-specific
software solutions. ECM stores all elec-
tronic records in a central location, regard-
less of their format. ECM transfers key
information from proprietary formats to a
generic format, facilitating review and
management of data through a central
browser interface, independent of the ini-
tial acquisition format of the data. ECM is
the answer to data storage, data organiza-
tion, and compliance needs.
ECM is fully web-based and scalable
from single laptop solutions to enterprise
system implementations for many users.
Its functionality combines the solution to
long-term archiving needs with the power
of a document management system. It pro-
vides full versioning and audit trails for all
human readable documents and reports,
including the MS Office applications
Word and Excel. It also provides a special
plug-in that enables Excel calculations to
run in a compliant fashion with restricted
access and template protection, as well as
revision control and audit trails of the cal-
culation templates.
Typically, the interface between the
CDS and LIMS automatically queries the
CDS at specified time intervals for
approved sample results. Any results that
have been approved by CDS since the time
of the last query are collected automatically
FEBRUARY 2005 FAST, FASTER, ULTRAFAST 33
and transferred to the LIMS. Typically, a
LIMS toolbar is used to display the current
status of the reporting process, showing
information such as the number of
approved samples found in CDS, how
many of those samples have been reported,
or how much time remains until the LIMS
will query the CDS again for approved
results (19,20). An example of such a tool
bar is given in Figure 27.
Recommendations for data handling:
Ultrafast LC can generate as many as a few
hundred chromatograms overnight. This
requires considerations of how to handle
the huge amount of data. HPLC systems,
which come as complete solutions, are best
suited for fast and ultrafast LC in routine
analysis. This solution should include
sophisticated sequencing and integration
of data as well as customized reporting.
Advanced data management with storage,
archiving, and data transfer to a LIMS
must be available.
Conclusions
This primer has examined many ways to
be more productive using optimized con-
ventional, fast, and ultrafast HPLC. All of
these methods are achievable in every lab,
and the resulting improvements in produc-
tivity can be realized easily in your day-to-
day work. This is accomplished using cur-
rently available improvements in column
technology, HPLC instrumentation, and
data system technology. This includes:
New 1.8-m particle size column tech-
nology is available in a variety of column
dimensions, including 4.6- and 2.1-mm
i.d. columns, which enable ultrafast analy-
sis with run times down to less than 40 s.
The combination of advanced, 1.8-m
particle size columns and high-speed,
high-capacity HPLC equipment (such as
high-pressure mixing pumps, high-speed
34 FAST, FASTER, ULTRAFAST FEBRUARY 2005
autosamplers, and optimized detectors)
enables cycle times
60 s for ultrafast LC
in 24  7 operation.
The combination of these elements with
fast, sophisticated data acquisition and reli-
able evaluation enables rapid and reliable
data review of huge amounts of chro-
matograms and results.
Table II provides a summary view of the
recommendations that have been pre-
sented in this primer. In the table, recom-
mendations are given as to what instru-
ment combinations will provide best
results for different cycle times. The table is
related to 4.6-mm i.d. columns. If 2.1-mm
i.d. columns are used for cycle times
5
min, the flow capillaries should have an
internal diameter of 0.12 mm to minimize
peak dispersion.
As a result, the methodology enables up
to 20 higher throughput. The column
and detector technology plus the mini-
mized dispersion are key factors to achieve
in addition up to 2 the resolution and an
increased sensitivity.
References
(1) A.D. Jerkovich, J.S. Mellors, and J.W. Jorgen-
son, Recent developments in LC Column Tech-
nology, Supplement to LCGC Eur., June 2003,
pp. 20–23.
(2) B. Yan, J. Zhao, J.S. Brown, J. Blackwell, and
P.W. Carr, Anal. Chem. 72, 1253–1262
(2000).
(3) J.D.Thompson and P.W. Carr, Anal. Chem.
74, 4150–4159 (2002).
(4) D. Lubda, K. Cabrera, W. Kraas, C. Schaefer,
D. Cunningham, and R.E. Majors, LCGC
Eur. 14(12).
(5) G. Rozing, Recent Developments in LC Column
Technology, Supplement to LCGC, June 2004,
pp. 12–17.
(6) J.D. Loyd, “Advances in HPLC: Monolithic
Silica Columns,” http://bama.ua.edu/~chem/
seminars/student_seminars/spring03/papers-
s03/Loyd-s03.pdf, University of Alabama,
Mobile, Alabama, 2003.
(7) A.M. van Nederkassel, A. Aerts, A. Dierick,
D.L. Massart, and Y. Vander Heyden, J.
Pharm. Biomed. Anal. 32(2), 233–249 (2003).
(8) R.E. Majors, LCGC 21(12), 1124–1133
(2003).
(9) Y. Yang and D.R. Lynch Jr., Recent Develop-
ments in LC Column Technology, Supplement
to LCGC, June 2004, pp. 34–39.
(10) A.D. Broske, R.D. Ricker, B.J. Permar, W.
Chen, and M. Joseph, Agilent Appl. Note
5988-9251EN, http://www.agilent.com, Agi-
lent Technologies, Wilmington, Delaware,
2003.
(11) M. Stahl, Agilent Appl. Note 5988-9914EN,
Agilent Technologies, Wilmington, Delaware,
2003.
(12) W.E. Barber and M.J. Joseph, Agilent Appl.
Note 5989-0540EN, Agilent Technologies,
Wilmington, Delaware, 2004.
(13) A. Fandino and S. Schuette, Agilent Appl.
Note 5989-1603EN, Agilent Technologies,
Wilmington, Delaware, 2004.
(14) W.E. Barber, Agilent Appl. Note 5989-
0542EN, Agilent Technologies, Wilmington,
Delaware, 2004.
(15) W.E. Barber and M.J.Joseph, Agilent Appl.
Note 5989-0026EN, Agilent Technologies,
Wilmington, Delaware, 2003.
(16) M. Stahl, Agilent Appl. Note 5988-9638EN,
Agilent Technologies, Wilmington, Delaware,
2003.
(17) M. Stahl, Agilent Appl. Note 5988-9863EN,
Agilent Technologies, Wilmington, Delaware,
2003.
(18) A. Gratzfeld-Huesgen, Agilent Appl. Note
5988-9340EN, Agilent Technologies, Wilm-
ington, Delaware, 2003.
(19) Agilent Brochure 5989-1384EN, Agilent
Technologies, Wilmington, Delaware, 2004.
(20) S. Bolton and U. Bober, Agilent Appl. Note
5988-8962EN, Agilent Technologies, Wilm-
ington, Delaware, 2003. 
Agilent’s HPLC & MS purification
Your first great discovery will be the system itself
The Agilent 1100 Series Purification Platform
Combines ruggedness, >97% purity, >98% recovery
and 24/7 operation. This is made possible by:
• Dedicated systems for lowest peak dispersion
• Precise and automated delay volume measurement
• Integrated system intelligence with real time data
processing
• ”Smart” fraction collection based on time, peak
and/or mass
u.s. and canada: 1-800-227-9770
www.agilent.com/chem/purification
©Agilent Technologies, Inc. 2005 MC5272
Agilent’s 1100 Series Purification Platform
has been enhanced with even more features
for you to discover. This includes signifi-
cant hardware and software innovations
for the highest purity and recovery, rugged
hardware for unattended 24/7 operation,
and a new dual loop autosampler PS for fast
injection of high sample volumes. What’s
more, the ChemStation Data Browser
provides high-throughput analysis and
purification data. It’s all accessible to you
with a wide range of software solutions
providing the option to configure a system
specific to your needs. In short, Agilent’s
1100 Series forges an unparalleled union
of reliability, safety and performance.
To learn more, Agilent offers a compre-
hensive, free e-seminar series with topics
for all levels of users. Register now on
www.agilent.com/chem/education
 Catalog No.
5989–2108EN