Buenas Prácticas de HPLC Agilent

Best Practices for
Efficient Liquid
Chromatography
(LC) Operations
F E B R U A RY 2 0 1 9
Making the Most of Your LC System

Mia Summers and Andreas Kugler
Taking Advantage of LC Column Characteristics

Anne Mack
Making Good LC Methods Even Better

William Long
Sponsored by
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© Agilent Technologies, Inc. 2019

INTRODUCTION
G
etting better, faster, more reliable results is a top-of-mind concern at busy
laboratories, many of which are often seeking new ways to increase efficiency.
What new techniques are available for ensuring the most reliable results and
more efficient laboratory operations?
This ebook on Best Practices for Efficient Liquid Chromatography (LC) Operations covers some
straightforward techniques that could immediately increase efficiency in the laboratory.
First, Mia Summers and Andreas Kugler describe how proper preventative maintenance can
avoid many common problems, and a methodical approach to dealing with them can reduce
the time and difficulty involved in diagnosing and fixing issues. This covers everything from
proper column selection to issues with autosamplers and injectors to signs of contaminated
solvent.
Next, Agilent Technologies’s Anne Mack reviews analytical method development challenges
and explores the importance of keeping the objectives of the separation in mind during method
development. She also describes which physical characteristics must be considered when
choosing an LC column, including particle pore size, particle technology, and the actual particle
size itself.
Readers will also learn from William Long, also of Agilent Technologies, how with existing
methods, simple adjustments, such as incorporating new column technologies, can often
result in enhanced resolution and better peak shape and ensure that the method will perform
reproducibly and provide reliable—even improved—results.

Diagnosing LC Method
Best Practices for Efficient Liquid
Problems Columns Development Chromatography
Images under license from stock.adobe.com

S P ONS OR E D C ONTE NT
Making the Most of

Your LC System
Mia Summers and Andreas Kugler
Overview system contamination, unexpected peaks,

Although modern liquid chromatography and repeat analysis.
(LC) is a mature technology, problems can Signs of a contaminated solvent can in-
still arise during analysis. When challenges clude ghost peaks, baseline drift, climbing
occur, it can be extremely frustrating. backpressure, frequent degasser failures,
This can be especially true when deal- or unusually short column lifetimes. Of
ing with system failures such as leaks, those, ghost peaks are often the first sign
bubbles, and clogs. Moreover, diagnos- of solvent contamination. They show up
ing chromatographic problems can be a as peaks that seem to move around the
time-consuming experience. Fortunately, chromatogram or appear and disappear.
proper preventative maintenance can Ghost peaks can be a result of low-level
avoid many common problems, and a mobile phase or sample-related impurities
methodical approach to dealing with them that build up in the system and are eluted
can reduce the time and difficulty involved inconsistently at different times, some-
in diagnosing and fixing issues. times triggered by the injection solvent
or gradient conditions. They can also be
The Mobile Phase negative peaks caused when a continuous
Having clean mobile phase is essential for baseline of a contaminant in the mobile
ensuring the LC system runs reliably and phase is interrupted by the injection of a
reproducibly. sample that offsets the wavelength detec-
While it may be tempting to cut corners, tion of the background contaminant.
using high-performance liquid chromatog- Some ghost peaks can be attributed to
raphy (HPLC) or mass spectrometry (MS)- the method conditions rather than sys-
grade solvents will save laboratories time tem or mobile phase contamination. The
and money in the long run by reducing most likely cause is that the run time is
4 FEB R U A RY 2 0 1 9 | L C G C SPO N SO R ED CO N TEN T

too short for the sample, and some late filter membrane material is compatible
eluting components from each injection with the solvent. If mobile phases contain-
are showing up in subsequent chromato- ing dissolved solids or salts have been
grams. When all components in a sample run through the LC system, then flushing
mixture are not with water after the
properly eluted off analysis is completed
the column, these “Small particles in the will be necessary
components remain mobile phase can cause to ensure they are
on the column for removed. Simply
slow and inconsis- serious problems for switching to a differ-
tent elution over LC components, from a ent solvent system
subsequent injec- for the next analysis
tions. If analysts malfunctioning degasser could result in precip-
suspect this is the to damaged pump seals or itation. When switch-
case, they should ing solvent systems,
wash the column even pistons. ” it is also important to
with a high percent- maintain miscibility at
age of the organic component of a re- all times. It is best practice to step to an
versed-phase mobile phase and then flush intermediate solvent that is miscible with
the column with several blank runs with mobile phases being used to the desired
method conditions. Finally, a single injec- mobile phase. Keep in mind that the pres-
tion with an extended run time should be ence of salt can change miscibility. For
performed to verify that all the peaks are instance, water and isopropanol (IPA) are
eluted within the programmed run time. miscible when pure, but not in high salt
Small particles in the mobile phase can concentrations.
cause serious problems for LC compo- Another source of particulates are mi-
nents, from a malfunctioning degasser crobes growing in the aqueous mobile
to damaged pump seals or even pistons. phase bottles. In multi-solvent systems, it
A common source of particulates comes is customary to mix aqueous and organic
from contamination of buffer salts, or components at the time of analysis us-
precipitation due to inattentiveness to ing an integrated solvent proportioning
solvent conditions. When using buffers on valve. This simplifies the analyst’s work,
a gradient system, one should never drive but increases the risk of microbial growth
the organics so high that the buffers are in the aqueous mobile phase bottle and
no longer soluble. line. Solutions with buffers may be an
Any mobile phase component prepared even more microbe-friendly environment.
with dissolved solids should be passed Microbial particulates cannot always be
through a filter with 0.45-μm pores or seen. They can collect in the inlet frit of
smaller. Care should be taken that the a column, causing high backpressure

and ghost peaks. They are also often There are times when one must flush
smaller than the column inlet frits pore out some or all of the system for non-use,
size, passing through and collecting onto storage, or troubleshooting. If salts are
the column bed. Again, an inline filter present in the system, then water is the
will help, but microbial growth can be obvious first choice to dissolve salts and
pervasive throughout an LC system once remove them from the system. Once the
introduced and difficult to remove after presence of salts can be ruled out, then
the fact. To avoid the challenging trouble- IPA works well as a final flush for the
shooting of microbial particulate, it is best system. It is miscible with most common
to take steps to prevent introduction into LC solvents, both normal and reverse
the system. Adding a small amount of phase. Its relatively high viscosity and low
organic to the aqueous mobile phase (and surface tension make it a good choice
adjusting the method conditions slightly for dislodging bubbles. It has a low vapor
to compensate) can prevent microbial pressure, which means components will
growth. Protecting the mobile phase from not dry out as quickly. It will also suppress
light using amber bottles will inhibit the microbial growth.
growth of cyanobacteria. Even with these
steps, it is a good idea to change out any
aqueous mobile phase with fresh water in
The Pump
a clean bottle every couple of days. When- Many of the problems frequently encoun-
ever the system is left untended, flush the tered with LC systems can be traced to
water line with the organic solvent used the pump. Indeed, pumps are expected
in reversed-phase methods. The degasser to provide a continuous and steady flow
is another area prone to microbial growth of liquid at hundreds of bar pressure. The
if left with water in it. Always be sure the pressure produced by the pump should be
lines are not stored in 100% aqueous and consistent. Monitoring a system’s pres-
stored in organic solvent when not in use. sure provides a valuable diagnostic tool.
Solvent evaporation will change the A steadily climbing back pressure is often
composition of a premixed mobile phase, an indication that a module component
in addition to being a safety concern. is being fouled, usually by particulates in-
Properly closed solvent bottles will troduced into the LC system. Particulates
prevent this, but the bottle must not be can also show up as frequent failures of
completely sealed to avoid vacuum form- the degasser, or premature column death.
ing. This can be as simple as a cap with As mentioned, the mobile phase is the
a hole drilled through it just big enough most common culprit, but once the pres-
for the line, or ideally a commercially sure has begun to climb, removing the
designed cap with an integrated filter obstruction becomes another priority.
and vent system, such as InfinityLab Stay When tracking the source of increased
Safe Caps by Agilent. back pressure, start downstream at the
detector. Because the column is typically

the strongest contributor to the system’s are reliable and steady most of the time,
back pressure, it is always a challenge to and can now be run remotely once set up.
differentiate between the column being Sometimes, other signals are the first sign
the root cause or other parts in the system. of a pump problem. If the detection base-
For troubleshooting, therefore, the column line becomes noisy or retention times
always should be removed and replaced start to shift noticeably, it is time to check
by a “restriction capillary” (e.g., Agilent p/n on the pump.
5022-2159 Stainless Steel Capillary, 0.12 Pump problems can most commonly be
mm ID, 2000 mm length). This capillary traced to one of three common causes:
provides a certain back pressure for reliable bubbles in the pump, pump seals, or the
troubleshooting. It is pump valves. An air
always good to know “Pump problems can bubble can typically be
what is the normal back most commonly be fixed by purging with an
pressure range of each organic solvent. Flushing
system. Disconnect tub- traced to one of three the system to remove air
ing to components mov- common causes: bubbles is best done with
ing steadily upstream IPA due to its high viscos-
toward the degasser bubbles in the pump, ity and with a restriction
until the backpressure pump seals, or the capillary installed to create
drops. The segment that a certain back pressure.
causes the pressure pump valves. ” The column should, of
to drop back to normal course, be uninstalled.
when disconnected is the culprit. Modern LC pumps offer special instrument
Over a series of runs, the system pres- settings (piston stroke) or even automated
sure should not change drastically, although flushing or purging procedures that can be
a small ripple (sometimes called “delta”) used. Care should be taken not to exceed
is normal. Advanced LC systems, such system pressures when using or switching
as the Agilent InfinityLab LC series, allow solvents to/from IPA. In a multi-line systems,
analysts to monitor the pump ripple. While flush all the lines simultaneously. This action
the ripple will vary from system to system may solve the problem.
and method to method, a drastic change Once air bubbles are ruled out, if system
or deviation from the expected value is problems persist, one can examine the seals
a strong indication of a problem with the or valves for wear and tear or damage. A
pump. As a best practice, it is good to be dirty seal or malfunctioning valve often mani-
familiar with and document the expected fests as unusual pressure ripple affecting
pressures for your LC system to quickly baselines, or unexpected lower or higher
identify any deviations. system pressures and shifting retention
Changes in pressure can sometimes go times. Salt buffers, harsh solvents, and
unnoticed. After all, modern LC pumps pH extremes can be hard on seals, which

FIGURE 1: Traditional pump head.
may require more frequent replacement. shown in red. It is a good idea to establish
While requiring some disassembly, it is a regular replacement schedule instead of
a relatively quick job to replace a seal or waiting for a problem to occur, especially in
valve on most systems. Many manufactur- a QA/QC environment where the instrument
ers provide pump head maintenance kits is under heavy use and system downtime
with all the replaceable components and must be kept at a minimum. Seals come in
detailed instructions. It is important to use different materials. Be sure to use a material
the proper supplies that are designated for that is compatible with your solvents.
the instrument or pump module. Quick Ref- The purge valve is a very useful tool for
erence Guides can offer quick identification flushing and priming the pump. The Agilent
of parts that go with specific modules. 1200 series LC systems have a purge
Figure 1 is a diagram of a traditional Agi- valve containing a PTFE frit that protects
lent HPLC system pump head. The seals are the rest of the system from particulates,

FIGURE 2: Replacing PTFE frits in the purge valve.
which is a good feature to protect your Also known as a gradient valve or solvent
LC investment. However, over time, this selection valve, it will fail over time and
PTFE frit can become clogged. When may need to be replaced. As with the
purging the system, most of the pressure degasser, flushing with water after us-
drop will be across this frit. If the pressure ing mobile phases with buffer salts will
exceeds 10 bar with water at 5 mL/min, it lengthen the lifetime of the valve. One
is time to change the frit. The frit is easily way of determining if the selection valve
accessible and quick to replace (see Fig- is the source of the problem is to premix
ure 2). As with the pump seals, it makes the mobile phase solvents used in an
more sense to replace the frit on a regular isocratic method and bypass the valve and
basis rather than wait for a problem. directly connect into the pump head. Use
As mentioned, shifting retention times only one line or pump head at a time to
can result from problems in the pump, confirm a standard separation. Moving the
but if the pump appears to be functioning solvent source then from one solvent line
properly (and the current pump settings or pump head to another is a good way to
match the current type of solvent), the is- isolate the problem.
sue could be with the proportioning valve.

FIGURE 3: Injection valve.
The Autosampler and Injector needle seat is replaced, it will instantly

Autosamplers and injectors have become become damaged by the old needle and
complex pieces of equipment that can be vice versa. In case of blockages at the
the source of some chromatographic is- needle and needle seat, it is important to
sues. A poor seal between the needle and check for the root cause. Debris could be
needle seat can cause carryover, resulting released from some other part upstream
in ghost peaks or irreproducible quantita- (e.g., vial caps or plate seals).
tion. The needle seat and
needle should be seen as S P ONS OR ’ S C ONT E NT
one “functional” piece and

in case of trouble, must Agilent InfinityLab: Mak-
always be replaced at the
ing Great Connections
same time. A damaged
Less stress, more
needle can damage the
reliable fittings
needle seat; if only the

Fitting
FIGUREConnections
4: Correct and incorrect fitting connection.
Bad Ferrule cannot seat properly→ leak Poor Fitting Connections

• will broaden or split peaks or cause tailing
• will typically affect all peaks, but especially
early eluting peaks
Too long
• can cause of carry-over
Bad Mixing chamber→ dead volume
mAU
140
One bad connection!
120
100
80
60
Too short 40
20
Good 0
0 0.1 0.2 0.3 0.4 min
Properly fitted tubing, no dead volume
mAU
210 Fixed!
180
150
120
90
60
30
0
0 0.1 0.2 0.3 0.4 min
January 29, 2019

1
Figure 3 shows exploded and cutaway voids. But, it can also result from having
views of an injection valve. The rotor seals incorrectly sized capillary tubing. The small-
will wear over time or be damaged by est diameter and shortest tubing suitable
particulates. Worn rotor seals can cause for the system should be used. Capillary
sample carryover and irreproducible injec- tubing that is too long or has an inner
tions and should be replaced. They come in diameter (ID) that is too wide can cause
a variety of materials. Analysts should be extra-column volume, which will contribute
sure to choose the correct material for the to the broadening of the peaks before they
solvent and/or pH. Instrument manufactur- even reach the column.
ers will often provide kits containing all the Different manufacturers may use different
consumable parts for autoinjector modules types of fittings. Using the wrong fitting
in a single package. can damage the instrument or result in
leaks or tailing peaks. The most common
The Column are Swagelok-style with a 10-32 thread and
Poor efficiency might be traced to poor col- one or more tapered ferrules. When install-
umn choice or an aging column developing

FIGURE 5: Agilent InfinityLab Quick Connect and Quick Turn Fittings.
ing a fitting for the first time, the tubing tion technology avoids these problems.
should be pushed all the way in before For instance, the Agilent InfinityLab Quick
tightening the fitting. Otherwise, the gap Connect Fitting addresses these issues
will create peak broadening, usually in the with a spring load function that automati-
form of consistent tailing of peaks, even cally adapts to the receiving port depth of
those that are less retained. Figure 4 the column (see Figure 5).
shows correct and incorrect installation of When experiencing problems, it is
fittings. However, newer column connec- also critical to examine the column con-
nection fitting because a
damaged fitting will also
S PO N SO R’S CO N T EN T
impair the receiving port
of the new column. When
Agilent InfinityLab Stay standard stainless-steel fit-
tings are used, use a new
Safe Caps – Reduction capillary when changing
of Solvent Evaporation to a new column because
either the new column will

have a slightly different swage depth these cases, the column may need to be
or the worn fitting on the old capillary replaced with a new one. Routine preven-
will damage the new column fitting. In tative maintenance on the system and the
addition, take care not to overtighten addition of sample preparation techniques
the column connection fittings (typically such as filtering samples or solid phase
the standard stainless steel fittings). Ap- extraction may be considered to reduce
plying excessive force can damage the costly troubleshooting and downtime of
fittings and the column receiving ports the LC system.
and cause leaks. Users might then try to
apply even more force to achieve a leak
tight connection. Sooner or later, the
The Detector
column will get replaced, either because There are only two components likely to
the leak persists or simply because the require attention in a UV detector: the lamp
column reached the end of its lifetime. and the flow cell. Most lamps contain deu-
Again, new removable fittings like the terium gas, which will leak over time. Since
Agilent InfinityLab Quick Connect Fit- method response is based on the ratio of
tings address these issues with features peaks, a weakening bulb may not be obvi-
like the spring load function to automati- ous at first, however the baseline noise will
cally adapt to different port depths and appear to grow relative to the peaks over
have the ability to replace ferrules in time. Poor detection signal or a noisy base-
case of damage. line are good signs that the lamp is due for
A variety of issues can arise with the replacement. When changing the lamp, be
column itself. One issue that was already sure to avoid touching the bulb.
mentioned is that columns can develop Flow cells are generally maintenance
voids through rough handling or age. If the free, but problems can arise. If using a
peaks coming out on or near the void are second detector downstream, be cogni-
broad or tailing, and extra-column broad- zant of the additional back pressure it cre-
ening has been ruled out, it may be time ates. Too much back pressure in the first
to change the column. High backpressure flow cell can rupture it. For these applica-
that is not attributed to the system may tions, high-pressure cells are available.
be due to column contamination if the Cells can also become fouled and must be
column has been heavily
used or many insoluble S P ONS OR ’ S C ONT E NT
components such as for-

mulation excipients have
Sample Preparation
been injected onto the col- Whitepaper
umn. Microbial contamina- Make the most out of your
tion can also build up on
LC system. Is your “good
enough” sample preparation
the column bed. In all of really good enough?

flushed clean.
Newer technologies with RFID tags
(such as Agilent InfinityLab flow cells and
Mia Summers
lamps) can help track usage and preventa- Former LC Columns
tively plan replacement to keep systems Product Manager
at maximal uptime and improve lab opera- Agilent Technologies
tional efficiency.
Conclusion
Several common problems can arise while
using an LC system, however, good main-
tenance and preventative measures will Andreas Kugler
help systems run for years without major Product Support Engineer
LC Supplies
problems. Avoiding microbial growth; Agilent Technologies
regularly replacing valves, seals and frits;
making proper column connections and
maintaining good mobile phase practice
will go a long way toward avoiding annoy-
ing, time-consuming, and expensive sys-
tem downtime. It is also helpful to have
some staff members trained specially for
troubleshooting. Customer training (such
as basic and troubleshooting classes) will
help them understand the special func-
tionalities and operation principles of the
instrument as well as how to troubleshoot
problems, reduce downtime, detect po-
tential problems upfront, and ensure the
instruments operate correctly.

Taking Advantage of LC Column
Problems Columns Development Characteristics
Taking Advantage
of LC Column
Characteristics
to Improve Analyses
Anne Mack
C
hromatographers make numer- phers have many different chromatographic
ous decisions during the liquid modes to choose from, the choice of which
chromatography (LC) method one to use can be overwhelming. However,
development process, many of since approximately 80% of all HPLC sepa-
which are important for developing a robust rations are performed by reversed phase, a
analysis. Column choice is one of those good end-capped C18 phase is a good place
decisions and is a significant factor for a suc- to start. Method transfer is another chal-
cessful analysis. This manuscript will explore lenge. Many companies have laboratories all
various column features and illustrate how over the world and must transfer methods
choosing the appropriate LC column can between them and require that the method
improve chromatographic results, including perform as well as it did in the destination
improved resolution and peak shape via lab as it did in the original lab. In addition,
contract laboratories—which are widely
alternate selectivities, the impact of mobile
used in the pharmaceutical industry—may
phase composition and pH, and extend col-
have many different HPLC instrument
umn lifetime.
brands. This diversity can complicate the
method transfer process because systems
from different vendors can have widely
Analytical Method Development Chal- different system volumes, tubing and fit-
lenges and Defining Objectives ting differences, and many other different
Today, several challenges can make high- operational characteristics. Consequently,
performance liquid chromatography (HPLC) a way to simplify the method development
method development more confusing than process is desirable.
ever. One significant challenge is choosing Before actually initiating the method de-
the mode of chromatography and the right velopment process, the objectives of the
HPLC column to use. Since chromatogra- separation should be addressed. Items that

FIGURE 1: Common separation goals and performance criteria.
should be considered include: side of Figure 1 lists method goals com-

• How many peaks are likely to be present? mon to most applications, many of which
• Will it be easy or challenging to separate are often included as system suitability test
the peaks? requirements. Resolution is one of the more
• Are efficiency and speed (run time or important criteria in method development,
inject-to-inject cycle time) important? especially when using nonspecific detectors
• What kind of instrumentation is available? like ultraviolet, evaporative light scattering
• Can mobile phase gradients be used? detector, or fluorescence. A resolution of at
• Are pressures above 400 bar available? least 2 is preferred, but at a resolution of 1.5,
• Who is going to run the routine samples? two adjacent peaks are considered baseline
What is their level of training? resolved. System suitability parameters are
These questions, and certainly others, can established during method validation, where
help decide the method requirements and method performance parameters, shown
how much effort should be put into develop- in the right side of Figure 1, are evaluated.
ment. When setting system suitability parameters
In addition to method objectives, it is and criteria, it is good to avoid criteria that
important to have a few goals in mind, limit the ability to modify the method in the
depending upon the application. The left future. For example, if efficiency or absolute

FIGURE 2: Efficiency, selectivity, and retention factor are variables that

affect resolution, with selectivity having the greatest impact
retention times are used as system suit- drive resolution. The scatter plot in Figure
ability criteria, a modification of the method 2 shows the effect that each of these
may require a full revalidation. three variables have on resolution. The al-
pha term, or selectivity, has a far greater
effect on resolution than efficiency and
Method Development: retention. Therefore, during method
Where To Begin? development, changing selectivity will
Once method development goals and ob- have the greatest impact on the separa-
jectives have been determined, method tion to achieve the desired resolution.
development begins by taking advantage Alpha or selectivity is changed by altering
of the resolution equation, shown in the the chemistry of the system, either the
upper right of Figure 2. As seen in Figure mobile phase (composition or pH), the
2, efficiency (N), selectivity (alpha), and column bonded phase (e.g., C18, C8,
retention factor (k') are the variables that phenyl-hexyl, or cyano), or both. Column

temperature can also affect alpha, espe- Superficially porous particles are an
cially if acids or bases are present. alternative new technology that offers
substantial increases in efficiency and
peak capacity over columns packed with
Physical Characteristics older, conventional 5-μm particles and can
of HPLC Columns be used with existing conventional HPLC
Many physical characteristics must be systems. It is important to recognize
considered when choosing an LC column that superficially porous particles are not
before initiating method development, magic; they obey the same chromato-
including particle pore size, particle technol- graphic principles as 5-μm particles so the
ogy, and the actual particle size itself. same method development processes
It is important to select the optimal pore can be used. Superficially porous particle
size for any HPLC analysis application. shave a solid core limiting diffusion to the
Smaller, 80–120-Å pore size column pack- outer shell. Figure 3 illustrates why these
ings are used for the separation of smaller particles are more efficient; shorter diffu-
molecules (i.e., molecular weights of less sion distances result in less peak broaden-
than 4,000). Smaller pore size columns will ing and higher efficiency (plates) while
also maximize loading capacity and reten- enabling higher flow rates/faster analysis
tion. The slightly larger 120-Å pore size is a times without compromising efficiency.
great choice for peptide mapping, and 300-Å The choice of particle size is somewhat
pore size columns are used for larger mole- driven by the complexity of the separation
cules like intact peptides and proteins. Even and therefore the resolution required. In
larger pore sizes of 1,000–4,000 Å are used general, smaller particle sizes are more ef-
for very high molecular weight proteins. ficient. However, this efficiency comes with
In the earlier days of liquid chromatog- a price: Because pressure is proportional to
raphy, particle sizes were well above 10 the column length over the square of the
μm. It was not until the early 1970s when particle size, higher system back pressures
small, totally porous silica particles were will be encountered as the particle size
made specifically for HPLC. In the 1980s, decreases. Superficially porous particles
5-μm particles were introduced, and in are available in three different particle sizes:
the 1990s, 3.5-μm particles made faster 1.9 μm, 2.7 μm, and 4 μm. The choice of
separations possible. A column particle particle size depends on the separation
technology that has become popular within requirements and the available instrumenta-
the last 10 years are the sub-2 μm totally tion. The overall diameter determines the
porous particles used for ultrahigh-perfor- pressure, so superficially porous particles
mance liquid chromatography (UHPLC) operate at pressures similar to the corre-
applications. However, sub-2-μm particles sponding totally porous particle (TPP) par-
require specialized instrumentation to take ticle size. The thickness of the porous layer
full advantage of their capabilities. determines the column efficiency: use of

FIGURE 3: Particle Technology: Superficially porous particles improve

mass transfer, resulting in higher efficiency, when compared to totally
porous particles
superficially porous particles can result in of undiluted freshly brewed green tea.
efficiencies up to 200% of a conventional
5-μm TPP.
HPLC Column Bonded Phase Options
In general, 1.9-μm superficially porous Different bonded phases emphasize differ-
particles are used for the highest UHPLC ent chemical interactions, so that the sepa-
performance, 2.7 μm for UHPLC perfor- ration conditions for a specific sample can
mance at lower operating pressures, and 4 be fine-tuned by choosing the appropriate
μm for improved HPLC performance. bonded phase. Polar versus non-polar com-
Another advantage of the 2.7-μm and pounds will behave differently with different
4-μm particles is that the larger overall size column chemistries. In cases of pi-bonding
means that 2-μm frits can be used at the in the analytes (e.g., aromatic compounds),
column inlet, the same as those used in a a phenyl-hexyl phase can be useful because
5-μm column, so the column is much less of the enhanced pi-pi interactions. Besides
susceptible to blockage than sub-2 μm par- improving selectivity, a change in the
ticle columns, as demonstrated in Figure 4 bonded phase can also sometimes reduce
for more than 1,600 injections of samples analysis time.

FIGURE 4: Long lifetime with dirty samples on 2.7 μm InfinityLab Poro-

shell 120 column
C18 and C8 bonded phases are a great often yielding unique selectivity for polar
first choice for method development. They compounds. Cyano phases can be used for
work great from pH 2 to 9. Endcapping, or both normal as well as reversed-phase sepa-
de-activation of Si-OH groups leftover on the rations and bare silica phases can be used
silica surface of a column during the bond- for hydrophilic interaction chromatography
ing process, can also affect selectivity. Many of very polar molecules. Pentafluorophenyl
C18 and C8 bonded phases are available phases are also often used as an orthogonal
both endcapped and non-endcapped and phase for polar compounds.
can be evaluated during method develop- To gain a more detailed understanding of
ment. Phenyl-hexyl is another excellent the selectivity of different chemistries, bond-
alternative phase that can be used to take ed phase characterization tests are avail-
advantage of pi–pi interactions with an ana- able. Examples of the characterization of
lyte and the stationary phase. The selectivity reversed-phase chemistries are the Tanaka
with phenyl–hexyl phases are very similar to Test and the Hydrophobic Subtraction Mod-
phenyl or diphenyl columns. Other phases, el (HSM). These tests classify chemistries
like those that have a polar group embedded according to various parameters and can
in the bonded phase, are also a good choice be used to calculate a similarity factor (Fs)

FIGURE 5: Separation of 8 Steroids with Methanol Gradient – Having a

choice of column phases enables easier method development
between two columns. A small Fs indicates eight steroids are baseline separated with
that two columns are very similar, while a a shorter run time than any of the other
large factor indicates that two columns are phases. There is no guarantee that any
very different. When developing methods, of the phases will separate all the com-
it is useful to evaluate columns that provide pounds in all samples without additional
different or orthogonal selectivity to improve work, but as demonstrated in Figure 5,
the separation. selectivity is affected with a change in sta-
An example of how column selectivity tionary phase, which is very helpful early
can be used to enhance a separation is in the method development process.
shown in Figure 5 for the separation of
eight steroids using a methanol gradient Altering Selectivity
mobile phase. With many of the phases, with Mobile Phase pH
changes in selectivity can be seen. In Mobile phase pH can also affect the reten-
addition, a reasonable separation is ob- tion and selectivity of ionizable compounds.
tained, but some peaks are not resolved. Many newer phases can operate over a
On the phenyl–hexyl phase, however, all wide pH range (e.g., 2–10), so pH can now

FIGURE 6: Varying mobile phase pH can offer very different selectivity,

shown by the poorly correlated retention times of various analytes at pH
3 and 10
be used to drive selectivity and improve tered data points represent compounds that
method development by improving both changed ionization state from pH 3 to 10,
retention and resolution. Figure 6 illustrates affecting their retention.
how an Agilent InfinityLab Poroshell 120
HPH-C18 2.7-μm column was used to
compare selectivity at both pH 3 and pH 10
for several compounds. The scatter of the
data points in this plot shows how much pH
can effect selectivity. The correlation coef-
ficient, R2, here referred to as the indicator
of orthogonality, is very low at 0.40. Non-
ionizable compounds, or those that do not
ionize over the range of 3–10, elute on the
light blue line, positioned at y=x. The scat-

FIGURE 7: Injection volumes contribute to overall system volume, and

therefore must be kept small to preserve the performance of high-efficiency
columns like Agilent InfinityLab Poroshell 120 1.9 μm columns.
Maximizing the Effect of The data collection rate is how often the
High-Efficiency LC Columns detector is recording information; lower data
rates generate artificially broad and short
To maximize the effect of high-efficiency
peaks, which decrease efficiency and reso-
LC columns, one must consider data col- lution. A general rule of thumb is to use a
lection rates, injection volumes, and the data collection rate that collects a minimum
effect of guard columns and fittings on of 20 data points across the chromatograph-
system dispersion. ic peak. Since high-efficiency separations
result in narrower peaks, data
S PO N SO R’S CO N T EN T rates must be increased pro-
portionately. Data rates as high
Agilent LC Column as 10 to 20 Hz or more are
Navigator commonly used when using
Easily find the best column high-efficiency LC columns.
for your analysis by method Injection volumes contribute
parameters, USP method, or to overall system volume,
compound. and therefore must be kept

small to preserve the performance of Conclusion

high-efficiency columns. Figure 7 shows Choosing a column from the wide selection
how decreasing injection volume, even of new particle technology can play a major
while maintaining the same mass load role in method development. The wide vari-
on the column, dramatically increases ef- ety of phases available to affect selectivity,
ficiency, even for later eluting compounds. and the use of pH as a selectivity tool—
Since low system dispersion is necessary while keeping in mind a few simple param-
to maintain the high efficiency of superfi- eters to maximize the use of these new
cially porous particle columns, it is important high-efficiency columns—allows the quick
to use sub-2-mm particle guard columns that and efficient development of new methods
are specially designed for use at high pres- or the update of existing methods.
sures especially for samples with complex
or “dirty” matrices, even when using an up-
front sample preparation. Analytical columns
are very expensive to replace, while guard
columns are not. Therefore, it is often a
good idea to use a guard column to extend Anne Mack
the life of the analytical column. When se- Applications Scientist
lecting a guard column, look for one that is
Agilent Technologies
compatible with the analytical column phase
and does not impart additional extra-column
volume to the system.
One common problem that surfaces when
installing LC columns involves the fittings
used to connect the column into the instru-
ment. Improperly swaged fittings with col-
umns can result in leaks, extra-column dead
volume, poor chromatography with broad
and tailed peaks and a lack of resolution. Dif-
ferent vendors use different swage depths,
different threading, and different ferrules, so
fittings should not be interchanged among
systems. Agilent InfinityLab fittings are de-
signed to properly seat against the column
inlet every time. They can be hand-tightened
for use with pressures up to 1,300 bar so
that the tubing is always properly swaged,
and used with many different manufactur-
ers’ instruments.

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recommend the best columns and supplies to optimize your results.
Find out more at www.agilent.com/crosslab

Diagnosing LC Method Making Good LC Methods Even Better
Problems Columns Development
Making Good LC
Methods Even Better
William Long
M
any existing liquid chro- Common performance metrics to define
matography (LC) methods are listed in Figure 1. Sufficient resolution
warrant a fresh look to (Rs) and reproducibility are fundamental.
ensure that they are Retention factor (k’) should be taken into ac-
yielding optimal separations and to see count. For example, if k' is too low, changing
if new column technologies or other solvent or increasing injection volume can be
modifications can be applied for improved useful, but later-eluting peaks with high k’ val-
results. Often, simple adjustments, such ues become broad and difficult to detect. The
as incorporating new column technolo- signal-to-noise (S/N) ratio generally should be
gies, can result in enhanced resolution greater than 10 and, ideally, closer to 100.
and better peak shape and ensure that Avoid defining column efficiency (theoreti-
the method will perform reproducibly and cal plate, N) or absolute retention time as
provide reliable—even improved—results. system suitability criteria because these
Many existing methods also can be made factors are easily altered and may prevent
“mass-spec-friendly” with relatively easy achieving a faster method if constrained.
tweaks.
Scouting Gradient
Separation Goals and Method Criteria Running a scouting gradient is an excellent
The first step in assessing an LC method is starting point for method development or
to establish separation goals and method for adjusting a known method. John Dolan,
performance criteria. General factors to LCGC Troubleshooting columnist, recom-
consider when setting goals are the impor- mends a gradient of 5–95% organic solvent
tance of efficiency and speed, the com- using a C18 column and low pH. Gradient
plexity of the sample, instrumentation avail- time can be varied depending on the column
able, and the skill set of the operator(s). length and particle size.1

FIGURE 1: Common separation goals and performance criteria.
The gradient scouting run will help deter- phase buffers. An experiment was carried
mine whether an isocratic or a gradient out separating a group of nonsteroidal
method is preferable. If the peaks are anti-inflammatory drugs using sub-2-μm
spread out over the gradient range, a rapid-resolution high-throughput columns
gradient method is indicated. If peaks are (Agilent ZORBAX StableBond [SB-C18,
clustered and elute in a narrow organic Eclipse Plus C18, Bonus-RP, and the
range, an isocratic method will likely be Eclipse Plus Phenyl-Hexyl) with three typ-
sufficient. Running a fast gradient, such ical low-pH buffers (0.1% trifluoroacetic
as a 15-min gradient on a shorter, effi- acid [TFA], 0.1% formic acid, and 0.1%
cient column, may indicate that additional acetic acid), ammonium acetate as a mid-
method time is either needed or wasted. level buffer, and water for column rinsing.
Scouting gradients were used starting at
4% MeCN for the SB-C18, 8% MeCN for
Column and Buffer Screening the Eclipse Plus C18, and 0% MeCN for
Column and buffer screening can be the Bonus-RP and Eclipse Plus Phenyl-
carried out by running scouting experi- Hexyl for a total of 20 combinations.
ments using various short, fast columns Nine of the 20 experiments resulted
in combination with different mobile in baseline resolution of all compounds,

FIGURE 2: Column choices and buffer screening for a separation

of analgesics.
but, as shown in Figure 2, the Eclipse modifiers. StableBond with acetic acid re-
Plus C18 clearly demonstrates the highest sulted in coeluting peaks, and results were
minimum resolution at 3.288. Further opti- marginal with formic acid. Phosphoric and
mization could be carried out with just a few TFA both yielded good separations. To further
experimental runs to reduce the run time. optimize the separation, TFA was increased
to 1.0%, which reduced peak tailing.
In summary, a scouting gradient followed
Selecting Mobile Phase Additive by simple adjustments to the mobile phase
and pH often results in arriving at improved
Choice of mobile phase additive can vary
separations quickly.
peak shape and position. A separation of six
components found in salicylic acid production
was run on an Agilent ZORBAX StableBond Method Robustness
C18 column using phosphoric acid, formic A method may be quite good in some cases,
acid, TFA, and acetic acid as mobile phase but show unfavorable results with minor

FIGURE 3: Nortriptyline and dipropyl thalate run on the Eclipse Plus and
a C18 column from an existing method.
changes in conditions. Ideally, a minimum of 2.05 for the separation, the other two
resolution of 2.0 should be maintained for the columns showed somewhat lower resolu-
method to ensure complete separation of tion: 2.03 and 1.99. One objective was to
peaks. Often, small tweaks can boost mini- ensure that the method would perform with
mum resolution and, thus, improve method a minimum resolution of 2.0, so slightly more
robustness. water was added to the mobile phase and
A separation of alkyl phenones was run on acetonitrile was reduced from 65% to 60%.
an InfinityLab Poroshell EC 4.6 mm x 50 mm, With this slight adjustment, resolution in-
2-μm C18 column at flow rates from 1.0 mL/ creased to 2.55, 2.54, and 2.56 for the three
min to 2.0 mL/min. Pressure increased, as columns, ensuring a robust method.
expected, from 99 bar to 204 bar, but a rea-
sonable resolution of 2.05 was maintained
throughout the runs. Taking Advantage of
The separation was carried out on three New Column Technology
different columns to test method robust- Testing newer technologies, such as new
ness. While one column yielded a resolution columns, is another productive avenue for

FIGURE 4: Separation of sulfa drugs scaled from a 5-μm Zorbax to a 2.7-

μm InfinityLab Poroshell 120 column.
improving methods. For example, Agilent resulted in better sensitivity, sharp peaks,
has developed new surface treatments for better resolution, and a flatter baseline.
C18 columns to reduce peak tailing and Agilent has developed an InfinityLab
also to improve their stability with high pH Poroshell column called “HPH” (high pH
without changing selectivity. The develop- stability). This column takes advantage of
ment of superficially porous columns, such using changes in pH to control selectivity in
as the InfinityLab Poroshell 120 EC-C18 and a separation. An offshoot of this column is
Eclipse Plus, also yields the advantages of the InfinityLab Poroshell HPH C18 column,
lower pressure, higher performance, and less which lasts longer in phosphate buffer than
sample preparation. previous columns and maintains excellent
Figure 3 illustrates a simple preparation performance. Even methods involving com-
of a customer’s sample of nortriptyline and plex samples can be switched to this column
dipropyl thalate run on the Eclipse Plus and with minimal adjustments while maintaining
another C18 column using their method and similar selectivity and with the added benefit
a 25 mmol, pH 7.4 phosphate buffer. The of excellent lifetime at mid-pH ranges.
improved bonded phase of the Eclipse Plus Rotor seal considerations. When operating

FIGURE 5: Allowable method adjustments for isocratic and gradient sepa-

rations according to the US Pharmacopeia.
using high-pH mobile phases, some parts of option for method improvement. Figure 4
the instrument may break down. The rotor shows a separation of sulfa drugs scaled
seals found in injector valves and on some from a 5-μm Agilent ZORBAX Eclipse Plus
other valves are made of a polyimide material column to a 2.7-μm InfinityLab Poroshell
(Vespel) and are susceptible to attack at pH 120 column. All 10 peaks elute in about 7
levels greater than 10 by compounds such as min on the InfinityLab Poroshell, which is
ammonium hydroxide or ammonium carbon- before the first peak elutes on the longer
ate. PEEK rotor seals can be used in place of column. Sensitivity is improved, and, even
Vespel if high-pH mobile phases will be used though less material is injected, the S/N
for extended periods. ratio is higher, resulting in sharper, com-
pressed peaks, which are ideal for further
optimization. No change in sample prepara-
Improvements Using tion was required, and both separations
Superficially Porous columns can be run at pressures less than 400 bar.
Simply changing from a totally porous col- Many compendial methods, such as those
umn to a superficially porous column, such in the U.S. Pharmacopeia (USP), can poten-
as InfinityLab Poroshell 120, is another tially be upgraded. The USP provides guide-
lines on allowable adjustments (Figure 5). For

isocratic methods, if the column length (L) to totally porous column. The 2.7-μm column
particle diameter (dp) ratio is kept to within is a workhorse that provides UHPLC per-
–25% to +50% of the original or if the num- formance at substantially lower pressures.
ber of plates (N) is within that ratio, no revali- These columns are now available in 1000 bar
dation is needed. This flexibility in method and show typical pressures that are about
adjustment is allowed as long as linear veloc- 50% of a sub-2-μm totally porous particle
ity is kept constant. Injection volume also can column and provide about 90% of the effi-
be changed to meet detection limits. ciency with no additional sample preparation
Very few changes are allowed for gradient needed. The 1.9-μm column gives extremely
methods without revalidation; however, the high UHPLC performance and pressures
USP currently is investigating possible allow- similar to those of sub-2-μm totally porous
able adjustments. particle columns and 120% of the efficiency.
Simple Scaling of Methods Converting LC Methods to LC–MS

The USP method for naproxen was scaled up
Mass spectrometry (MS) is widely used for
from a 5-μm totally porous material (Agilent
the analysis of polar to very nonpolar com-
ZORBAX Eclipse Plus) to a 2.7-μm superfi-
pounds. Electrospray ionization (ESI) can be
cially porous material (Agilent InfinityLab Po-
used in MS for both high and lower molecu-
roshell 120). Values for the standard method
lar weight compounds and is, thus, suitable
were N = 10,639, Rs = 13.7, and an L/dp ratio
for diverse samples. ESI should be avoided
of 30,000. Changing the method to a 4.6 mm
with more nonpolar samples because charge
x 100 mm column resulted in almost a 100%
induction will be inefficient and not much
improvement in N, but the L/dp ratio only
signal will be produced. Atmospheric pres-
23%, which meets the USP requirements for
sure photoionization (APPI) or atmospheric
an allowable method adjustment. A shorter
chemical ionization (APCI) can be used for
column (4.6 mm x 50 mm) column was tried,
these compounds.
which increased N from 10,639 to 11,281 and
ESI is used widely In many cases, iden-
maintained similar resolution as described by
tifying unknown compounds is a goal of
the method (Rs > 11.5).
method improvement. And, because LC–MS
Poroshell particles are scalable across the
equipment has become easier to use and
family. Agilent recommends
specific column pore sizes S P ONS OR ’ S C ONT E NT
for different types of applica-
tions. For a simple improve-
ment in HPLC performance, The LC Handbook: Guide
a 4-μm column might be to LC Columns and
sufficient and typically yields Method Development
about a 200% improvement
in performance over a 5-μm

is more readily available in laboratories, it is tive mode ionization

desirable to develop methods that are more o 0.1– 1% formic acid, 0.1–1% acetic
MS-friendly. acid, 0.05–0.2% TFA
o ammonium salts favor formation of am-
monium adducts
Method considerations for ESI-MS are:
o TFA causes ion suppression
• pH of the mobile phase (and analyte pKa)
o use a TFA “fix”—postcolumn addition
affects ion formation and, therefore, signal of acetic or propionic acid
• voltage applied to the electrospray probe • basic mobile phases favor negative mode
will induce ion formation ionization
• choosing the best mobile phase pH can
improve sensitivity
• organic solvent has little effect on ionization, A separation of 10 compounds found
but it affects evaporation, so more volatile in green tea was run using a phosphoric-
mobile phases can be advantageous acid-based mobile phase with acetonitrile
• ESI works best at flow rates less than 0.5 (Agilent ZORBAX SB-C18, 4.6 mm x 150
mL/min mm column). The separation was accom-
plished nicely within about 15 min on a 5-μm
• ESI is compatible with reversed-phase,
column. Phosphoric acid is not MS-friendly,
hydrophobic interaction, and normal-phase
however, and a method goal was to increase
HPLC
the speed of the separation.
By running the separation on an Agilent
Buffer considerations for ESI include: InfinityLab Poroshell 120 SB-C18 column,
• buffer concentrations below 25 mM are which has comparable selectivity, the analy-
advisable to avoid signal-dampening effects sis was accomplished at about 60% of the
(best below 10 mM) backpressure. The same type of the selectiv-
• ESI has poor compatibility with non-volatile ity could be achieved using 0.2% formic acid,
buffers due to deposit buildup and metal 0.2% acetic acid, 0.02% TFA, or an ammoni-
ion buffer interference with ionization um formate buffer adjusted with formic acid
could be used and get similar separations.
• acidic mobile phases generally favor posi- The MS sensitivity was significantly affect-
ed by these choices. Examining
S PO N SO R’S CO N T EN T one particular peak as a means
of evaluating the separations
revealed that using acetic acid
Automated LC Method gave S/N = 155 versus 68 using
Development Webinar TFA, and the buffered mobile
phase gave only S/N = 33, so
Series
acetic acid was a good choice
in this case, and the method

FIGURE 6: Transfer of an existing method for antibiotics to MS using a) a

methanol mobile phase, b) an acetonitrile mobile phase.

could be optimized quickly. increased to 0.42 mL/min to optimize the

Data collection rate should also be con- method for MS detection.
sidered in optimization. Fast data collection This method was successfully scaled to a
yields the narrowest peaks, resulting in op- 3 mm x 50 mm column, which can be used
timal peak capacity, but it also means more for UV or MS detection. The separation is fast
baseline noise and reduced S/N sensitivity. and uses much less solvent than the original
Optimal S/N is achieved at slightly slower method.
data collection rates, but at the cost of
reduced peak capacity. Data collection rate,
therefore, should be optimized according to Conclusion
the goals of the particular method. Most laboratories will benefit from review-
Developing a new method suitable for ing their LC methods to determine if simple
MS is another approach. An existing UV adjustments can be made to improve chro-
method for the separation of antibiotics used matographic results or to make methods
to treat animals showed, for example, 10 MS-friendly. Column choice is a significant
compounds that were nicely separated. The factor in successful analyses, and newer
method, however, used a very high concen- column chemistries often can be applied for
tration of phosphoric acid, and the separa- improved resolution and better peak shape
tion had a run time of 35 min. Because an through taking advantage of alternative selec-
equivalent column was not available, a new tivities.
method was developed beginning with run-
ning generic gradients of 10% to 40% meth-
anol or acetonitrile. The use of a short 4.6 Reference
x 50 mm, 2.7-μm InfinityLab Poroshell 120
1. J. Dolan, LCGC 31(1), 30-35 (2013).
EC-C18 column and UV detection allowed
for rapid screening of different mobile phase
combinations with formic acid, ammonium
formate, acetic acid, and ammonium acetate
as mobile phase buffers.
As shown in the methanol runs in Figure
6a, peaks shifted substantially depending on
the type of buffer used. Peak shifting in the
acetonitrile runs (Figure 6b) was less and
pressure was much lower. pH was adjusted
to vary elution order and peak spacing. The William Long
best mobile phase combination was found LC Columns
to be pH 3.8 ammonium formate with Applications Scientist
acetonitrile, which resulted in a good separa- Agilent Technologies
tion of these 10 compounds. Flow rate was

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Best Practices for

Making the Most of Your LC System

Taking Advantage of LC Column Characteristics

Making Good LC Methods Even Better

Learn more about ensuring the best analytical outcomes:

© Agilent Technologies, Inc. 2019

more efficient laboratory operations?

straightforward techniques that could immediately increase efficiency in the laboratory.

reproducibly and provide reliable—even improved—results.

Images under license from stock.adobe.com

Making the Most of

Overview system contamination, unexpected peaks,

4 FEB R U A RY 2 0 1 9 | L C G C SPO N SO R ED CO N TEN T

5 FEB R U A RY 2 0 1 9 | L C G C SPO N SO R ED CO N TEN T

6 FEB R U A RY 2 0 1 9 | L C G C SPO N SO R ED CO N TEN T

7 FEB R U A RY 2 0 1 9 | L C G C SPO N SO R ED CO N TEN T

FIGURE 1: Traditional pump head.

8 FEB R U A RY 2 0 1 9 | L C G C SPO N SO R ED CO N TEN T

FIGURE 2: Replacing PTFE frits in the purge valve.

9 FEB R U A RY 2 0 1 9 | L C G C SPO N SO R ED CO N TEN T

FIGURE 3: Injection valve.

The Autosampler and Injector needle seat is replaced, it will instantly

one “functional” piece and

10 FEB R U A RY 2 0 1 9 | L C G C SPO N SO R ED CO N TEN T

Bad Ferrule cannot seat properly→ leak Poor Fitting Connections

January 29, 2019

11 FEB R U A RY 2 0 1 9 | L C G C SPO N SO R ED CO N TEN T

FIGURE 5: Agilent InfinityLab Quick Connect and Quick Turn Fittings.

12 FEB R U A RY 2 0 1 9 | L C G C SPO N SO R ED CO N TEN T

components such as for-

13 FEB R U A RY 2 0 1 9 | L C G C SPO N SO R ED CO N TEN T

14 FEB R U A RY 2 0 1 9 | L C G C SPO N SO R ED CO N TEN T

15 FEB R U A RY 2 0 1 9 | L C G C SPO N SO R ED CO N TEN T

FIGURE 1: Common separation goals and performance criteria.

should be considered include: side of Figure 1 lists method goals com-

16 FEB R U A RY 2 0 1 9 | L C G C SPO N SO R ED CO N TEN T

FIGURE 2: Efficiency, selectivity, and retention factor are variables that

17 FEB R U A RY 2 0 1 9 | L C G C SPO N SO R ED CO N TEN T

18 FEB R U A RY 2 0 1 9 | L C G C SPO N SO R ED CO N TEN T

FIGURE 3: Particle Technology: Superficially porous particles improve

19 FEB R U A RY 2 0 1 9 | L C G C SPO N SO R ED CO N TEN T

FIGURE 4: Long lifetime with dirty samples on 2.7 μm InfinityLab Poro-

20 FEB R U A RY 2 0 1 9 | L C G C SPO N SO R ED CO N TEN T

FIGURE 5: Separation of 8 Steroids with Methanol Gradient – Having a

21 FEB R U A RY 2 0 1 9 | L C G C SPO N SO R ED CO N TEN T

FIGURE 6: Varying mobile phase pH can offer very different selectivity,

22 FEB R U A RY 2 0 1 9 | L C G C SPO N SO R ED CO N TEN T

FIGURE 7: Injection volumes contribute to overall system volume, and

23 FEB R U A RY 2 0 1 9 | L C G C SPO N SO R ED CO N TEN T

small to preserve the performance of Conclusion

24 FEB R U A RY 2 0 1 9 | L C G C SPO N SO R ED CO N TEN T

Find out more at www.agilent.com/crosslab

© Agilent Technologies, Inc. 2018

26 FEB R U A RY 2 0 1 9 | L C G C SPO N SO R ED CO N TEN T

FIGURE 1: Common separation goals and performance criteria.

27 FEB R U A RY 2 0 1 9 | L C G C SPO N SO R ED CO N TEN T

FIGURE 2: Column choices and buffer screening for a separation

28 FEB R U A RY 2 0 1 9 | L C G C SPO N SO R ED CO N TEN T

29 FEB R U A RY 2 0 1 9 | L C G C SPO N SO R ED CO N TEN T