Starting Out Right, Parti-Selecting The Tools: Feature Index Home Main Index

Thu Aug 30 15:28:35 2001.
updated 09/18/2000
LC-GC - December 1999
Feature Index - Home - Main Index
Starting Out Right,

Part I -
Selecting the Tools
This first part of a multipart series tells you how to
put your best foot forward and maximize your
chances for a successful separation.
John W. Dolan
LC Troubleshooting Editor
Liquid chromatography (LC) problems tend to fall into

one of two categories. The first type of problem is
associated with instrumentation. Problems with check valves, mixers, detector lamps, and
other hardware can be irritating and tedious to isolate, but the solution usually is simple
after the source has been identified. Many instrument problems can be prevented through
disciplined preventive maintenance. The second type of problem is associated with the
separation itself. Poor peak shape, inadequate separation, and drifting retention are some
examples of separation problems. Unfortunately, the symptoms of separation problems can
be obvious, but identifying the problem source can be difficult and correcting it can seem
impossible.
One of the key factors in minimizing separation problems is to start out with a good method.
By using a good method, it is much easier to maintain operation within specifications and to
correct problems when they arise. For the next few months, “LC Troubleshooting” will focus
on the components that make up a good separation and discuss how to put them into
action. The staff at my company has been teaching chromatographers these skills in short
courses for more than 25 years. Over time, a fairly simple approach to method development
emerged - we use this approach in our courses and contract laboratory - and I will use it as
the core of this series.
OPEN YOUR CUPBOARD
If you were about to cook a gourmet dinner, you wouldn’t just walk into the kitchen and start
cooking. First you’d consult a recipe, check your supplies, and go shopping to obtain the
ingredients. You’d make sure the pots and pans were clean and the knives were sharp.
Sure, you may need to cut some corners and make some substitutions, but you would use
the right ingredients and follow the recipe to get the best results. The same approach holds
true for developing an LC method. You need specific tools and reagents to obtain the best
results. If you start developing a method without proper planning, you may get an adequate
separation but the likelihood of a robust method that is tolerant of small changes is less than
if you had used a systematic approach.
COLUMN SELECTION
One of the most important choices in method development is column selection.

Chromatographers can choose from hundreds of columns, so where do you start? One tool
I use to keep tabs on trends in column usage is Ron Majors’ annual Pittsburgh Conference
review in his “Column Watch” columns in the March and April issues of LCGC each year.
Since 1984 (1), Ron has shared information about new columns, the changes in column
technology, and column usage habits.
For example, most separations performed today use reversed-phase columns, and C8 and
C18 phases are the most popular of these. Chromatography, however, is not just a
popularity contest. The reason why these two phases are so popular is that they obtain the
desired separation in most cases. Although some standard sample types don’t point to
these columns as the first choice (see Table I), the C8 or C18 columns usually offer the best
stationary phase with which to start. There’s not enough difference between the C8 and
C18 phase for me to strongly recommend one over the other - I prefer C8 for most
applications, but I’ll leave that choice up to you.
TABLE I: Alternative Sample and Method Combinations

Sample Characterstics Preferred HPLC Method or Column
High molecular weight Special columns usually required;
size-exclusion and ion-exchange high
performance liquid chromatography
(HPLC) often preferred
Optical isomers Special chiral columns required
(enantiomers) present
Other isomers Normal-phase often best, especially
(stereoisomers or position with unmodified silica
isomers) present
Mixtures of inorganic salts Ion chromatography
Carbohydrates Amino bonded-phase columns with
reversed-phase conditions; ion-
exchange resins
Biological samples Special conditions often required for
life-science samples; may not require
different approach
Most samples encountered by average chromatographers will be amenable to reversed-

phase separation; however, some compound types likely will require another LC method for
successful separation. The sample and method combinations listed in Table I are
suggested starting points. For more information on these samples, see reference 2 for a
general treatment of method development.
The production of low-metal, high-purity silica is perhaps the most important practical
column development in the last 15 years. Detailed descriptions of the silica surface are
available elsewhere (2,3). These publications describe the traditional chromatographic silica
as a heterogeneous, acidic surface. After attaching the bonded phase to the surface, the
resulting columns could be characterized by tailing peaks for basic analytes and often
lower-than-desired column-to-column reproducibility.
Recent advances in silica production have resulted in a nearly metal-free silica with a very
homogeneous distribution of low -acidity surface silanol groups. The practical result of these
developments is columns that are more stable, more reproducible, and produce much better
peak shapes. This improved silica often is called Type B silica. Because of the benefits of
these phases, most manufacturers prominently distinguish these with names touting their
superiority such as Inertsil (GL Sciences Inc., Tokyo, Japan), BDS (base-deactivated silica,
Hypersil Inc., Needham Heights, Massachusetts), Symmetry (Waters Corp., Milford,
Massachusetts), and YMC-Basic (YMC, Inc., Wilmington, North Carolina). Most column
manufacturers make a Type B silica column, so check with your favorite column supplier for
the one you should use. Because of the superiority of these phases, I strongly recommend
that you set aside your older, Type A phases and start every new method development
project with a Type B phase.
COLUMN SIZE
Another important choice is the column dimensions and packing particle diameter (dp ). The
most popular particle size is 5 -µm dp, but 3.0 - and 3.5-µm particles also are widely used.
Any of these particles are suitable for starters, but most chromatographers still prefer the 5 -
µm particles because of their long usage history. Smaller particles produce columns with
higher plate numbers as well as higher back pressure. The 3.0-µm particles are packed with
smaller frits that are prone to fouling, so some manufacturers produce 3.5-µm particles that
enable the use of traditional 2.0-µm frits, which are less susceptible to fouling.
I prefer the 150 mm X 4.6 mm column size because it generates a sufficiently high plate
number ( N) with 5-µm particles to obtain adequate separations with most samples. An
alternative configuration is the 75 mm X 4.6 mm, 3.5-µm dp column, which provides
separations similar to those of the 150-mm long, 5-µm dp column but in half the time. An
added benefit of either of these column configurations is that the flow rate can be set at 1.5-
2.0 mL/min with reasonable back pressures (for example, less than 2000 psi). Higher flow
rates mean shorter run times, and as the saying goes, “time is money.” Columns as long as
250 mm generate a few more plates, but the benefit is marginal ( N grows only with the
square root of the length increase) and the penalties are longer run times and higher back
pressures.
Some workers suggest 30- or 50-mm column lengths for initial screening, but these
columns often do not generate a sufficiently high plate number for a reasonable separation.
Another option is narrow-bore (2-mm i.d.) or microbore (<1-mm i.d.) columns. These
columns use less solvent, but during the method development stage they place
unnecessary demands on the LC system, such as requiring smaller sample volumes and
minimizing extracolumn plumbing and detector cell sizes. My preference for using any of
these short or narrow diameter columns is to wait until the fine-tuning stage at the end of
method development.
In other words, I’ll stick with my 150 mm X 4.6 mm, 5-µm dp column as the workhorse for
method development. My alternate is the 75 mm X 4.6 mm, 3.5 -µm dp column. Typically I
recommend running these columns with a 1.5-mL/min flow rate.
ORGANIC SOLVENT SELECTION
Another choice that can have a bearing on the success of the separation is the organic
solvent. Reversed-phase separations provide three choices: methanol, acetonitrile, and
tetrahydrofuran. Each solvent has unique selectivity advantages, but chromatographers
seldom can predict which solvent will be the best choice on this basis. So you must choose
the starting solvent based on other considerations.
Most of the work in my laboratory requires analyzing pharmaceutical compounds. Many of
these samples have very poor UV absorbance, so analysts often find themselves working
with detector settings of 220 nm or less. Because of its high background absorbance,
tetrahydrofuran is not very useful below about 240 nm. Although low concentrations of
methanol can be used at low wavelengths, gradients with methanol usually drift off scale at
wavelengths shorter than 220 nm.
You want a solvent that is nonreactive with samples and the atmosphere. Tetrahydrofuran
can degrade and form peroxides, so workers must take special care if they use it. Some
workers have found that adding water to tetrahydrofuran greatly diminishes this problem (4).
Tetrahydrofuran also is much slower to equilibrate with the column and LC system than is
acetonitrile or methanol - it may take twice the solvent volume for initial equilibration with
tetrahydrofuran. And, of course, tetrahydrofuran’s unpleasant odor is another negative
factor.
A final beneficial property of acetonitrile and methanol is their relatively low back pressure at
flow rates of 1 -2 mL/min when compared with tetrahydrofuran.
When I consider the various desirable solvent properties - low viscosity, low UV
absorbance, low reactivity, and convenience - my first choice for an organic solvent is
acetonitrile. However, I would not argue with anyone who preferred methanol as a starting
solvent.
AQUEOUS PHASE SELECTION
If the samples are neutral compounds, you may be able to use water as the aqueous
phase. However, ionic compounds generally are present in pharmaceutical analysis and
many other applications. Ionic compounds require analysts to maintain pH control to obtain
reproducible methods. You will get the best results if the compounds are fully protonated or
fully ionized. Thus, the pH of the mobile phase should be at least 1.5 units above or below
the p Ka if possible. For organic acids, working at pHs lower than pH 3 generally will be
satisfactory. However, bases will require buffers with pHs higher than pH 8, which exceeds
the recommended working pH of most silica-based columns. Silica columns with extended
pH ranges are available now, but few have extensive experience with these materials at
high pH, so I recommend organic buffers to reduce silica dissolution. These factors make
me wary of routine use of high-pH mobile phases with silica columns.
Sample characteristics and column pH stability combine to suggest that a low pH is

preferable to a high pH for routine operation. Most C8 and C18 columns are stable down to
a pH of approximately 2. Under these conditions, most acidic samples will be protonated,
bases will be fully ionized, and the ionization of surface silanols will be minimized. For many
routine methods, 25 mM phosphate buffer at pH 2.5 will be a satisfactory starting aqueous
phase. If the method is destined for use with a mass spectrometer, you will need to use a
volatile buffer. Although not as useful as true buffers, 0.1% trifluoroacetic acid or formic acid
will provide satisfactory pH control for many LC -mass spectrometry applications.
OTHER FACTORS
You should consider a few other factors before embarking on developing a new LC method.
Because retention changes by 1 -3% for a 1 °C change in temperature, it is important to
control the column temperature. Changes in temperature also can affect selectivity, which
should give you additional incentive to control the temperature. I generally operate the
column oven at a temperature just higher than room temperature (for example, at 35 °C) for
ease of control and to reduce the solvent viscosity for lower-pressure operation.
Sometimes you’ll need to use additional reagents such as ion-pairing reagents or
triethylamine for special sample types. I recommend, however, that unless you know that
you will need these additives, you should start with an additive-free mobile phase.
Remember the KISS principle - Keep It Simple, Stupid - and don’t complicate the mobile
phase unless it is necessary.
Finally, I strongly recommend that you select a column that will be available for years to
come. Chances are that your method will be used for several years, and the heart of
reproducible separation is obtaining a column of similar chemistry year after year. Use a
column vendor that you are confident can provide controlled column chemistry over the long
haul.
IN OTHER WORDS
I cannot overemphasize the importance of choosing starting conditions carefully. The

choices of column and mobile phase are ones that you - and likely others, too - will live with
for years. Make those choices wisely. I’ve summarized my recommendations for starting
conditions in Table II.
TABLE II: Recommended Starting Conditions for Reversed-Phase LC

Method Development
Separation Variable Preferred Initial Choice
Column
Dimensions 150 mm X 4.6 mm
Particle size 5 µm
Stationary phase C8 or C18
Mobile phase
Solvents A -B Water-acetonitrile
% B solvent Variable
Buffer 25 mM phosphate (pH 2.5) or 0.1%
trifluoroacetic acid or formic acid
Additives such as ion-pair As necessary
reagents and amines
Flow rate 1-2 mL/min
Temperature 35 °C
The 150 mm X 4.6 mm, 5 -µm dp Type B silica column is a workhorse, and when operated
at low pH under controlled temperature it will be an excellent place to start method
development with most samples.
Now that I’ve described the tools to use, next month I’ll begin looking at the steps toward
obtaining the best separation in the minimum amount of time and effort.
REFERENCES
(1) R.E. Majors, LC Magazine 2(3), 202-208 (1984).
(2) L.R. Snyder, J.J. Kirkland, and J.L. Glajch, Practical HPLC Method Development (John
Wiley & Sons, New York, 2nd ed., 1997).
(3) U.D. Neue, HPLC Columns. Theory, Technology, and Practice (Wiley-VCH, New York,
1997).
(4) J. Zhao and P.W. Carr, LCGC 17(4), 346-352 (1999).
John W. Dolan
“LC Troubleshooting” editor John W. Dolan is president of

LC Resources Inc. of Walnut Creek, California, and a
member of LCGC’s editorial advisory board. Direct
correspondence about this column to “LC Troubleshooting,”
LCGC, 859 Willamette Street, Eugene, OR 97401, e-mail
John.Dolan@LCResources.com. For an ongoing
discussion of LC troubleshooting with John Dolan and other
chromatographers, visit the Chromatography Forum
discussion group at www.chromforum.com.
Feature Index - Home -Main Index
Copyright Advanstar Communications

Thu Aug 30 15:31:42 2001. updated 08/30/2001
LC-GC - January 2000
Starting Out Right,

Part II -
Measuring Satisfaction
Just how good is good enough?
John W. Dolan
Last month’s “LC Troubleshooting” discussed selecting

appropriate starting conditions when developing a new
liquid chromatography (LC) method (1). Choosing the
proper column and mobile phase is an important step in
increasing the likelihood of achieving a successful separation. Another important part of the
method development process is obtaining the tools that quantitatively measure the quality of
the separation. Although most chromatographers can look at a chromatogram and provide a
qualitative opinion about the quality of the separation, it is important to be able to measure
the separation quality. This measurement ability is especially important because it allows
chemists to assess the separation when intentional or unintentional changes occur.
This month, I will cover the measurement of retention, peak shape, and resolution. These
quantitative measurements of separation quality are important from the first injection
through method validation and on into the application stage of an LC method.
Retention
The retention time (tR ) is measured as the time between sample injection and the apex of
the peak (Figure 1). Retention time probably is the most used chromatographic parameter.
Retention is said to be characteristic of a compound but not unique. It is characteristic
because if all conditions are held constant, an analyte will be eluted at the same time in
every run. For this reason, retention time often is used as a qualitative tool to identify a
compound. If a standard of the compound is injected, and the retention time is the same as
a peak in the sample, it is likely that the two compounds are the same. However, retention
is not unique because more than one compound can be eluted at the same retention time.
This potential for coelution is the basis of the separation challenge in LC.
Figure 1: Chromatogram illustrating retention time, column dead time, and
retention factor.
Retention Factor
Although retention time is a very useful measurement, the retention factor ( k) often is a
more useful parameter for method development. The retention factor provides analysts with
information about the quality of the separation. For example, chromatographic conditions
that generate values of 1 , k , 20, or better yet 2 ,, k , 10, are more likely to yield an
acceptable separation than those that generate k values outside these limits.
Chromatographers know from experience that if k is too small, the retention is short and the
peaks of interest tend to have problems with interferences at the injection front; if k is too
large, peaks become broad and hard to detect and the run time is excessive.
The k value can be calculated from equation 1
[1]
where t0 is the column dead time, usually noted by the first disturbance in the baseline or
the elution of a solvent peak (Figure 1). Although k can be calculated, usually it is easier to
estimate the value of k, and an estimate is good enough for method development purposes.
Note that the numerator of equation 1 is the corrected retention time - the time after t 0
required for the peak to be eluted. The denominator defines the units for k - units of t0. So to
estimate t0 , just start measuring at t0 and see how many t0 units the peak requires to be
eluted. Figure 1 illustrates this measurement below the time axis. The peak eluted at 6 min
comes out three t 0 units after t 0, so the k value is approximately 3. For method development
purposes, if k is estimated within 0.5-1 units, it will be satisfactory. It should be noted that k,
as defined in equation 1, is useful only for isocratic separation.
Let’s pause for a moment and compare tR and k. Retention is directly affected by changes
in the flow rate and column size, whereas k remains constant when either of these
parameters changes. Both t R and k change when the mobile-phase composition or the
column temperature changes. Thus, anything that affects t 0 and tR proportionally will not
affect k. The practical implication of this situation? After you obtain a separation that you like
on one column, you can change to a column of different dimensions, even if the flow rate is
changed, and the k value will remain unchanged, which means that the quality of the
separation, in terms of retention, is unchanged.
So when examining a chromatogram, you should make a quick estimate of the k values of
the first and last peaks to see if they fall within the 1 , k , 20 range. If everything is bunched
at the beginning (small values of k) or is strongly retained (large k values), a change in the
mobile-phase composition probably will be necessary to obtain a satisfactory separation.
Peak Shape
One of the first things experienced

chromatographers notice when looking at
chromatograms is the shape of the peaks.
Ideally, chromatographic peaks are
Gaussian shaped, but in practice, most
peaks show some peak tailing. Peak tailing
is measured using the asymmetry factor
(As) or the U.S. Pharmacopeia (USP)
tailing factor ( Tf) as defined in Figure 2.
The pharmaceutical industry uses the USP
tailing factor as a standard, whereas most
Figure 2: Measurement of peak tailing other chemical applications use As to
using the USP tailing factor and peak measure peak shape. From a practical
asymmetry factor. standpoint, it doesn’t matter which
measurement you use, as long as you use
one technique to measure peak tailing. The
Table I: Peak Asymmetry and Peak two measures are roughly comparable if
Tailing Factor Relationship* minimal tailing is present, as Table I shows
(2).
Peak Asymmetry Peak Tailing
Factor (at 10%) Factor (at 5%) Tailing peaks indicate that more than one
retention mechanism is present in the
1.0 1.0 interaction of that peak with the stationary
phase, a situation that is undesirable. A
1.3 1.2 more practical concern is that when tailing
peaks are present, analysts must use
longer run times for baseline separations,
1.6 1.4 and because some of the area is under the
tail, the peak heights are smaller, which
can compromise detection limits. Usually
1.9 1.6
peak tailing can be minimized by using a
column based on Type B silica and
2.2 1.8 operated with a low-pH mobile phase, as
was discussed in last month’s “LC
2.5 2.0 Troubleshooting” (1). Sometimes mobile-
* Reprinted from reference 2 with phase additives such as triethylamine can
permission. be used to reduce peak tailing.
Column Plate Number
In addition to peak shape, it is important to

examine the peak width to determine
whether the column is performing in a
reasonable manner. Although peak width
can be expressed in time units, the plate
number ( N) is a more useful measure of
peak width. Most analysts prefer to
calculate the plate number as shown in
Figure 3 using the half-height method, in
which they use the width at half the peak’s
height to calculate N. It is easier to
determine the width at half-height than at
the baseline if the baseline is noisy or if the
peak tails or is not fully separated from
neighboring peaks.
A new column containing 5-µm particles

Figure 3: Calculation of the column plate will generate roughly 80,000 plates/m using
number using the half-height method. the column manufacturer’s test conditions,
whereas a 3-µm particle column will yield
approximately 100,000 plates/m. These measurements under standardized test conditions
are useful when the column is new to determine if it passes initial quality tests, but this
testing is inconvenient when the column is in routine use. Furthermore, the test compounds
used by column vendors often provide much higher N values than real compounds under
normal operating conditions. It is much more convenient to test the column during each use
by using a system-suitability sample to verify acceptable performance. For these purposes,
the plate number can be estimated as
[2]
where L is the column length in centimeters and dp is the particle diameter in micrometers.
Thus, a 150-mm column containing 5-µm particles should have a plate number of
approximately 9000 under practical operating conditions. Using this guideline, you should
examine peaks in the chromatogram to see if they have reasonable plate numbers.
Chromatographers should consider corrective action if the plate number for a peak of
interest is more than about 20% below the guideline of equation 2.
Putting It Together
So now you have three tools to help you examine the quality of a chromatogram. First, you
will want to estimate k values for the first and last peaks in the chromatogram to see if
retention is in the region most likely to yield a good separation. Next, examine the individual
peaks to see if they are well shaped and if the peak widths are reasonable. This set of
measurements will determine if the peaks are well behaved, increasing the likelihood of a
successful separation. However, these observations of the chromatogram are secondary to
the real goal of chromatography - obtaining a reasonable separation. To determine if the
separation is acceptable, you must make one more measurement - resolution.
Resolution
Resolution is the separation between two peaks in a chromatogram, defined as
[3]
where t1 and t 2 are the retention times and w1 and w2 are the baseline widths of the two
peaks. An alternative formula uses the half-height peak widths
[4]
where w0.5,1 and w0.5,2 are the half-height peak widths. For Gaussian shaped peaks, the
valley between two peaks hits the baseline at Rs ' 1.5. To have a safety margin that allows
for some deterioration of a method, most workers strive for separations with a minimum
resolution of 1.7 -2.0. When resolution gets much larger than 2, no particular separation
improvement is achieved for most applications and run times can be excessive.
Although equations 3 and 4 are good tools for measuring resolution, during method
development resolution is more usefully defined using
[5]
where a is k2 /k1 , which is the ratio of k values for two adjacent peaks. Equation 5 comprises
three major components. Part i relates to the column quality. Columns with larger plate
numbers result in better resolution. However, resolution improves only as the square root of
the plate number, so to double the resolution, chromatographers must increase N fourfold.
This change is impractical. For example, connecting four columns in series would mean a
fourfold increase in run time and pressure plus a significant financial investment for a
twofold increase in resolution. The plate number is easily calculated from first principles,
and it is easy to predict how a change in particle size, column length, or flow rate will affect
N. Because plate number changes are so easily predicted, it is best to leave these changes
to the end of the method development process. Start with a column that will generate a
reasonable number of plates for most separations. Last month I suggested that a 150-mm,
5-µm dp or 75-mm, 3.5 -µm dp column was a good starting point.
Part ii of equation 5 relates to selectivity - the ability of the column-mobile phase

combination to separate two peaks. Selectivity is related primarily to chemical factors, but,
as I will describe next month, selectivity and retention are closely related as well. Whereas
plate number changes are readily predicted, changes in selectivity are more difficult to
anticipate.
Part iii of equation 5 is the retention term (in iii k is the average k value of the two peaks
under consideration). As retention (or k) increases, resolution also improves. An interplay
between the retention and selectivity terms exists, because both contain k; that is, a change
in k ( iii) generally will change a (ii) as well.
Putting the Tools to Work
Last month, I looked at the importance of selecting a column and the mobile-phase
conditions that were most likely to provide a successful separation. Although success under
those conditions is not guaranteed, the chances are improved for obtaining an acceptable
separation with a minimum investment in method development time.
This month, I presented the tools for measuring the quality of the chromatogram. These
tools allow you to look at a chromatogram, make a few calculations, and determine if the
separation is satisfactory or not. An unsatisfactory peak shape or column plate number
indicates problems with the basic chromatographic process, and they should be addressed
before further method development. Generally, chromatographers can adjust retention ( k
value) and resolution by changing either the mobile- or stationary-phase conditions. Now
that you have these tools at your disposal, you can move into the method development
process and use them to move quickly to an adequate separation. Next month I’ll examine
how to control retention and selectivity, using equation 5 to help guide us.
References
(1) J.W. Dolan, LCGC 17(12), 1094-1097 (1999).
Wiley & Sons, New York, 2nd ed., 1997), p. 210.
John W. Dolan

Thu Aug 30 15:33:59 2001. updated 08/30/2001
LC-GC - February 2000
Starting Out Right,

Part III -
The Role of the Solvent
in Controlling Selectivity
The chromatographic challenge is
to separate two peaks
John W. Dolan
In the first installment of this series (1), the discussion

centered on choosing a set of starting conditions that
would be likely to obtain a successful liquid chromatography (LC) separation. Although no
single set of starting conditions will guarantee success, I explained that most samples will
benefit from starting with a C8 or C18 column packing based on Type B silica using a
mobile phase of acetonitrile or methanol modified with water or a low-pH buffer.
There’s a saying to the effect that if you don’t know where you’re going, you’ll never know
when you get there. Addressing a similar concern with LC separations, the second
installment (2) emphasized the need to be able to quantitatively measure the attributes of
the separation. The specific measurements of interest were retention time ( tR ), retention
factor ( k), peak asymmetry (As ) or U.S. Pharmacopeia tailing factor (Tf), column plate
number ( N), and resolution (Rs).
This month, I’ll show you how these tools can be used to guide analysts to a successful
separation. In particular, the fundamental resolution equation (equation 1) will help evaluate
the effect of various selectivity-controlling parameters during method development.
Resolution is the Key
The goal of the chromatographic process is to separate one or more sample components
from other materials in a sample. Last month, I described the measurement of resolution. At
a resolution of approximately 1.5, the valley between two peaks reaches the baseline. Most
workers like to have separations with resolution values of more than 1.7 to provide a little
leeway for loss in separation without making the separation unusable. Similarly, when
resolution is much greater than roughly 2.0, little is gained for most applications - run times
increase without any additional quantitative advantage.
For purposes of method development, the fundamental resolution equation below is a

useful tool to guide the process of achieving a satisfactory separation.
[1]
where
a = k 2/k1…… [2]
and k1 and k2 are the retention factors of the two peaks of interest. For the balance of this
installment of “LC Troubleshooting,” we’ll concentrate on terms ii and iii in equation 1.
Before getting into the specifics of

controlling selectivity, it is useful to
examine the relationship between term iii of
equation 1 and resolution. If these terms
are plotted against each other, as in Figure
1, the justification for some retention
guidelines given last month becomes
obvious. The data of Figure 1 confirm what
most workers already know from
experience. If k is quite small, the
resolution is poor because peaks get
crowded together, especially near the
unretained material in the sample. At the
Figure 1: Plot of retention factor (part iii other end of the separation, when retention
of equation 1) versus resolution. times are quite long, little resolution is
gained; however, run times increase and
peaks get broader, shorter, and generally harder to detect. The remaining portion of Figure
1 represents conditions that give reasonable chromatography (acceptable retention times,
peak widths, and peak heights), and a change in retention ( k) in this region can make a
significant change in resolution. This information is the basis of the recommendation that for
good chromatography and increased likelihood of an acceptable separation, retention
should be kept in the 1 , k , 20 span, or better yet, 2 , k , 10.
Finally, equations 1 and 2 show that as retention changes, peak spacing (a) also changes.
This relationship is the source of the general improvement in resolution as retention
increases (Figure 1). (Note that k in iii of equation 1 is the average of k1 and k2 of equation
2.)
Adjust Solvent Strength First
Before you worry too much about selectivity, it is important to get the retention time within a
reasonable range. With isocratic separations, one simple way to adjust retention is to start
with 90% or 100% solvent B (organic solvent) and reduce the concentration 10% at a time.
So run 100% solvent B, then 90%, 80%, and so on until retention times fall in the 1 , k , 20
range desired for good chromatographic behavior. Figure 2 illustrates three steps of this
series of runs. As the organic solvent concentration is reduced from 80% to 70% to 60%,
the retention and resolution increase, as expected from equation 1 and Figure 1. The
column used for the runs of Figure 2 was a 150 mm X 4.6 mm column operated at a 1.5-
mL/min flow rate, so t0 is approximately 1 min. A quick examination of the chromatogram for
the 60% B mobile phase allows a retention factor estimation of 2 , k , 7, which is very good.
The peaks are baseline resolved and the run time is short, so the separation doesn’t need
much optimization.
Figure 2: Simulated chromatograms generated using (a) 80% B, (b)
70% B, and (c) 60% B solvent concentrations.
Note that the peaks in Figure 2 move in a regular fashion as the organic content of the
mobile phase is changed. The Rule of Three will guide us in the general peak movements.
The general rule states that retention factors will increase roughly threefold for each 10%
reduction in the organic-solvent content of the mobile phase. The k values are 1.1, 2.7, and
6.9 for the last peak in the runs of Figure 3, so the rule for this particular sample is more like
the Rule of 2.5, but as a general guideline, the Rule of Three is quite handy. Finally, note
that it is threefold for each 10% change, so 20% solvent B change would cause a 3 X 3 or
roughly ninefold change in retention factor.
The (Not So) Special Case
All the peaks in Figure 2 follow Figure 1’s predicted pattern. That is, retention and resolution
increase as the mobile-phase strength is reduced. This pattern of change is a generality,
but fortunately it does not hold for every compound. The exceptions to the rule are
important, because they allow chromatographers to fine-tune the mobile-phase composition
and gain selectivity in a separation.
Figure 3 illustrates how you can take advantage of fine-tuning the mobile -phase
composition to improve a separation. From the top to the bottom of Figure 3, the mobile
phase is made weaker, much as it was in Figure 2. And in the same manner, you can see
that the resolution of peaks 2 and 3 improves, and retention time increases with a lower
percentage of solvent B. However, notice the movement of the last two peaks in each run.
The resolution of this peak pair changes in the opposite way. These changes are common
in samples submitted for LC analysis; that is, although the general pattern of longer
retention and better resolution with weaker mobile phases holds for many compounds, it is
quite common for resolution to decrease under the same circumstances for other
compounds.
Figure 3: Simulated chromatograms generated using (a) 48% B, (b)

47% B, and (c) 46% B solvent concentrations.
When you observe peak movements as in Figure 3, you may need to compromise to
achieve acceptable separation. In the present case, the resolution of peaks 2 and 3 is better
at 46% solvent B, but the last two peaks are better resolved at 48% solvent B. Midway
between these two conditions, 47% solvent B, compromises the separation of both peak
pairs but gives the best overall separation of this sample.
Figure 3 shows one other separation feature that you should look for in method
development - robustness. Robustness is the ability of a separation to withstand small
changes in conditions without compromising the results. The separation at 47% solvent B in
Figure 3 certainly is not robust - a 61% change in organic solvent concentration significantly
degrades the separation. In routine use, this method would require undue care in mobile -
phase preparation, so these results should alert chromatographers to look for alternate
conditions that would produce a more satisfactory separation.
Solvent Type
The adjustment of solvent strength alone can allow you to find satisfactory separation
conditions for many samples. For other samples, however, no concentration of acetonitrile
will generate a suitable separation. At this point, you should take a different approach for
changing a. One powerful way to change a for many samples is to change to another
organic solvent in the mobile phase.
For the sample of Figure 4, acetonitrile-water mobile phases were unsuccessful in achieving
the desired separation. My next choice in mobile -phase solvents is methanol. Figure 4a
shows the best separation with methanol-water (55% methanol). The resolution for most of
the peaks in the chromatogram is satisfactory, but peaks 2 and 3 are poorly separated.
Fine-tuning the concentration of solvent B, as was done in the previous example, was
unsuccessful, so I tried still another solvent.
Figure 4: Simulated chromatograms obtained using (a) methanol-

water, (b) tetrahydrofuran-water, and (c) blended methanol-
tetrahydrofuran-water mobile phases.
I substituted tetrahydrofuran for methanol for the separation of Figure 4b, and this figure
shows that the resolution of the later peaks in the run is satisfactory. However, the
beginning of the run still has trouble. Peaks 1 and 2 and peaks 4 and 5 are poorly resolved.
Once again, fine-tuning the concentration of solvent B provided no better results.
At this point, you might want to give up - acetonitrile, methanol, and tetrahydrofuran all
failed to provide a satisfactory separation. However, the separations of Figure 4 illustrate
another practice in vigilance that should be the habit of successful chromatographers. Note
that the critical peak pairs (those most poorly resolved) change between the runs of Figure
4a and 4b. Just as was the case for the sample of Figure 3, an intermediate set of
conditions should give peak separations and retention times falling between those observed
for the best methanol-water and tetrahydrofuran-water conditions. Indeed, this situation is
what I observed. When the isocratic solvents of Figure 4a and 4b were blended, I obtained
the separation of Figure 4c. This condition gives the desired baseline resolution of all peaks
in the chromatogram.
Summary
Guidelines for Adjusting The organic solvent plays a major role in controlling
selectivity in reversed-phase LC separations. I’ve
the Concentration of
summarized several guidelines that you should
Solvent B remember from the examples above.
• Adjust the concentration of
solvent B for acceptable First, adjust the concentration of solvent B
retention range (acetonitrile as the first choice) until all the peaks fall
in the 1 , k , 20 region. In general, resolution will
• Fine-tune the concentration of increase with increasing retention, but watch the
solvent B for desired resolution movement of individual peaks to help determine
when fine-tuning of the concentration of solvent B is
•Change solvent type to change justified.
selectivity
•Blend solvents, if necessary If acetonitrile is ineffective, try methanol and repeat
the optimization experiments; if methanol fails, use
•Selectivity changes between tetrahydrofuran. Keep relative peak movements in
runs can help you evaluate the mind as you make these changes, because changes
usefulness of intermediate in selectivity between conditions often indicate that
conditions an intermediate condition or solvent blend will be
useful to pull apart hard-to-separate peak pairs.
Solvent strength and type are two powerful variables that affect chromatographic selectivity,
but they are by no means the only variables available. Next month’s “LC Troubleshooting”
column will highlight some other variables that can be used to control selectivity. I will
provide some guidelines about when the use of these variables is appropriate.
References
(1) J.W. Dolan, LCGC 17(12), 1094-1097 (1999).
(2) J.W. Dolan, LCGC 18(1), 28-32 (2000).
John W. Dolan

Thu Aug 30 15:34:19 2001. updated 09/18/2000
LC·GC · March 2000
Starting Out Right,

Part IV -
Additional Variables
to Control Selectivity
Variables such as pH, temperature, and column type can
be useful tools to help control peak spacing.
John W. Dolan
Last month’s installment of “LC Troubleshooting”

described the role of solvent strength and solvent type in
controlling selectivity in liquid chromatography (LC) separations (1). I explained that by
making systematic variations in solvent strength, chromatographers can obtain dramatic
changes in selectivity and retention. Changing the solvent type, from acetonitrile to
methanol, for example, gave additional selectivity leverage during method development. In
some cases, blending more than one organic solvent is useful.
Although varying solvent strength and type provides powerful tools for controlling the
separation, these variables may be insufficient to obtain the desired resolution for a
particular sample. For this situation, chromatographers must use other parameters to adjust
selectivity. This month’s “LC Troubleshooting” column will concentrate on three additional
parameters: pH, temperature, and column type.
Mobile -Phase pH
For separations discussed in previous installments in this series, I suggested aqueous-

organic solvent mobile phases in which the aqueous portion was water for neutral samples
or low-pH buffer for ionic samples (1-3). Using low -pH mobile phases (pH 2 -3) is a good
starting point because these conditions suppress the ionization of both acidic compounds
and the residual silanol groups on the silica surface. However, when ionic compounds are
present, pH can have a marked influence on retention and selectivity, so if the initial low-pH
mobile phase is unsatisfactory, the exploration of pH effects is a natural next step in method
development.
Figure 1 illustrates the power of using pH to control selectivity (4). The figure shows that
acids will be protonated, hydrophobic, and thus more strongly retained at low pH than at
high pH. Conversely, bases will be neutral and well retained at high pH, whereas low pH
results in ionization and poor retention in a reversed-phase system. Neutral compounds are
unaffected by pH, so the retention will vary little, if at all, when the mobile-phase pH is
changed.
Figure 1 also illustrates that a mixture of acids, bases, and neutrals will cause many
retention reversals when the pH is changed. This effect can be a powerful tool to control
selectivity and obtain a separation impossible through the adjustment of other parameters,
but it also indicates that chromatographers must take care to control the pH and avoid any
unintended changes in selectivity.
Figure 1: The influence of pH on retention of a

mixture of acids (curves 1 and 2), bases
(curves 4 and 5), and neutral (curve 3)
compounds. (Reprinted from reference 4 with
permission.)
The plot of retention versus pH for compound 4, which is a base, in Figure 1 shows a shape
closely resembling a titration curve. Not surprisingly, the midpoint of this curve is at the p Ka
for this compound, which is approximately pH 7. Acids show the same curve shape in mirror
image, although the pH range in Figure 1 is insufficient to show the entire curve for any
compound except number 4.
Figure 1 shows that an adequate separation of all sample components can be obtained at
pH 5 or 7. However, at pH 7, the method is much more likely to be sensitive to small
changes in pH, particularly for compounds 4 and 5. For maximum method robustness, it is
best to work at a pH at which the compound or compounds of interest are either fully
ionized or ionization is suppressed. This condition means that chromatographers should
operate at more than ±1.5 pH units from the pKa value. As usually is the case, workers may
need to compromise for the best results, but operation at pH 5 will be less sensitive to pH
changes than that at pH 7 for this sample.
As mentioned earlier, I recommend starting the initial run with most ionic samples at low pH;
for example, pH 2.5. Under these conditions, adjust the organic solvent content of the
mobile phase to obtain a good retention range for neutral and nonionized samples. To
screen for gross effects of pH, chromatographers can change the mobile-phase pH while
keeping the organic solvent at a constant concentration. For example, make the initial run at
pH 2.5, then change to pH 7.0 for another run to see if dramatic changes occur. However,
to fine-tune the mobile-phase pH, it is best to vary the mobile phase in steps not exceeding
0.5 pH units because retention and peak spacing can change dramatically with small
changes in pH. These dramatic changes can make peak tracking difficult, especially if peak
areas change, which can happen with pH changes.
Figure 2 shows an example of fine-tuning the mobile -phase pH for a group of benzoic acids
(5). In each case, only the pH is varied. Whenever examining a set of chromatograms such
as in this figure, it is useful to concentrate on the critical (least resolved) peak pairs. Peaks
2, 6, and 9 move dramatically in relation to the other peaks in the sample. A satisfactory
separation of all nine components can be obtained at pH 3.0. As is expected with a group of
organic acids, lower pH will reduce the degree of ionization and make the components more
hydrophobic and thus more strongly retained.
Figure 2: Simulated separation of nine benzoic acids at (a) pH 2.5, (b) pH 3.0, (c) pH
3.5, and (d) pH 4.0. Peaks: 1 = 2 -nitrobenzoic acid, 2 = phthalic acid, 3 = impurity, 4 5 2 -
fluorobenzoic acid, 5 = 3-cyanobenzoic acid, 6 = 2 -chlorobenzoic acid, 7 = 3 -nitrobenzoic
acid, 8 = 3-fluorobenzoic acid, 9 = 2,6-dimethylbenzoic acid. The conditions are listed in
Table I. The simulated chromatograms were generated from the data of reference 5.
Table I: Conditions for Figures 2 and 3
Variable Figure 2 Figure 3
Column 250 mm X 4.6 mm, 5 µm dp C8 Same
Flow rate 1 mL/min Same
Methanol (%) 35% Same
Buffer 25 mM phosphate Same
pH Varies pH 3.2
Temperature 35 °C Varies
When using pH to control an LC separation, it is especially important to obey the

fundamental rules of buffer usage. Remember that a buffer is effective in the range of ±1
pH unit from the pKa of the buffer. Outside this range, buffering is marginal, meaning that
methods using those conditions will be less robust. This rule means that phosphate buffer is
a good choice in the pH 2.0 -3.1 and 6.2-8.2 regions (below pH 2, bonded-phase stability is
a problem), whereas acetate buffer is a better choice in the pH 3.8 -5.8 region. Although the
ideal buffering ranges for these two buffers don’t overlap, stretching the range of each
buffer by less than one-half a unit will allow a blend of acetate and phosphate to make a
buffer useful throughout the entire pH 2-8 range.
Column Temperature
Another variable that can provide useful changes in selectivity is column temperature. Most
workers consider temperature only in terms of retention time changes. As a general rule,
retention in reversed -phase LC can be expected to change 1 -3% for each 1 °C change in
temperature. Figure 3 illustrates this rule; in this figure retention changes roughly 25% (19
min to 25 min for the last peak) with a 15 °C change in temperature, or approximately
1.7%/°C. Table I lists the conditions for this separation. Temperature control is important for
reproducible retention times.
Figure 3: Simulated separation of nine benzoic acids at (a) 30 °C, (b) 35 °C, (c) 40 °C,
and (d) 45 °C. Peaks: Same as in Figure 2. The conditions are described in Table I. The
simulated chromatograms were generated from the data of reference 5.
It is less appreciated that changes in temperature can cause changes in selectivity in LC
separations. This condition is illustrated in Figure 3 for the same benzoic acid sample that
was shown in Figure 2. Although the general trend is shorter retention times with increased
column temperature, important selectivity changes take place, too. Notice the movement of
peak 5 as the temperature is increased, changing from coelution with peak 6 at 30 °C to
beginning to merge with peak 4 at 45 °C. Because peaks move in a regular fashion with
changes in mobile-phase conditions, peaks 4 and 5 would completely merge when the
temperature is increased to higher than 45 °C (overlap is complete at 53 °C). Similarly,
peaks 8 and 9 move relative to each other when the column temperature is changed. For
this sample, a temperature of 40 °C will provide baseline separation of all sample
components.
Why does temperature provide such dramatic changes in selectivity for this sample? In this
case, the changes to a large degree are due to the ionic character of the sample. The
temperature will influence the ionization characteristics, hydrophobic retention of ionized
versus nonionized compounds, silanol interactions with ionic species, mobile -phase pH, and
the p Ka s of sample components. With all of these influences, it would be surprising if a
change in temperature did not influence selectivity.
The practical result for this benzoic acid sample is that workers can obtain a satisfactory
separation under more than one set of conditions. The similarity between the pH 3.0 (35 °C)
run of Figure 2b and 40 °C (pH 3.2) run of Figure 3c is striking. Both conditions provide
baseline separation of all components in approximately 25 min. When ionic species are
present in the sample, column temperature and mobile-phase pH will play an important role
in peak spacing.
Column Selectivity
Up to this point in the discussion, I have talked about changes in selectivity accomplished
by making changes in the mobile-phase composition. This approach usually is the most
convenient and least expensive way to change selectivity. An alternative approach, usually
best left until mobile-phase alternatives have been exhausted, is to rely on changes in
column selectivity to control the separation. As with changes in mobile -phase selectivity,
chromatographers should make significant changes in column selectivity to take maximum
advantage of this parameter. Therefore, changing from one brand of C18 column to another
or from a C18 to a C8 column will not be very fruitful. These kinds of changes may result in
selectivity variations, but they tend to be minor when compared with changes in the
stationary-phase chemistry.
For reversed-phase separations, three column types - C18 or C8, cyano, and phenyl -
traditionally are selected. Figures 4 and 5 illustrate the changes in selectivity that occur
between a C8, cyano, and phenyl bonded phase for two different samples. The sample of
Figure 4 (6) is a group of substituted benzoic acids, some of which are in the sample of
Figures 2 and 3. The most prominent change is in the retention order of the 4-5-6 triplet, in
which compound 4 comes out first with the C8 column, but last with the other two. Also, the
peak spacing between peaks 1 and 2 changes markedly.
An herbicide mixture was used for the separations of Figure 5 (6). Peaks 1 and 2 run
together on the cyano column and the separation of 6 and 7 is reduced as well. No retention
order changes are observable for the herbicide mixture on these columns.
It is apparent that column chemistry changes may yield dramatic selectivity changes, as is
the case for the benzoic acids in Figure 4, or the changes may be much more subtle, as
with the herbicides in Figure 5. It is impossible to accurately predict changes in selectivity
between columns, and no software is available to assist in fine-tuning selectivity changes
such as the software used for mobile -phase modifications, so column changes tend to be
trial-and-error in nature. For this reason, I recommend trying the easier and often more
effective mobile-phase changes before exploring column selectivity.
Figure 4: Separation of a substituted Figure 5: Separation of an herbicide

benzoic acid sample using 250 mm X 4.6 mixture using a 40% acetonitrile –water
mm (a) C8, (b) phenyl, and (c) cyano mobile phase on (a) C8, (b) phenyl, and
columns. Mobile phase: (a) 25% methanol– (c) cyano columns. Peaks: 1 = atrazine, 2
buffer, (b) 35% methanol–buffer, (c) 35% = metribuzin, 3 = fen -amiphos sulfoxide,
methanol–buffer. Peaks: 1 = phthalic acid, 2 4 = fenamiphos sulfone, 5 = diuron, 6 =
= 2-nitrobenzoic acid, 3 5 2 -fluorobenzoic propanil, 7 = propanamide metabolite, 8
acid, 4 = 3-nitrobenzoic acid, 5 = 2- = swep. (Reprinted from reference 6 with
chlorobenzoic acid, 6 = 3 -fluorobenzoic acid, permission.)
7 = m-toluic acid. (Reprinted from reference
6 with permission.)
In my discussions with other users, I have found that the phenyl column is used infrequently
today as a tool to enhance selectivity. In the last few years, the polar embedded phases
have come into favor. These phases tend to be C18 or C8 phases with an amine-containing
polar function attached near the base of the C18 chain, typically on the third carbon from
the base. The result is bonded phases that often have distinctly different selectivity
characteristics than traditional C18 or C8 phases. In my laboratory, we have found these
phases to be a good third member to go with the C18-C8 and cyano phases when exploring
column selectivity. Some brand names are Discovery Amide (Supelco, Bellefonte,
Pennsylvania), Symmetry Shield (Waters Corp., Milford, Massachusetts), and Zorbax
Bonus RP (Agilent Technologies, Wilmington, Delaware). Check with your favorite column
supplier for other columns.
Other Variables
The discussion thus far has concentrated on the variables that will give the most leverage in
changing the selectivity of a separation. Many other variables can be explored. For
example, ion-pair reagents often are used to enhance selectivity, especially when a mixture
of acids and bases is present in the sample, but ion pairing tends to be more problematic
than simple pH control. Chromatographers can exploit special chemical characteristics of
the sample - such as the presence of chelators, chiral compounds, or shape differences - to
gain selectivity. When a good effort has been made at obtaining a separation with the
traditional reversed-phase variables, workers may want to explore modes of
chromatography such as ion exchange, normal phase, or size exclusion. Reference 7 is a
good resource for method development for these and other samples.
Conclusions
By the time chromatographers have explored mobile-phase solvents, pH, temperature,

column type, and perhaps ion pairing, they likely will have obtained a satisfactory reversed-
phase separation. Sometimes the selectivity, or peak spacing, is satisfactory, but the peaks
still are not quite baseline separated. In other cases, the peaks may be overseparated,
which is a sign of wasted time. This is the stage in the method development process when it
is appropriate to see if changes in the column size, mobile-phase flow rate, or packing
particle diameter can be used to improve the results. These changes in column parameters
will be the subject of next month ’s “LC Troubleshooting” column.
References
(1) J.W. Dolan, LCGC 18(2), 118-125 (2000).
(2) J.W. Dolan, LCGC 17(12), 1094-1097 (1999).
(3) J.W. Dolan, LCGC 18(1), 28-32 (2000).
(4) P.J. Twitchett and A.C. Moffat, J. Chromatogr. 111, 149 (1975).
(5) J.W. Dolan, D.C. Lommen, and L.R. Snyder, J. Chromatogr. 535, 55-74 (1990).
(6) J.J. DeStefano, J.A. Lewis, and L.R. Snyder, LCGC 10(2), 130-139 (1992).
Wiley & Sons, New York, 2nd ed., 1997).
John W. Dolan
“LC Troubleshooting” editor John W. Dolan is president

of LC Resources Inc. of Walnut Creek, California, and a
member of LCGC ’s editorial advisory board. Direct
correspondence about this column to “LC
Troubleshooting,” LCGC, 859 Willamette Street,
Eugene, OR 97401, e -mail
Thu Aug 30 15:34:38 2001. updated 08/30/2001
LC-GC - April 2000
Starting Out Right,

Part V -
Changing Column Conditions
John W. Dolan
Resolution, pressure, and run time - choose any

two.
In the four preceding installments of “LC

Troubleshooting” (1-4), I provided guidelines that progressed from initial injection to using
the mobile phase to obtain a successful liquid chromatography (LC) separation. Hopefully,
by the time these steps in the method development process have been completed, the
method will separate all the peaks in the sample. When the chemical factors in the
chromatographic process have been fully exploited, the separation often can be improved
further by adjusting the column conditions - flow rate, column size, and packing particle
diameter. This month’s “LC Troubleshooting” will focus on selecting column conditions to
increase resolution or to improve sample throughput.
Back to the Basics
In February’s column (3), I introduced the fundamental resolution equation as a guide for
the method development process:
[1]
where resolution ( Rs) is a function of the column plate number (N), the i portion of the
equation; the selectivity factor (a), the ii portion of the equation; and the retention factor ( k),
the iii portion of the equation. The February and March installments (3,4) discussed
adjusting solvent strength and mobile-phase chemistry to control k and α and obtain the
best possible separation for one or more pairs of peaks in the sample. After these
parameters are optimized, chromatographers can do nothing else using that column,
solvent system, or temperature to improve the separation. However, at this point in the
method development process, the column plate number has not been optimized.
There are several schools of thought about the best column size to use when starting
method development. Some workers contend that a short, 30-50 mm column packed with 3-
µm particles should be used because it allows very fast runs and, thus, the chance to
explore many different mobile-phase conditions quickly. At the other extreme, others would
choose a 250-mm, 3 -µm dp column, so that they could obtain the maximum resolution from
every run. I tend to sit in the middle of the pack - my first choice would be a 150-mm, 5-µm
dp or 75-mm, 3.5-µm dp column. I feel that although the short columns allow more
separations to be run in a shorter time, the plate numbers aren’t large enough to provide
reasonable separations of anything but simple samples. The long, small-particle columns
require low flow rates for reasonable back pressures, so the run times are excessive. The
intermediate column lengths generate sufficient separation power for most separations, yet
they can be operated at flow rates of 2 mL/min or higher without excessive back pressure.
No matter which column size you select for the initial work, you may improve the overall
separation with a change in the column conditions, including the mobile-phase flow rate,
column size, and packing particle size. Sometimes these changes can transform a marginal
separation into a satisfactory one. At other times, changes in column conditions can trade
excess resolution for improved sample throughput. For many samples, the separation is
good enough already, so it isn’t worth trying to adjust the column conditions. If you have
access to chromatography modeling software, you can model changes in column conditions
to help you decide if this avenue of method improvement is worthwhile.
Flow Rate - Simple Things First
As it is with mobile -phase parameters, analysts always have a choice of which column
parameter to vary first. I usually choose to vary flow rate first, although flow rate is not likely
to make a large change in resolution, because it is easy and it will not change selectivity
due to the absence of potential chemical changes. The flow rate’s importance as a
parameter influencing resolution is decreased as the column packing’s particle size is
reduced. Figure 1 illustrates this relationship by comparing 10 -, 5-, and 3-µm dp column
packings. Compare the results of a change in flow rate for each packing size. In these
comparisons, it is easy to focus on the resolution, which is expressed as the depth of the
valley between the two peaks. With the 10-µm dp material, a dramatic improvement in
resolution occurs as the flow rate is reduced from 4 mL/min (Rs 0.8) to 2 mL/min to 1
mL/min ( Rs 1.1). The change for the 5 -µm dp column is less obvious, and the resolution
with the 3 -µm dp column changes only slightly. Another way to look at this relationship is
that increasing the flow rate from 1 mL/min to 4 mL/min will reduce the run time by a factor
of four in all cases, but it costs less than 10% in resolution for the 3-µm dp column, whereas
resolution drops by 25% with the 10-µm dp column.
Figure 1: Separations showing the effect
of flow rate on resolution for columns
packed with (a) 3 -, (b) 5 -, and (c) 10-µm dp
particles. Flow rates (from left to right) are
1, 2, and 4 mL/min.
For practical purposes, flow rate is not a very important factor with the 3-5 µm dp columns
commonly used today. Most workers choose to adjust the flow rate for the shortest run time
while keeping the system pressure within reasonable limits. In cases in which resolution is
marginal, a drop in flow rate sometimes can be advantageous, but the effect will be small,
as is illustrated in the examples of Figure 1. However, changes in flow rate are easy to
make by resetting the LC pump, and, because no chemical change is made, the relative
peak spacing will remain unchanged.
Column Size - Too Many Choices

My next choice of column parameters to change is the column size. I choose the column
size before the particle diameter because the likelihood of system chemistry changes is
minimal. That is, if I switch column lengths for the same type of column from the same
manufacturer, it is likely that only the column length will change. On the other hand, if I
change the particle size, I know that the base silica was prepared using a slightly different
process, and although the column chemistry allegedly is the same, the possibility of minor
chemistry changes exists.
Where do you start when considering column size changes? Most manufacturers have a
large variety of column configurations from which to choose. For example, I made a quick
check of the catalog on the Waters Corp. (Milford, Massachusetts) web site
(www.waters.com) for the XTerra columns packed with the company’s MS phase. The
listing includes 60 separate part numbers distributed between combinations of 2.5-, 3.5-,
and 5-µm dp packings in columns of 20-, 30-, 50-, 100-, 150-, and 250 -mm lengths and 2.1-,
3.0-, 3.9-, and 4.6-mm inner diameters. The assumption here is that chromatographers
could freely change between any of these columns and have identical selectivity, as long as
the mobile -phase composition and column temperature didn’t change. Let’s take a look at
the influence of column diameter and length on the separation; particle size is covered in
the next section.
Column inner diameter should have no effect on the separation, if workers take two
important factors into account. First, if the linear velocity of the mobile phase is kept
constant, the separation should be identical for columns of the same length and packing
content. Therefore, if the column diameter is changed, the flow rate must be adjusted
according to the square of the diameter change. For example, changing from a 4.6 -mm to a
2.1-mm i.d. column results in approximately a fivefold change in cross-sectional area, so the
flow rate must be reduced by fivefold to maintain the same linear velocity.
Second, a potential problem related to column diameter occurs when a column

configuration generates very narrow peaks. Because extracolumn volume is more
detrimental to small-volume peaks, early eluted peaks from a 50 mm X 2.1 mm column are
much more likely to be adversely affected than well-retained peaks on a 150 mm X 4.6 mm
column packed with the same particles. For this reason, it is much easier to operate a larger
column near its theoretical efficiency than a small, narrow -bore column. When using
narrow-diameter columns, chromatographers must take care to use short lengths of narrow-
bore connecting tubing and take other precautions to minimize extracolumn band
broadening.
Column length, on the other hand, is a convenient tool to adjust the method either to
increase the resolution or to reduce the run time. Consider an increase in resolution. The
second two peaks in Figure 2a are marginally separated with a resolution of roughly 1.4.
Let’s assume that our method goal is resolution of at least 1.7. By increasing the column
length from 150 mm to 250 mm, resolution is improved to 1.8 (Figure 2b). (Table I lists
conditions for these separations.) According to equation 1, resolution increases with the
square root of the plate number, and the present case demonstrates that relationship. The
plate number increases in proportion to the column length, so (250/150) 0.5 1.3, and
resolution improves by (1.8/1.4 ) 1.3. However, the increase in resolution is not without
cost. Note that the pressure increases in proportion to column length, as does the run time.
If you want to keep the system pressure near 2000 psi, the flow rate must be reduced by
one-third, which would further increase the run time by one-third to approximately 10 min.
Because of the square-root relationship between N and Rs (equation 1), you can make only
minor gains in resolution by changing column length - perhaps 30-40% gain is possible in
favorable conditions.
Figure 2: Simulated separations generated using (a) 150- and (b)
250-mm long columns. Pressures were (a) 1800 and (b) 3000 psi.
Other conditions are summarized in Table I. The computer
simulations are based on data from reference 5.
The runs of Figure 3 illustrate another case in which adjustment of the column length will be
useful. In this case, the initial separation (Figure 3a) used a 150-mm long column operated
at 1 mL/min. The desired separation for the second two peaks was a resolution of at least
1.7; the actual resolution is 2.1. Although many workers would leave the separation as is, if
you need a resolution of only 1.7, this separation is wasting time. Reducing the column
length from 150 mm to 100 mm (Figure 3b) reduces the resolution to the 1.7 target, but it
also shortens the run time and results in lower back pressure. Doubling the flow rate has no
practical effect on the resolution, yet the run time is halved while maintaining a reasonable
system pressure (Figure 3c). Thus, in this example, shortening the column by one-third
allows the run time to be reduced by two-thirds.
Figure 3: Influence of packing particle size, column length, and
flow rate on resolution. Conditions: (a) 150-mm long, 3-µm dp
column operated at a 1 -mL/min flow rate; (b) 100-mm long, 3-µm dp
column operated at a 1 -mL/min flow rate; (c) 100-mm long, 3-µm dp
column operated at a 2 -mL/min flow rate. Other conditions are
listed in Table I. Same sample as in Figure 2.
To take advantage of adjusting column length to throw away excess resolution, you must
start with a separation that has excess resolution. This is one argument for doing the extra
work to get the maximum possible resolution with a separation through adjustment of the
mobile-phase chemistry - any excess resolution can be traded for shorter run times by
adjusting the column conditions.
Particle Size - Is Smaller Better?
It is tempting to think of reducing packing particle size as a universal way to improve

separations. A satisfactory separation almost always is a compromise between resolution,
run time, and back pressure; therefore, reduced particle size may not provide the best
results. By examining Figures 2-4 and the data of Table I you can see some examples of
the advantages and disadvantages of changing particle size. The resolution for the run of
Figure 2a using a 150 -mm long, 5-µm dp column definitely improved by changing to a 3-µm
packing material, as shown in Figure 3a. However, the smaller particles generated higher
back pressure, so the flow rate was reduced and the run time was increased. When the 3 -
µm dp column used in Figure 3b was replaced with the 1.5-µm dp one generating the
chromatogram of Figure 4a, resolution again increased but at the cost of trebling the
pressure. Shortening the column and adjusting the flow rate (Figure 4b) moved the
separation close to the target resolution with a reasonable back pressure, but this
separation has no improvement in run time.
Figure 4: Influence of packing particle size, column length, and

flow rate on resolution. Conditions: (a) 100-mm long, 1.5 -µm dp
column operated at a 1 -mL/min flow rate; (b) 75-mm long, 1.5 -µm
dp column operated at a 0.7 -mL/min flow rate. Other conditions are
listed in Table I. Same sample as in Figure 2.
Table I: Summary of the data from Figures 2-4

Length Particle Size Flow Rate Pressure Run Time
Figure (mm) (µm) (mL/min Resolution (ps) (min)
2a 150 5 4 1.4 1800 5
2b 250 5 4 1.8 3000 8
3a 150 3 1 2.1 1250 18
3b 100 3 1 1.7 825 12
3c 100 3 2 1.7 1650 6
4a 100 1.5 1 2 3300 12
4b 75 1.5 0.7 1.6 1750 13
My observations from my laboratory and those I visit lead me to believe that the 5 -µm
particles still are the workhorse packing for routine use. Many times analysts can improve
resolution or shorten run times by using 3 -µm packing material. When available, I prefer the
3.5-µm particles over the 3.0-µm ones because they allow use of column hardware that is
less prone to blockage and provide nearly the same chromatographic performance as 3.0 -
µm particles. Smaller particles, such as 1.5-µm dp, are available, but they are best used for
specialty applications such as separation of proteins or fast runs when a short, low-plate
number column can be traded for speed.
Putting It All Together
You can think of resolution, pressure, and run time

as representing three corners of a triangle, as shown
in Figure 5. To make gains in one parameter, you
must sacrifice the performance of one or both of the
other parameters. Figures 2-4, as summarized in
Table I, illustrate these tradeoffs. If resolution is held
constant, run time can be reduced only at the
expense of pressure (compare Figures 3b and 3c). If
pressure is kept approximately constant, resolution
can be increased only at the expense of run time
(compare Figures 2b and 4a). If run time is held
Figure 5: Illustration of the constant, resolution can be increased only at the
relationship between expense of pressure (compare Figures 3b and 4a).
resolution, run time, and So you can set goals for resolution, run time, and
system pressure. pressure before starting the method development
process, but in most cases, you must make
compromises before the final method is complete.
You can fine-tune a separation by making adjustments in the flow rate, column dimensions,
and packing particle size. My preference is to start with the flow rate, because it is easy and
leaves the selectivity unchanged. Next, I’d change the column length, because it is unlikely
that any significant column chemistry changes will occur between columns from the same
manufacturer packed with the same size and description of packing material. Finally, I’d
look at changing the packing particle size. Although particle size is a powerful way to
change the plate number, it carries the most risk of selectivity changes because the
different particle sizes were made in a slightly different synthesis process. Fortunately,
today’s column manufacturers work very hard to give chromatographers a variety of column
configurations that have as little variability in column chemistry as possible.
Next month ’s “LC Troubleshooting” will wrap up the “Starting Out Right” series by
considering the use of gradient scouting runs to speed the method development process.
References
(1) J.W. Dolan, LCGC 17(12), 1094-1097 (1999).
(2) J.W. Dolan, LCGC 18(1), 18-32 (2000).
(3) J.W. Dolan, LCGC 18(2), 118-125 (2000).
(4) J.W. Dolan, LCGC 18(3), 286-294 (2000).
(5) L.R. Snyder, J.W. Dolan, and M.P. Rigney, LCGC Mag. 4(9), 921-929 (1986)
John W. Dolan

Thu Aug 30 15:34:56 2001. updated 08/30/2001
LC-GC - May 2000
Starting Out Right,

Part VI -
The Scouting Gradient
Alternative
Gradient scouting runs can speed the development of
both isocratic and gradient methods.
John W. Dolan
The five preceding installments of “LC Troubleshooting”

presented a strategy for developing liquid
chromatography (LC) separations (1-5). Using this strategy, a chromatographer would start
with conditions that provide a high statistical probability for success with most samples. By
exploring a series of isocratic conditions and logical modifications of the mobile phase, you
can find suitable separation conditions for a large proportion of the samples that you are
likely to encounter. Fine-tuning the separation for improved resolution or higher throughput
can be accomplished by adjusting the column conditions - column size, particle diameter,
and flow rate. Following the steps presented in this series is a good way to approach LC
method development, but I think an even better way to begin method development is by
using scouting gradients.
A scouting gradient is a separation run under a standardized set of conditions to determine

the complexity of a sample and quickly get some information about the difficulty of a
separation. Scouting gradients have several inherent advantages that make them my
favorite starting point in method development. First, with gradient elution, all the peaks will
be eluted either during the gradient or within a few minutes after the gradient is complete.
Therefore, the run time is known in advance, which is in contrast to isocratic separation in
which analysts never know how long the run will require unless previous chromatographic
data are available. Second, it is possible to design a scouting gradient that will provide good
general chromatographic behavior for most compounds without any prior knowledge of a
sample. For isocratic separation, you may need to perform several runs before you can find
conditions that yield a retention factor ( k) in the desired 1 , k , 20 region. Third and maybe
most important, scouting gradients allow analysts to determine if a final isocratic method will
be possible or if a gradient will be necessary. With this knowledge, chromatographers can
use scouting gradients to predict effective isocratic conditions. This final column in the
“Starting Out Right” series will consider these three advantages of gradient scouting runs in
the LC method development process.
When Will It Be Eluted?
When I presented the strategy of using step-wise mobile -phase strength changes to identify
effective isocratic conditions (3), I acknowledged a major weakness of this technique.
Wasted runs are almost guaranteed. The first run usually is made with 100% organic
solvent (solvent B) or perhaps 90% solvent B. Under these conditions, nearly every
compound will come off the column very quickly. In my previous discussion, I reduced
mobile-phase strength in 10% increments until a separation began to appear (3). I used the
Rule of Three as a guideline that a 10% change in the percentage of organic solvent
generates an approximately threefold change in retention. Even with experience and luck,
chromatographers generally need four to six runs before they can obtain a separation in
which the peaks are eluted in the desired 1 , k , 20 region. Next, they undertake
adjustments to improve selectivity.
One advantage of gradient elution is that runs can be designed to end at a high percentage
of organic solvent, often 100% solvent B. At 100% solvent B in isocratic separations, most
compounds are eluted with k , 1. In a similar manner, when the gradient reaches 100%
organic solvent, compounds that haven’t been eluted yet generally will be eluted in less
than 5 min. So now for a scouting run ending at 100% organic solvent, in most cases we ’re
guaranteed that all sample peaks will be eluted within 5-10 min after the gradient is
complete. For example, a 20 -min gradient followed by a 10-min hold should be sufficient for
the elution of all components from nearly any sample.
From a practical standpoint, you can compare the use of a scouting gradient with the
sample used for Figures 1-3. If you use the isocratic step procedure - starting with 100%
acetonitrile, 90% acetonitrile, and so forth - and allow 5 min after the last peak is eluted
before changing to the next solvent plus a 10-min solvent changeover, it will take just less
than 3 h before you find conditions under which the first peak is eluted with k . 1. Contrast
this situation with a scouting run of 20, 30, or even 60 min, in which all the peaks would be
eluted within the defined gradient time.
Another advantage of the gradient as a scouting run is that it automatically flushes the
column between each run. You don’t have to wait for an undetermined amount of time after
each run and hope that all the sample and matrix components have been eluted before
setting up for the next run.
What is the Best Starting Gradient?
With isocratic separations, I demonstrated that conditions for good chromatography were
those that generated 1 , k , 20, or better yet 2 , k , 10 (3). These conditions were found by
step-wise exploration of isocratic conditions. For gradient separations, the solvent strength
changes within the run, which means that k values change within the run, so the isocratic
expression of retention factors doesn’t apply with gradients. Instead, I can use the average
retention factor ( k*) to express retention factors in gradient elution. It turns out that k* values
in gradient elution are roughly the same for all peaks in the run. A target for good
chromatography in gradient elution sets is 1 , k* , 10. I like k* to be approximately 6 for a
scouting run. Fortunately, gradient elution theory is developed well enough that it is possible
to predict separation conditions that can generate any desired k* values; for example, see
chapter 8 of reference 6. For k* approximately equal to 6, theory tells us that for a full-range
(5-100%) gradient:
t = 30V /F ……[1]
G M
where t is the gradient time in minutes, V is the column volume in milliliters, and F is the
G M
flow rate in milliliters per minute. The recommended initial conditions I’ve been using so far
in this series are a 150 mm X 4.6 mm column with a column volume of approximately 1.5
mL at a flow rate of 1.5 mL/min. These conditions mean that a 30 -min gradient should, with
high probability, yield a good separation on the first injection. Obviously, adjustments will be
necessary to get the best separation, but as a starting point, these conditions are likely to
generate a useful chromatogram with the first injection.
Is Isocratic Possible?
The conditions shown in Table I are recommended starting conditions, according to the
guidelines of equation 1. These conditions are the gradient version of the isocratic starting
conditions recommended in Table II of reference 1. Under these conditions, you should
allow at least 10 min to equilibrate a column under the initial solvent conditions (5% B)
between each run and place a 5 -min hold at 100% B at the end of the gradient. If phosphate
buffer will be used, it is wise to verify the solubility of the buffer in acetonitrile on your LC
system before making the first gradient run. Some systems have problems mixing
phosphate and acetonitrile when the acetonitrile composition exceeds 80%. Problems can
be minimized by dropping the phosphate concentration to 10 mM or choosing a more
soluble buffer such as trifluoroacetic acid.
Table I: Recommended starting conditions for a scouting run with

reversed -phase LC method development
Separation
Peferred Initial Choice
Variable
Column
Dimensions 150 mm X 4.6 mm, 5-µm dp, or 75 mm X 4.6 mm, 3.5 -µm dp
Stationary Phase C8 or C18
Mobile Phase
Solvent A Water or buffer
Solvent B Acetonitrile
Buffer 25 mM Phosphate (pH 2.5), 0.1% trifluoroascetic acid, or
formic acid
Flow Rate 1-2 mL/min
Gradient Range 5-100% solvent B
Gradient Time 30 min with a 5-min hold for a 150-mm long column or 15 min
with a 5-min hold for a 75-mm long column
Temperature 35° C
Figure 1 shows an example of the results of a scouting run using the conditions of Table I
for a 14-component polynuclear aromatic hydrocarbon sample. By examining the
chromatogram of Figure 1, you can see the complexity of the sample and get an idea of
how difficult it will be to obtain a satisfactory separation. Although all the peaks are not
baseline separated, all 14 peaks are present in the sample, so I would feel confident that
fine-tuning the conditions will allow me to obtain a separation with baseline resolution of all
peaks.
Figure 1: Simulated chromatogram for a separation of 14
polynuclear aromatic hydrocarbon compounds. See text for details.
Most chromatographers desire an isocratic method, if it is possible. Gradient methods often

can be more problematic, especially in routine production or quality control environments.
One advantage of using a scouting gradient is that analysts can determine whether an
isocratic run is possible. The process is simple. First, determine the retention time range
(Dt ) by subtracting the retention time of the first peak (t ) from that of the last peak (t ) .
g i f
Divide the retention time range by the gradient time (t ) to find the portion of the run
G
occupied by peaks. If this value is less than 25% of the run, an isocratic method should be
successful. If the retention-time envelope exceeds 40% of the run time, an isocratic method
probably will be impossible, so you should pursue gradient optimization. If the retention time
range is 25-40% of the run time, an isocratic separation may be possible and should be
explored if an isocratic method is the first choice.
For the example of Figure 1, the retention times of the initial and final peaks are
approximately 18.5 and 28.5 min, respectively, so Dtg is 10 min. This time period can then
be used to calculate Dt /t ' 10/30 ' 0.33 . This amount falls between the clear guidelines
g G
for isocratic and gradient methods, so you could explore both methods, if desired.
Selecting Isocratic Conditions
Continuing with the example of Figure 1, first assume that you desire an isocratic
separation. You can use the information from the scouting run to find conditions for an
isocratic separation with the aid of Figure 4. First, determine the average retention time for
the scouting run - (t 1 t )/2 ' (28.5 + 18.5)/2 ' 23.5 min . Then, select the column at the
f i
top of Figure 4 corresponding most closely to the column length (assuming an inner
diameter of 4.6 mm) and the system dwell volume (V ). The figure shows typical dwell
D
volumes for low-dwell volume systems, such as Agilent Technologies’ HP 1100 LC system
(Wilmington, Delaware) and Waters’ model 2690 LC system (Milford, Massachusetts), high-
pressure mixing systems (2.3 mL), and low-pressure mixing systems (4.5 mL).
The current example was modeled on a 150 mm X 4.6 mm column with a high-pressure
mixing system, so choose data under the 150/2.3 heading. Find the retention time below the
heading that corresponds closely to the average retention time for the scouting run and read
the recommended isocratic mobile phase to the left. The average retention of 23.5 min falls
between 21 and 25 min, so I’d choose 65% acetonitrile for the first isocratic run. This mobile
phase should provide an isocratic k value of approximately 4 for the peaks in the middle of
the run. Because Figure 4 is based on several generalizations, it is likely that the first
isocratic run will be a bit long or a bit short, but it should give you a reasonable starting point
for further isocratic optimization.
Figure 2 shows a simulated run under the recommended conditions of Figure 4. Only 12 of
the 14 sample components are clearly visible in the separation. The peak at 20 min
obviously is a doublet, but the location of the second overlapped peak pair (peaks 1 and 2)
is not apparent. The k range for the separation is 1 , k , 22, which is a bit broad but usable.
Earlier “LC Troubleshooting” columns in this series showed that resolution generally
increased with increased overall retention. Improving this separation would require even
longer run times. With these results, I would be pessimistic about obtaining an isocratic
method that yields baseline separation of all the peaks in the sample.
Figure 2: Simulated isocratic

separation of the sample in
Figure 1 using a 65%
acetonitrile mobile phase.
The Next Gradient
With discouraging results for the prospects of developing a good isocratic separation, you
could explore a gradient method next. By examining the separation of Figure 1 again, you
see that all 14 components are nearly baseline resolved, but the first half of the
chromatogram is completely wasted. A simple way to think about peak movement in
gradients is that a given analyte sits at the head of the column until the arrival of a solvent
strong enough to push the analyte through the column. With this in mind, it seems that
almost half of the gradient is unused, so perhaps starting at 50% B would work. Similarly,
you should be able to stop the gradient as soon as the last peak is eluted. Rather than
guess at the starting conditions, you can use Figure 5 to guide the selection of new initial
and final gradient conditions.
Use Figure 5 in the same manner as Figure 4 to find the data set corresponding to the
column and LC system - a 150-mm long column and a 2.3 -mL dwell volume for this
example. Look down in the table until you find the retention time of the initial peak and read
the recommended initial percentage of solvent B at the left. In this case, 18.5 min falls
between 17 and 20 min, so I would select 45% acetonitrile for the new starting percentage
of solvent B. Similarly, you can find the retention time of the final peak (28.5 min) and read
to the right to find the recommended final percentage of solvent B (90% acetonitrile). This
figure tells us that we can get rid of the wasted time in the run of Figure 1 by using a
gradient of 45 -90% acetonitrile instead of the 5 -100% range selected for the scouting
gradient.
In gradient elution, peak spacing and k* values are controlled by the steepness of the
gradient in a manner similar to changing solvent strength (percentage B solvent) in isocratic
separation. For this reason, it is good to make the next run with the refined gradient range
under conditions that give the same gradient steepness (percentage B solvent per minute)
as in the initial run. This new gradient rate is calculated as a simple proportion based on the
scouting run:
……[2]
So the next gradient run should be 45-90% acetonitrile in 14 min, with all other conditions
the same as the scouting run. Figure 3 shows the results of this new run. The separation is
roughly the same as that in Figure 1, but the run time is approximately 12 min shorter. The
chromatogram still has a 6 -min region at the beginning of the run in which no peaks are
eluted, so it may be possible to increase the starting percentage of solvent B a little more,
perhaps to 50%. Even so, peaks occupy approximately two-thirds of the separation time
instead of the one-third used in Figure 1. You could continue from here by trying different
gradient times to optimize the gradient separation.
Tying It All Together
This month’s column ends the “Starting Out Right”

series, which was intended to provide some useful
tips for developing successful LC separations. By
following the guidelines presented in these “LC
Troubleshooting” columns, you can avoid many of
the problems of trial-and-error method development.
As I pointed out in this installment, I prefer to start the

Figure 3: Simulated gradient method development process with a scouting
separation of sample in Figure gradient. The scouting gradient gives me information
1 using a gradient of 45 about the complexity of the separation, the likelihood
of a successful separation using the selected solvent
system, and a way to determine whether it is best to
develop an isocratic or gradient separation.
After the initial scouting run is out of the way, Figures

4 and 5 will help guide you to appropriate conditions
for the next runs. Whether optimizing isocratic or
Resources Inc. of Walnut Creek, California, and a member of
LCGC’s editorial advisory board. Direct correspondence about
this column to “LC Troubleshooting,” LCGC, 859 Willamette
Street, Eugene, OR 97401, e-mail John.Dolan@
LCResources.com.
For an ongoing discussion of LC troubleshooting with John

Dolan and other chromatographers, visit the Chromatography
Forum discussion group at www.chromforum.com.

Resources Inc. of Walnut Creek, California, and a member of
LCGC’s editorial advisory board. Direct correspondence about
this column to “LC Troubleshooting,” LCGC, 859 Willamette
Street, Eugene, OR 97401, e-mail John.Dolan@
LCResources.com.
For an ongoing discussion of LC troubleshooting with John

Dolan and other chromatographers, visit the Chromatography
Forum discussion group at www.chromforum.com.

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Thu Aug 30 15:28:35 2001.

Starting Out Right,

Liquid chromatography (LC) problems tend to fall into

OPEN YOUR CUPBOARD

One of the most important choices in method development is column selection.

TABLE I: Alternative Sample and Method Combinations

Most samples encountered by average chromatographers will be amenable to reversed-

ORGANIC SOLVENT SELECTION

AQUEOUS PHASE SELECTION

Sample characteristics and column pH stability combine to suggest that a low pH is

I cannot overemphasize the importance of choosing starting conditions carefully. The

TABLE II: Recommended Starting Conditions for Reversed-Phase LC

(1) R.E. Majors, LC Magazine 2(3), 202-208 (1984).

(4) J. Zhao and P.W. Carr, LCGC 17(4), 346-352 (1999).

“LC Troubleshooting” editor John W. Dolan is president of

Feature Index - Home -Main Index

Copyright Advanstar Communications

Starting Out Right,

Last month’s “LC Troubleshooting” discussed selecting

The k value can be calculated from equation 1

One of the first things experienced

Column Plate Number

In addition to peak shape, it is important to

A new column containing 5-µm particles

Resolution is the separation between two peaks in a chromatogram, defined as

Part ii of equation 5 relates to selectivity - the ability of the column-mobile phase

Putting the Tools to Work

(1) J.W. Dolan, LCGC 17(12), 1094-1097 (1999).

“LC Troubleshooting” editor John W. Dolan is president of

Starting Out Right,

In the first installment of this series (1), the discussion

Resolution is the Key

For purposes of method development, the fundamental resolution equation below is a

Before getting into the specifics of

Adjust Solvent Strength First

The (Not So) Special Case

Figure 3: Simulated chromatograms generated using (a) 48% B, (b)

Figure 4: Simulated chromatograms obtained using (a) methanol-

(1) J.W. Dolan, LCGC 17(12), 1094-1097 (1999).

(2) J.W. Dolan, LCGC 18(1), 28-32 (2000).

“LC Troubleshooting” editor John W. Dolan is president of

Starting Out Right,

Last month’s installment of “LC Troubleshooting”

For separations discussed in previous installments in this series, I suggested aqueous-

Figure 1: The influence of pH on retention of a

Table I: Conditions for Figures 2 and 3

Variable Figure 2 Figure 3

Column 250 mm X 4.6 mm, 5 µm dp C8 Same

Flow rate 1 mL/min Same

Methanol (%) 35% Same

Buffer 25 mM phosphate Same

When using pH to control an LC separation, it is especially important to obey the

Figure 4: Separation of a substituted Figure 5: Separation of an herbicide

By the time chromatographers have explored mobile-phase solvents, pH, temperature,

(1) J.W. Dolan, LCGC 18(2), 118-125 (2000).

(2) J.W. Dolan, LCGC 17(12), 1094-1097 (1999).

(3) J.W. Dolan, LCGC 18(1), 28-32 (2000).

“LC Troubleshooting” editor John W. Dolan is president

Starting Out Right,

Resolution, pressure, and run time - choose any

In the four preceding installments of “LC

Back to the Basics

Flow Rate - Simple Things First