Section 1

What's Wrong With My HPLC Column

A Quick Reference

This section offers quick diagnostic guidelines on how to identify and prevent problems related
to your HPLC column. Many of the subjects presented here are discussed in more detail in later
sections. References to these sections are noted where appropriate.

The HPLC column is the heart of a separation and analysis. But when a problem arises in HPLC
analysis, it can be dfficult to determine if it is caused by the column or another part of the
system. Often the HPLC column is identified as responsible for the problem. More often than
not, these problems are due to other factors, such as changes in the mobile phase, shifts in flow
rate, variations in temperature, differences in the sample, or problems with the instrumentation.

When a chromatographic problem results in column failure, proper corrective action should be
taken to avoid another failure. Identifying the cause of the problem may seem like a difficult,
time-consuming task. Fortunately, there are only a limited number of reasons why a column
would perform poorly.

The following will summarize several common problems that can result in HPLC column
failure. After the description of each problem there is a list of possible problem causes along with
the recommended corrective action. By referring to these guidelines when a problem arises, you
can be well on your way to isolating and correcting any HPLC column problem you might
encounter.

PROBLEM: An unacceptable increase in system back pressure.

Possible Cause: Corrective Action to Take:

Column developed a void Fill in the void with similar packing
material, or replace column
"Worn-out" guard column Replace guard column

Non-column causes: extra column effects (damaged or improperly made fittings/connections),

increase in injected sample volume, increase in strength of sample solvent, changes in mobile
phase composition.

PROBLEM: Peak tailing or peak splitting

Possible Cause: Corrective Action to Take:

Column fouling Clean or replace column
Particulates on column inlet frit Replace inlet frit
Loss of bonded phase Replace column
"Worn-out" guard column Replace guard column
Bad sample distribution Change to a column with a better
inlet design

Non-column causes: extra column effects (damaged or improperly made fittings/connections),

injected sample overload, changes in mobile phase composition (additives, pH, buffer
concentration).
PROBLEM: Mystery peaks

Possible Cause: Corrective Action to Take:

Column fouling Clean or replace column
"Worn-out" guard column Replace guard column

Non-column causes: impurities in the mobile phase, additional components in the sample, air
bubbles in the sytstem, electronic problems.

Section 2
Steps for Ensuring Good Column Performance

This section provides some general information on conditions that affect column lifetime. The
information is divided into four practical steps that help ensure good column performance.

Step 1 shows you several methods to reduce column failure and increase the usable lifetime of
your HPLC columns.

Over the years, advances in column technology have resulted in improved column performance
and extended column life. Depending upon the sample and sample matrix, chromatographic
conditions, and the column packing itself, an HPLC column can support anywhere from 50 to
2000 injections. Step 2 provides guidelines that can help you achieve the maximum column
lifetime for your conditions. Step 3 shows how and when to use a guard column. This one action
can dramatically improve column lifetime.

Even when you practice all of these good habits columns will eventually need to be replaced.
Step 4 tells you which HPLC parameters to monitor so that you can spot column failure early
and avoid a crisis.

Maximizing the number of analyses you can achieve with an HPLC column saves time and
money. If the purchase price of a column is $400, the cost of analysis attributed solely to the
column can vary from $0.20 to $8 per injection depending upon the durability of the column.
Extending column life maximizes sample throughput and reduces time troubleshooting
chromatography problems.

Follow these four steps and you will save time and money in your laboratory.
 Step 1: Prevent the Three Most Common Causes of Column Failure

Most people would like to extend column lifetime. This can be done by minimizing the causes of
column failure. The three most likely reasons for reversed-phase column failure are:

 loss of bonded phase

 column voiding
 column contamination

Loss of Bonded Phase

As bonded phase strips off the silica support, retention times and resolution will change, and
peaks may tail more.This is the most common reason that columns "wear out."

Cause:

Hydrolysis of the siloxane bond of the bonded phase or endcapping reagents.

It is most severe with a mobile phase pH of less than 3.

Prevention:
 Use stabilized bonded phases (i.e. StableBond? when you need to operate below pH 3
(e.g. phosphate buffered mobile phases pH 1-3). StableBond column materials are more
resistant to acid hydrolysis. Operate other types of columns within a pH range of 3 to 7.
 Operate at a mobile phase temperature of less than 60 캜. A StableBond SB-C18 column
can be used up to a temperature of 90 캜 at low pH.

Monitor:
Capacity factor, selectivity and tailing factor. (See Step 4)

Column Voiding

When a void develops at the head of an HPLC column, efficiency drops (low N); resolution
between peaks decreases, and peaks broaden, tail, or split into doublets.

Causes:

 Poorly packed columns that settle during use.

 Dissolving the silica column packing by operating at high pH.
 Excessive system back pressure or pressure surges due to poorly operating pumps or
sample injection valves.

Prevention:
 Keep the operating pressure under 5000 psi and eliminate any large pressure surges.
 Purchase quality columns from suppliers with the expertise to pack columns well.
 When using a mobile phase pH greater than or equal to 7, operate columns at less than 40
캜 to reduce silica solubility in the mobile phase.
 Operate with a mobile phase pH of less than 7. If you need to operate at or above pH 7
use a silica saturator column between the pump and the injector. The silica in this column
will dissolve first, saturating the mobile phase with dissolved silica, extending column
 life. Use columns with dense, hydrophobic bonded phases, such as the Eclipse™ XDB-
C8, to protect silica from dissolution.

Monitor:
Theoretical plates and tailing factor. (See Step 4)

Column Contamination

Column fouling results from a buildup of retained material on the stationary phase. Buildup can
lead to shifts in peak retention and loss of resolution. Peak shape may also be adversely affected
by column fouling. Particulate matter or precipitated samples and buffers will also foul a column
by plugging column frits and column packing.

Causes:

 Samples containing compounds that do not elute from the column with the mobile phase
being used. This is common when using low organic mobile phases.
 Impurities in the mobile phase that adsorb to the column's stationary phase. This is
common when using ion-pair chromatography and other mobile phase additives with low
organic isocratic mobile phases.
 Particulate matter in the samples or mobile phase plugging the column inlet frit.

Prevention:
 Clean up samples before injection. This may include filtering the samples to remove
particulates and solid phase extraction techniques to remove highly retained sample or
matrix components.
 Use HPLC grade solvents for the mobile phase and filter buffer solutions.
 Routinely flush the column with a strong solvent (i.e. 100% acetonitrile) to elute retained
material off the stationary phase.
 Place a guard column in-line between the sample injection valve and the analytical
column to collect non-eluting; compounds and protect the analytical column. Change the
guard column frequently.

Monitor:
Theoretical plates, capacity factor, selectivity, and back pressure.
(See Step 4)
 Step 2: Treat Your Reversed-Phase Column Well

Comments
Minimize pressure Make rapid injections to reduce pressure changes during
surges sample injection. Maintain pumps to minimize pressure swings
resulting from inconsistent flow rates.
Use a guard column Place each of these before the column and after the injector. An
and/or an in-line 0.5 in-line filter will catch large particulates and a guard column
탆 filter. will prevent strongly adsorbed materials from reaching your
analytical column.
Frequently flush Flushing with 100% acetonitrile is often adequate, but if
columns with a stronger solvents are needed, consider methylene chloride
strong solvent. (CH2Cl2). Less-polar solvents, like CH2Cl2, are strong
solvents in reversed-phase chromatography. Many strong
solvents are immiscible with aqueous containing mobile
phases. Remember to flush the column and HPLC system with
isopropanol prior to and after the use of CH2Cl2.
Pretreat "dirty" Solid phase extraction, filtering sample through 0.45 탆 filters,
samples to minimize or high-speed centrifugation are useful pretreatment
strongly techniques.
Use column Check column manufacturer specifications.
temperatures of less
than 60 캜.
Keep mobile phase If operating outside the 3 - 7 pH range, choose a column
pH between 3 and 7 designed for low pH (StableBond) or high pH (Eclipse XDB).
for silica based
columns.
Add 100-200 ppm Stagnant aqueous reservoirs readily generate bacterial growth
sodium azide to which can cause dramatic baseline disturbances and plug
prevent bacterial columns.
growth.
When storing This prevents precipitation of buffer salts in the column.
columns, purge out Acetonitrile is a good storage solvent because aqueous and
salts and buffers. alcohol mobile phases can increase the rate of stationary phase
Leave column in hydrolysis
pure acetonitrile

Step 3: Know When to Use and Change a Guard Column

The main purpose of the guard column is to act as a trap for highly retained sample
components and for particulate matter. It is installed after the injector and before the analytical
column. Protecting your analytical column by using guard columns can substantially increase
column lifetime. But it is important to know when to change the guard column for the best
protection of the analytical column. To be effective the guard column must be replaced often
enough to prevent contaminants from reaching the analytical column.
Monitoring Chromatographic Parameters:
A "quantitative" way of knowing when to replace a guard column

The best way to determine the right time to replace a guard column for a specific set of sample
and mobile phase conditions is through experience. However it is valuable to have some
quantitative measure to help make the replacement decision. By monitoring plate number (N),
pressure (P), and resolution (Rs), the performance of the guard and the analytical column, can be
closely watched to determine when a guard column should be replaced.

When N, P, or Rs changes by more than 10%,

the guard column should be replaced.

IMPORTANT NOTE:
Even though monitoring N, P, and Rs provides clues as to when guard columns should be
replaced, you cannot always be certain if the guard column is adequately protecting the
analytical column. Fouling of the analytical column can still take place (due to a saturated guard
column) before a 10% change in N, P, or Rs is observed. It is always better to replace the guard
column too soon rather than too late.

In the absence of other information, a good rule-of-thumb is to replace the guard column after
every 150 sample injections or 1,000 analytical column volumes of mobile phase, whichever is
reached sooner.

Step 4: Quickly Determine When a Column is Going Bad

Eventually all columns lose performance and must be replaced. Timing depends on the
chromatographic conditions used as well as the column itself. By monitoring column
performance you can tell when your column is starting to "wear out" and predict how often your
columns will need to be replaced. Also by understanding the major factors that limit column
lifetime, steps can be taken to extend the usable lifetime of a column. The important column
parameters to monitor are theoretical plates (N), capacity factor (k), selectivity ( ), tailing factor
(Tf), and back pressure. Many data systems allow these parameters to be printed as a part of the
chromatographic report, simplifying the job of monitoring column performance.
 Parameter What to Look For
Theoretical Theoretical plates is a measure of the efficiency, or resolving
plates, N power, of a column. The most common reasons for losing
(efficiency) theoretical plates are column voiding and column fouling. Peaks
in your chromatogram broaden with decreasing efficiency. By
monitoring N, you can detect these problems long before they
affect your chromatographic separation.
Capacity Capacity factor is a measure of retention independent of flow
factor, k rate and column dimension. Changes in capacity factor under
constant chromatographic conditions may indicate either
problems with loss of bonded phase or problems with column
fouling due to non-eluting compounds. However, changes in
capacity factor may also be caused by changes in mobile phase
composition that give the false impression of a column problem.
Selectivity, Selectivity is a measure of the relative retention of two
compounds. Shifts in selectivity are an additional indication,
along with capacity factor, of problems with loss of bonded
phase, column fouling, or changes in mobile-phase conditions
Tailing factor, Tailing factor is a measurement of peak symmetry. An increase
Tf in the tailing factor may indicate a problem with column
voiding but may also result from an interaction between polar
solutes and silanol sites, permitted by the loss of bonded phase.
If column back pressure increases, it is almost always due to
Column back particulates that have collected on the column inlet frit.
pressure, P However, column voiding induced by column packing collapse
can cause a large surge in pressure. Monitoring these HPLC
parameters helps you decide when it is time to replace your
column.

Section 3
What is the Best Column
Configuration for Your Method?

Many chromatographers establish the initial separation on a 4.6 x 250 mm column

because it gives excellent resolution and reasonable analysis time for method development.
When it is time to make a method routine, it is useful to consider other column configurations to
do the analysis as quickly and inexpensively as possible.

Will a Shorter Column Work for Your Application?

It is not necessary to actually install, equilibrate and run a sample on a shorter column to
determine if it will provide sufficient resolution for your sample mix. if you already have data
from a longer column, a few quick calculations will tell you if a shorter column having the same
particle size will work.
Estimating Change in Rs Estimating Changes in Analysis Time
and Back Pressure
Resolution, Rs, changes directly with In similar fashion, we can predict how
the square root of the change in column analysis time (T) and back pressure (P)
plate number. Since plate number will change with column length.
changes directly with column length, we
know that resolution will change T2 = T1 x L2/L1
directly with the square root of the ratio P2 = P1 x L2/L1
of column lengths (L).
Where:
Rs2 = Rs1 x (L2/L1)0.5 T1 = Analysis time with column #1
T2 = Analysis time with column #2
Where: P1 = Pressure with column #1
Rs1 = Resolution with column #1 P2 = Pressure with column #2
Rs2 = Resolution with column #2
L1 = Column #1 length
L2 = Column #2 length

An Example

Figure 3-1

Column: 4.6 x 150 mm; Pressure: 1050

psi
Analysis time: 18 min; Rs(3,4) = 3.7

Column: 4.6 x 150 mm; Pressure: 630 psi

Analysis time: 11 min; Rs(3,4) = 2.9
 Suppose that you are using a 25 cm column. The resolution between the least resolved
peak pair is 3.7, the total analysis time is 18 minutes and the back pressure is 1050 psi. If
resolution greater than 2.5 is required for this analysis, could a 15 cm column be used in place of
the 25 cm column? How would the analysis time and pressure change?

Rs2 = 3.7 x (15/25)0.5 = 2.9

T2 =18 min x 15/25 = 10.8 min
P2 =1050 psi x 15/25 = 630 psi

Where:
Rs2 = Resolution with 15 cm column
T2 = Analysis time with 15 cm column
P2 = Pressure with 15 cm column

It is clear from the analysis that a 15 cm column could be substituted and perform satisfactorily.
This change would result in using 40% less solvent and reducing analysis time by 7 minutes.

The example above shows that you can quickly determine if a shorter column will work for a
particular analysis without having to actually install and run the column. This can save you many
hours of trial-and-error work in the laboratory.

Will a Smaller-Bore Column Work for Your Application?

The availability of analytical HPLC columns with internal diameters of less than 4.6 mm
provides the chromatographer with options for developing more cost efficient
methods. Solvent Saver™ (3.0 mm i.d.) and narrow-bore (2.1 mm i.d.)
columns provide several important benefits for chromatographers. These
include:

Reduced Mobile Phase Expenses

- less solvent usage
- lower solvent disposal costs
Enhanced Sensitivity for Mass-Limited Samples
- Solvent Saver columns can increase sensitivity by a factor of 2
- Narrow-Bore columns can increase sensitivity by a factor of 5
Reduced Sample Size Requirements
-smaller sample sizes can be used effectively
-sample preparation requires smaller volumes of reagents
Increased Compatibility with Special Detectors
-LC/MS
 - LC/NMR

These potential benefits are all a result of the reduced internal column volume of smaller
i.d. columns.

Solvent Consumption is Directly Related to Column Volume

One way to reduce solvent consumption in HPLC is to use columns with a smaller
internal diameter than "standard" 4.6 mm i.d. columns Solvent Saver and narrow-bore columns
can reduce solvent consumption by 60-80%. Table 3-1 shows the relationship between column
internal diameter, column volume, and solvent waste reduction.

Table 3.1

Column Internal
Dimensions Calculated Solvent % Solvent
Volume Peak Volume Consumption
mm mL 킠 mL Reduction
4.6 x 250 2.50 550 18.9 19,000 0
4.6 x 150 1.50 430 11.4 11,000 40
3.0 x 250 1.06 240 8.0 19,000 58
3.0 x 150 0.64 188 4.8 11,000 74
2.1 x 150 0.30 94 2.4 11,000 87

NOTE: k of last eluting peak is 6.6

Reduced column internal diameters result in smaller column volumes. It is

this reduction in column volume that allows for an increase in detection
limits.When the column volume is decreased the peak volume is decreased
(Table 3-1). This means that when the same sample volume is injected on a
4.6 mm i.d. column and a 3.0 mm i.d. column, the peak volume will be
smaller on the 3.0 mm i.d. column. The same sample is more concentrated in
the smaller peak volume and the result is enhanced sensitivity. [NOTE: Be
sure to read the discussion on Extra-Column Volume - Section 4]

Optimum Efficiency with Small-Bore Columns is Directly

Related to Peak Volume

Optimum efficiency (N) requires that your HPLC be compatible with small-
bore columns. The peak volume of each eluting band decreases significantly
as the diameter of the column is decreased (Table 3-1). Peaks with low k
values are especially critical, as they have the lowest peak volume in the
chromatogram.

A well-designed standard HPLC system may tolerate column peak volumes

as low as 80 킠 and still provide excellent efficiency. However, peak volumes
below 80 킠 will require careful system design. Standard 4.6 mm i.d. and 3.0
mm i.d. Solvent Saver columns can be used on most HPLC systems with
optimum efficiency, but 2.1 mm i.d. narrow-bore columns may require HPLC
system optimization to be used effectively (see Section 4, Correcting
Excessive Extra Column Volume). Columns below the 2.1 mm i.d. size
require sophisticated HPLC hardware systems not commonly found in today's
laboratories.

Small-Bore Columns Require Reduced Flow Rates

Many chromatographers use columns of reduced column diameter (< 4.6

mm i.d.) to reduce solvent waste or increase sensitivity when sample is
limited. No matter what your goal is for switching to a reduced diameter
column, it is important to know that a simple column substitution without
modification of flow rate will compromise resolution. Substituting a column
having the same packing, the same length, but a smaller diameter requires
that you reduce the flow rate in order to retain the same retention time and
resolution for peaks in the original chromatogram.

Figure 3-2
Separation of Nitrobenzenes on Columns of Different Diameters
with the Same Flow Rate

Figure 3-2 illustrates what you can expect if you substitute columns of
smaller inner diameter without reducing now rate. In this example, the same
sample is separated using identical experimental conditions on columns
having 4.6 mm, 3.0 mm and 2.1 mm inner diameter (i.d.).The flow rate for
each separation is 1.0 ml/min.The resultant chromatograms are very
different.The retention and critical resolution between peak pair 2,3 on the
4.6 mm i.d. column decreases significantly on the 3.0 mm i.d. column and
even more so on the 2.1 mm i.d. column.

As the column diameter decreases, the column void volume (Vm) decreases.
The column void volume for the three columns used in Figure 3-3 is listed in
the Table below.
 Table 3-1
Column Internal
Dimensions Calculated Solvent % Solvent
Volume Peak Volume Consumption
mm mL 킠 mL N Reduction

4.6 x 250 2.50 550 18.9 19,000 0

4.6 x 150 1.50 430 11.4 11,000 40
3.0 x 250 1.06 240 8.0 19,000 58
3.0 x 150 0.64 188 4.8 11,000 74
2.1 x 150 0.30 94 2.4 11,000 87

NOTE: k of last eluting peak is 6.6

Reduced column internal diameters result in smaller column volumes. It is this reduction in
column volume that allows for an increase in detection limits.When the column volume is
decreased the peak volume is decreased (Table 3-1). This means that when the same sample
volume is injected on a 4.6 mm i.d. column and a 3.0 mm i.d. column, the peak volume will be
smaller on the 3.0 mm i.d. column. The same sample is more concentrated in the smaller peak
volume and the result is enhanced sensitivity. [NOTE: Be sure to read the discussion on Extra-
Column Volume - Section 4]

Optimum Efficiency with Small-Bore Columns is Directly Related to Peak Volume

Optimum efficiency (N) requires that your HPLC be compatible with small-bore columns. The
peak volume of each eluting band decreases significantly as the diameter of the column is
decreased (Table 3-1). Peaks with low k values are especially critical, as they have the lowest
peak volume in the chromatogram.

A well-designed standard HPLC system may tolerate column peak volumes as low as 80 킠 and
still provide excellent efficiency. However, peak volumes below 80 킠 will require careful
system design. Standard 4.6 mm i.d. and 3.0 mm i.d. Solvent Saver columns can be used on most
HPLC systems with optimum efficiency, but 2.1 mm i.d. narrow-bore columns may require
HPLC system optimization to be used effectively (see Section 4, Correcting Excessive Extra
Column Volume). Columns below the 2.1 mm i.d. size require sophisticated HPLC hardware
systems not commonly found in today's laboratories.

Small-Bore Columns Require Reduced Flow Rates

Many chromatographers use columns of reduced column diameter (< 4.6
mm i.d.) to reduce solvent waste or increase sensitivity when sample is
limited. No matter what your goal is for switching to a reduced diameter
column, it is important to know that a simple column substitution without
modification of flow rate will compromise resolution. Substituting a column
having the same packing, the same length, but a smaller diameter requires
that you reduce the flow rate in order to retain the same retention time and
resolution for peaks in the original chromatogram.

Figure 3-2
Separation of Nitrobenzenes on Columns of Different Diameters
with the Same Flow Rate

Figure 3-2 illustrates what you can expect if you substitute columns of smaller inner diameter
without reducing now rate. In this example, the same sample is separated using identical
experimental conditions on columns having 4.6 mm, 3.0 mm and 2.1 mm inner diameter
(i.d.).The flow rate for each separation is 1.0 ml/min.The resultant chromatograms are very
different.The retention and critical resolution between peak pair 2,3 on the 4.6 mm i.d. column
decreases significantly on the 3.0 mm i.d. column and even more so on the 2.1 mm i.d. column.

As the column diameter decreases, the column void volume (Vm) decreases. The column void
volume for the three columns used in Figure 3-3 is listed in the Table below.
 Table 3-2
Changing Column Diameter Requires Flow Rate Adjustment

Column Column Void Relative Flow

Volume (mL) Rate (mL/min)

4.6 x 150 mm 1.5 1.0

3.0 x 150 mm 0.6 0.4

2.1 x 150 mm 0.3 0.2

k = [tr - (Vm / F)] / (Vm / F)

If the mobile phase and stationary phase is unchanged, k is unchanged. To maintain the same
retention time (tr), the flow rate (F) must decrease to compensate for a decrease in column void
volume (Vm).

For example:
Vm1 / F1 = Vm2 / F2 ; 1.5 / 1.0 = 0.6 / F2
1.5 F2 = 0.6
F2 = 0.4 mL/min

To keep retention time unchanged, the ratio Vm/F must be held constant for each separation.
Therefore, a decrease in Vm, must be followed with a proportional decrease in F (see Table 3-2
for example calculation). Figure 3-3 shows that running the 3.0 and 2.1 mm i.d. columns at 0.4
mL/min and 0.2 mL/min respectively, retrieves the original separation with the promised
reduction in solvent usage.

Figure 3-3
Separation of Nitrobenzenes on Columns of Different Diameters with the Adjusted Flow
Rate