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Gradient HPLC
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Contents Page
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1. Aims and Objectives
To explain the problems that can be encountered when using isocratic HPLC
To demonstrate how these problems can be overcome using gradient HPLC
To define the parameters of gradient elution HPLC
To explain and interactively illustrate how gradient elution HPLC works and how the
retention and elution processes differ from isocratic HPLC
To demonstrate why peak shapes in gradient HPLC are more efficient than those
obtained from isocratic HPLC
To examine the effects of gradient steepness and show how the various gradient
parameters can be practically determined
To interactively illustrate the use of scouting gradients in HPLC method development
and optimization
Examine the pitfalls and advantages of gradient elution HPLC in a practical situation
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2. Isocratic HPLC Analysis
When the composition of the mobile phase does not change during an analysis (i.e. the composition
is constant), the method is said to be isocratic.
Several potential problems are associated with isocratic analysis. These are listed below.
When the range of analyte polarities is broad, some analytes may be poorly retained and
resolution is lost with peaks eluting at or near the void volume (t0)
Alternatively, other analyte components may be significantly more hydrophobic and show
unacceptably long retention times
Due to the various band broadening processes, these late eluting peaks will be broad and
show reduced sensitivity due to reduced peak height
It is possible that some components will be irreversibly adsorbed on the column and cause
contamination
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3. Gradient HPLC Analysis
Many of the problems associated with Isocratic HPLC analysis can be overcome using gradient HPLC.
In this mode of analysis, the mobile phase composition is altered during the analysis – normally by
increasing the amount of organic modifier.
The initial composition is chosen so that the strength is appropriate to retain and resolve
early eluting analytes
The elution strength is then increased in a predetermined way to elute compounds with
optimum resolution
The final mobile phase composition is chosen to ensure elution of all compounds of interest
from the column within a reasonable time
It is possible to increase the organic modifier concentration to wash strongly retained,
potentially contaminating components from the column
Gradient elution is best suited to analyses carried out using reversed phase, normal phase
separations using bonded stationary phases, and for ion exchange chromatography. Particular
pumps are required to carry out gradient HPLC analysis, which allow on-line mixing of the mobile
phase components.
Improved resolution
Increased sensitivity
Ability to separate complex samples
Shorter analysis times
Decrease in column deterioration due to strongly retained components
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Re-equilibration time adds to analysis time
Instruments vary in their dwell volume (VD), which can cause method transfer problems
Column cleaning
Scouting runs for method development (more later)
Case Study 1
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Case Study 2
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Case Study 3
Figure 4: Gradient HPLC analysis of high molecular weight mixtures. Top - effect of % organic on
retention factor, bottom - chromatogram of high molecular weight separation.
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k* = gradient retention factor (target value of 5 for average separation)
tG = gradient time (min)
F = flow rate (mL/min)
ΔΦ = change in volume fraction of organic (Final %B – Initial %B) expressed as a decimal
Vm = column void volume (mL)
S = constant determined by strong solvent and sample compound (small molecules < 500 Da the
value is between 2 and 5; a value of 4 is used by convention when the value is not accurately known.
Proteins have much higher values, typically between 50-100, and need longer gradient times for
separation)
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4. Gradient Elution Parameters
Typically, two solvents are used, as indicated by solvent reservoirs A and B in the chromatographic
conditions shown. Reservoir A usually contains the weaker solvent, in this case, water with 0.1% TFA
(pH 2). Solvent reservoir B contains the stronger solvent, acetonitrile.
The elution strength usually increases with time, as shown above, where the gradient starts at 20%B
and ends at 60%B over 30 minutes. The gradient shown is a linear gradient that changes at a rate of
1.33% solvent B per min.
The mixing of the mobile phase composition is achieved using the HPLC pump, and two methods of
mixing are common. The first is low pressure mixing, in which the solvents are proportioned on the
low pressure side of the pump using solenoid valves. The second approach is known as high pressure
mixing and occurs when two or more pumps are used to deliver solvents at differing flow rates into a
mixing chamber.
To aid with solvent mixing, sometimes solvents are premixed or doped (i.e. solvent A contains 5%
solvent B and vice versa).
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5. Optimizing Gradient Parameters
Several parameters are required to be specified and optimized in a gradient HPLC method (Figure 1).
Initial %B - starting mobile phase composition (described in terms of the % of the strong solvent B).
Isocratic hold - a period within the gradient in which the eluent composition is held at the initial %B.
This achieves a degree of analyte focusing but also crucially enables easy transfer of gradients
between different instruments based on the specific instrument gradient dwell volume (VD).
Gradient time (tG) - the time during which the eluent composition is changing.
Purging - usually achieved using a short ballistic gradient ramp to high %B in order to elute highly
retained components (of no analytical interest) from the column. There may be an isocratic hold at
this composition to ensure elution of all analytes and strongly absorbed components of no analytical
interest.
Conditioning - returning the system (specifically the column) to the initial gradient composition. In
practice, with modern instruments, this step is programmed to occur very rapidly.
Equilibration - the time taken to ensure the whole of the analytical column is returned to initial
gradient composition. This is an important step and if not properly considered can lead to retention
time and quantitative variability. The equilibration time used is dependent on the column
dimensions and flow rate. It is recommend that 10 column volumes (Vm) of eluent at the initial
composition are used (calculate the time for the method flow rate and remember to add V D). The
re-equilibration time can be reduced empirically; the method should be monitored for any retention
time irreproducibility. The eluent flow rate can be increased during re-equilibration BUT ensure
stabilization prior to injection of the subsequent sample.
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The rate of change of the mobile phase composition during the gradient can be calculated from
equation 1.
(%Bfinal − %Binitial )
∆%B/min = (1)
tG
Optimization of the initial and final %B can be carried out using Equation 2 and 3.
VD
Optimized %Binitial = (%Binitial + (t i x ∆%B/min)) − ( x ∆%B/min) − 10%B (2)
F
VD
Optimized %Bfinal = (%Binitial + (t f x ∆%B/min)) − ( x ∆%B/min) − 10%B (3)
F
Where:
%Binitial = initial starting %B from scouting gradient
ti = elution time of the initial peak
tf = elution time of the final peak
Δ%B/min = rate of change of the mobile phase composition (Equation 1)
VD = dwell volume
F = flow rate
1. Schellinger, A.P; Stoll, D.R.; Carr, P.W. J. Chromatogr. A 2005, 1064, 143-156.
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6. Gradient Elution Principles
Gradient elution is most useful for reversed phase and ion exchange liquid chromatography.
The gradient is formed by increasing the percentage of organic solvent. Consequently, at the
beginning of the analysis, when the mobile phase strength is low, the analyte will be partitioned
wholly into the stationary phase (or focused) at the head of the column. (Region A in the movies
below)
As the mobile phase strength increases, the analyte will begin to partition into the mobile phase and
move along the column. As the mobile phase strength is increasing continuously, the rate at which
the analyte moves along the column accelerates. (Region B in the movies below)
At some point within the column elution, the analyte may be wholly partitioned into the mobile
phase, and will be moving with the same linear velocity as the mobile phase. (Region C in the
movies below)
One cannot assign a fixed retention factor (k) value to a compound when gradient elution is applied,
as this value changes during elution.
Calculating k using the formula k = (tr-t0) / t0 is only correct during isocratic elution.
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The relationship between the gradient retention factor and the mobile phase composition depends
upon molecular properties. Band spacing may change as column length is altered.
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7. Peak Shape in Gradient HPLC
In isocratic elution the peaks are relatively broad, the peak width increasing with retention time. In
gradient elution, the peaks are narrow with almost equal peak widths.
The main reason for the narrow peak shape is the velocity of the peak as it leaves the column.
During gradient elution, all compounds accelerate through the column and thus elute at a high
velocity. The retention time difference between compounds is a consequence of the percent organic
modifier at which each starts to accelerate. All compounds should have approximately the same
speed when they leave the column.
Another reason for peak focusing is the fact that the front and tail of a peak are residing in different
concentrations of organic modifier. The tail will experience a higher percentage of organic modifier
than the heart of a peak. The velocity of the tail will thus be slightly higher than the heart of the peak
and vice versa for the front. This results in peak focusing. Asymmetric peaks are less frequently a
problem in gradient elution. In practicality, the narrow peaks obtained in gradient elution provide
better detection limits and higher loading capacities.
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8. Scouting Gradients
When developing either gradient or isocratic separations – a scouting gradient analysis is a good
starting point.
The scouting gradient is a linear gradient from 5-10%B to 100%B over a set time (20 minutes is
standard). The elution strength is held at 100%B for a few minutes to make certain that all sample
components have eluted. For reversed phase gradient elution, water (or buffer if there are ionizable
analytes) and acetonitrile are typically the eluents of choice. The use of volatile buffers, for example
ammonium formate, will result in a method which is LC-MS compatible.
The chromatogram obtained can reveal a lot about the required mobile phase composition for the
separation.
If compounds elute more than 25% of the gradient time after the end of the gradient (i.e. after 25
mins. in our example), then a stronger B solvent is required for the analysis – or a less retentive
column.
Equation 1 can be used to determine if an isocratic separation is possible - this is always preferred as
it increases sample throughput (no re-equilibration time) and simplifies the analysis.
The mobile phase composition, at which the first peak elutes, should be used as the initial mobile
phase composition when developing a gradient.
Figure 1: Scouting gradient used to assess viability of performing an isocratic separation and to
calculate the initial %B for a gradient separation.
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Worked Example
Where:
tG = gradient time
ΔtG > tG therefore, isocratic analysis is NOT POSSIBLE for this analysis.
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Figure 2: Scouting gradient used to calculate the isocratic eluent composition if isocratic elution is
feasible.
If isocratic analysis is found to be possible, the isocratic mobile phase composition can be simply
estimated.
From the scouting gradient, calculate the mobile phase composition for the average retention time
of the eluted compounds; that is:
To calculate the isocratic mobile phase composition, first calculate the rate of change of %B (i.e.
Δ%B/min)
Then calculate the eluent composition at the average analyte elution time as follows (remember to
take into account the initial eluent composition of 5%B):
Therefore, the mobile phase composition that should be used for the isocratic analysis is:
14% water (or buffer at the correct pH and concentration) / 86% acetonitrile.
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9. Optimizing Initial Gradient Conditions
∆t G < 0.25t G
∆t G = t f − t i
Where:
ti = time of the initial peak (12.786 min in this case)
tf = time of the final peak (21.142 min in this case)
ΔtG > 0.25tG therefore, isocratic analysis is NOT possible for this separation.
3. Calculate the rate of change of the mobile phase composition using Equation 1.
(%Bfinal − %Binitial )
∆%B/min = (1)
tG
(100 − 5)
=
20
= 4.75 %B/min
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4. Optimize the initial %B required using Equation 2.
VD
Optimized %Binitial = (%Binitial + (t i x ∆%B/min)) − ( x ∆%B/min) − 10%B (2)
F
5.5
= (5 + (12.786 x 4.75)) − ( x 4.75) − 10%B
2
= 42.67 %B
VD
Optimized %Bfinal = (%Binitial + (t f x ∆%B/min)) − ( x ∆%B/min) − 10%B (3)
F
5.5
= (5 + (21.142 x 4.75)) − ( x 4.75) − 10
2
= 82.36 %B
The initial gradient conditions can then be programmed to 42-83 %B in 20 minutes. From this point
the gradient can be further optimized to produce the desired separation.
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10. Gradient Steepness
Gradient steepness is controlled by the mobile phase starting and ending composition and the
gradient time.
The steepness of the mobile phase gradient can have a significant effect on the separation – below
are some examples.
The retention factor (k*) in gradient HPLC is different from isocratic elution. In gradient HPLC the
retention factor of each analyte is constantly changing as the eluotropic strength of the mobile
phase is altered. k* can be thought of as an average k value throughout the separation.
Values of 2 < k* < 10 are desired. A good starting value for k* is 5. Gradient conditions should always
be checked (using the equation for k*) to ensure a reasonable value of k* can be achieved.
Usefully, the equation may be rearranged to obtain an expression that predicts the gradient time,
based on a scouting gradient as previously described. Each of the terms in the equation is defined
below. All terms in the equation are simply defined which makes the equation particularly useful in
practical terms.
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tGF
𝑘∗ =
SΔΦVm
Where:
k* = target value of 5 for average separation
tG = gradient time (minutes)
F = flow rate (mL/min)
Δφ = change in volume fraction of organic (final %B – initial %B) expressed as a decimal
Vm = the interstitial volume of the column (µL)
dc 2
Vm = π ( ) L0.68
2
Where:
dc = column diameter (mm)
L = column length (mm)
S = shape selectivity factor (5 is a good estimate for small molecules) (Equation 3)
S = 0.25MW 0.5
Where:
S = shape selectivity factor
MW = compound molecular weight
Below is a worked example for the application presented. Gradient times above those estimated by
the equation shown are unlikely to produce better resolution.
𝑘 ∗ SΔΦVm
tG =
F
5 x 4 x 0.95 x 1.7
=
2
= 16.15 min
Where:
k* = 5 for average separation
S = 4 for small molecules
Δφ = 5-100% = 95% = 95/100 = 0.95
Vm = 1.7 mL (4.6 x 150 mm column)
F = 2 mL/min
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11. Optimizing Gradient Analyses
It is worthwhile taking a further look at the terms in the equation for the gradient retention factor,
as there are several ways in which a gradient separation may be improved. Also, the manipulation of
the gradient retention factor is counter intuitive from knowledge gained about isocratic HPLC.
tGF
𝑘∗ =
SΔΦVm
Where:
Δφ = change in volume fraction of organic (final %B – initial %B) expressed as a decimal
S = constant determined by strong solvent and sample compound (small molecules < 500 Da the
value is between 2 and 5; a value of 4 is used by convention when the value is not accurately known)
F = flow rate (mL/min.)
tG = gradient time (min.)
Vm = column void volume (πr2L x 0.68)
1/k* = gradient steepness
From the equation it follows that in order to INCREASE the gradient retention factor you can:
The use of shorter columns and higher flow rates to GAIN retention in reversed phase HPLC will be
counter intuitive. Here are some of the more important terms with a further explanation on how
these changes will increase the gradient retention factor and how they may be usefully manipulated.
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To increase k*:
Gradient slope for a given column dimension is however more correctly given as %B/mL. It is flow
and not time that transports a compound through the column. An increase in flow rate consequently
means a decrease in gradient slope, i.e. typically an improvement in resolution and vice versa. The
change in plate number with flow rate may counteract this. The net effect is case dependent.
However, changes in band spacing may occur as with any alteration of a gradient slope.
Analyte elution times are less flow rate sensitive than in isocratic analysis. This is best understood by
considering a simplified case: Take a molecule that does not migrate when the organic modifier
percentage is below 30% but when it is above 30% it moves at the same velocity as the mobile
phase. The gradient reaches 30% at 20 minutes.
At a flow rate of 1 mL/min the compound elutes at 22 minutes, i.e. it does not move for 20 minutes
and then it goes through the column in two minutes.
Increasing the flow rate to 2 mL/min will results in an elution volume of 21 minutes - a much less
significant change in retention behavior compared to isocratic analysis.
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Use a shorter column (Vm)
This example shows the increase in resolution gained for this separation of proteins and peptides by
decreasing Vm, using a shorter column, and therefore increasing the gradient retention term. It is
counter intuitive compared to isocratic HPLC and something similar may happen with changes in
flow rate. The effect may not improve resolution depending on changes in the efficiency of the
separation.
The tradeoff between flow rate, column length, and time is similar for isocratic and gradient elution.
When separation time is a vital issue, use a short column (5-15 cm) and run at high flow rate. When
maximum resolution is crucial it is advisable to use a long column (20-30 cm) at normal flow rate.
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Compounds with a very steep velocity curve (e.g. proteins) do not benefit from long columns. These
compounds quickly reach (after a few centimeters of acceleration) a k* = 0 i.e. no stationary phase
interaction.
When applying steep gradients it is actually counterproductive to use a long column as the
compounds reach k = 0 (no partitioning) before leaving the column. The last part of the column then
only adds band broadening.
In this example the gradient time has been extended, therefore, reducing the gradient steepness.
This can affect the gradient retention factor as well as the selectivity of the separation.
1 SΔΦVm
∗
∝ gradient steepness = b =
𝑘 tGF
If b is kept constant from run-to-run peaks will elute in the same relative pattern, but overall analysis
time can be shortened.
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In this separation the gradient time and column length have been altered in order to retain the same
gradient steepness value. In this case the order of peaks remains constant.
It should be highlighted that although k* is increasing, this will not necessarily improve the
resolution - decreases in efficiency may counteract any benefit. This will depend upon the parameter
being altered and the specific application type.
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12. Practical Gradient HPLC
Due to long column equilibration times, applications using strongly retained components
such as triethylamine are not suitable. For the same reasons, ion pair applications are not
suited to gradient elution.
Normal phase HPLC applications utilizing bare silica columns are not suitable either. Long
equilibration times lead to retention time imprecision.
Solvents utilized in gradient elution must be pure. Water quality is of particular importance.
Impurities are retained on the column while the composition of the mobile phase is weak. As
the elution strength is increased, the impurities appear as peaks in the chromatogram. The
chromatogram shown is a real-life example of these ghost peaks – the impurities are from
the water, which has been inappropriately stored.
To avoid precipitation problems within the instrument, test the buffer to make certain it is
soluble in the final mobile phase composition.
Finally, to increase retention time precision, make certain that adequate re-equilibration
time is allowed between each chromatographic run. This is usually around 10 column
volumes but may vary widely with different columns and applications. To be certain,
retention time reproducibility should be verified during the analytical development.
Ghost peaks in gradient analysis can be caused by impurities in the solvents used - water is
particularly susceptible to this phenomenon.
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Column: C18
Gradient: 100% water to 100% acetonitrile in 20 min.
Flow Rate: 1 mL/min.
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13. Changing Method Parameters
Increased throughput can be achieved by speeding up gradient analyses. However, when changing
any parameter in order to decrease analysis time the effect on the chromatography (retention
factor, selectivity etc.) must be taken into account. For example, under isocratic conditions
increasing the flow rate will decrease the retention factor (k) resulting in a faster analysis.
Retention factor in isocratic HPLC is determined by Equation 1. An increase in flow rate under
isocratic conditions will decrease both tR and t0 by the same factor, hence, k will remain constant.
tR − t0
𝑘= (1)
t0
Where:
tR = retention time
t0 = void time
However, if flow rate alone is changed in gradient HPLC the retention factor k* will be altered, which
in turn can have an impact on selectivity and resolution, often leading to a change in elution order in
the chromatogram. This is unsurprising when the equation for calculating k* is considered (Equation
2).
tGF
𝑘∗ = (2)
SΔΦVm
Where:
tG = gradient time (minutes)
F = flow rate (mL/min)
Δφ = fractional change in eluent composition (i.e. 0.4 for a 20 to 60 %B gradient)
Vm = the interstitial volume of the column (µL) (Equation 3)
dc 2
Vm = π ( ) L0.68 (3)
2
It can be seen from Equation 2 that the retention factor (k*) in gradient HPLC is different from
isocratic elution. In gradient HPLC the retention factor of each analyte is constantly changing as the
eluotropic strength of the mobile phase is altered. k* can be thought of as an average k value
throughout the separation.
The retention factor is dependent upon the eluent flow (F) as well as the interstitial volume within
the HPLC column (Vm, and hence the column length and internal diameter). The Fundamental
Resolution Equation (Equation 5) shows that altering retention can alter resolution, therefore, the
ability to alter retention by changing only flow rate or column dimensions in gradient HPLC has a
direct effect, albeit limited, on resolution.
√N α − 1 k
RS = (5)
4 α k+1
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Equation 2 also allows gradient separations to be modified in order to decrease analysis time,
without incurring changes in analyte selectivity. In order to maintain selectivity the gradient
steepness 1/k* (often termed ‘b’) must be maintained (Equation 6).
SΔΦVm
b= (6)
tGF
Most modern applications of fast gradient HPLC arise when moving from traditional HPLC to UHPLC
formats. This usually involves a change in column dimensions and gradient time as well as alterations
to the flow rate.
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14. Gradient Suitability
Ideally for any method retention factors (k*) of between 2 and 10 are desirable for method
robustness. The use of UHPLC instrumentation and columns does allow these limits to be altered to
0.5 < k* < 5. If k* is too low there is a risk of interference from other sample components or
analytes, and if k* is too high the analysis time will be unnecessarily long. The suitability of a
method can be determined using Equation 1.
tGF
𝑘∗ = (1)
SΔΦVm
Where:
tG = gradient time (minutes)
F = flow rate (mL/min)
Δφ = fractional change in eluent composition (i.e. 0.4 for a 20 to 60 %B gradient)
Vm = the interstitial volume of the column (µL) (Equation 2)
dc 2
Vm = π ( ) L0.68 (2)
2
Example Calculations
dc 2
Vm = π ( ) L0.68
2
4.6 2
= π ( ) x 150 x 0.68
2
= 1695 μL = 1.7 mL
tGF
𝑘∗ =
SΔΦVm
7 x 1.5
=
5 x 0.45 x 1.7
= 2.75
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Fast HPLC Method
Column: C18 50 x 2.1 mm, 1.8 µm
Flow: 0.9 mL/min
Gradient: 20-65% acetonitrile (0.1% formic acid) in 2 minutes
dc 2
Vm = π ( ) L0.68
2
2.1 2
= π( ) x 50 x 0.68
2
= 118 μL = 0.12 mL
tGF
𝑘∗ =
SΔΦVm
2 x 0.9
=
5 x 0.45 x 0.12
= 6.67
A k* value of above 6 indicates that this method will be suitable, although, the method could be run
a little faster without loss of robustness.
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15. Method Transfer Between HPLC Systems
One of the most common problems encountered when transferring a gradient HPLC method
between instruments are irreproducible gradients and, therefore, inconsistent chromatographic
results.
One cause of irreproducible gradients is that instruments will have different dwell volumes. The
dwell volume is the volume between the point at which the gradient is mixed, to the point at which
that composition enters the HPLC column; this will differ depending on how the gradient is formed
(high or low pressure mixing), the tubing volume, internal volume of the autosampler and loop etc.
Differences in this system dwell volume (VD) can make large differences to not only the retention in
gradient HPLC but also to selectivity and, hence, resolution. In order to account for dwell volume
differences an isocratic hold can be included in the method or an injection delay can be used.
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Even though the system dwell volume has been catered for by altering the gradient start time (either
starting the gradient prior to injection or inserting an isocratic hold at the start of the gradient),
there can still be problems with irreproducible separation selectivity.
This problem results from differences in the way that gradients are formed and delivered and the
inherent pump volumes associated with solvent mixing. It is important to note that this problem can
be seen when transferring methods between different quaternary HPLC systems and is not only
associated with method transfer between high and low pressure mixing systems.
UHPLC systems have markedly different technologies to mix the required gradient composition
online and, as well as having different gradient composition delay characteristics, there will be
differences in the slope and wash out characteristics of the gradient delivery profiles as shown in
Figure 1.
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Unless one can compensate for the differences in the gradient delivery characteristics, then
successful method translation may be difficult. Whilst some UHPLC systems are equipped with
software which can simulate differences from certain manufacturer’s equipment, not all systems are
similarly equipped.
The following approximation can be used to adjust the gradient profile between systems, once the
gradient slope or wash out volume (wash out time x flow rate) has been characterized:
Adjusting the gradient segment times as outlined above will adjust the slope of the gradient
delivered by the new system. When combined with a similar adjustment to account for dwell volume
differences at the beginning of the analysis, this can help to more closely match the solvent
composition at the head of the analytical column at any given time within the gradient.
The wash out volume is the volume required for the instrument to wash out the strong solvent
channel and can be as much or more than the dwell volume. This can affect both method transfer
and re-equilibration time i.e. re-equilibration cannot occur until the initial eluent conditions are
reached. An estimate of the wash out volume is when the eluent at the column inlet contains 3% of
the final eluent (i.e. 97% flushed).
The wash out volume can be measured using a step change from 100%B to 100%A. B is water with
0.1% acetone and A is water (monitor at 254 nm).
1. Schellinger, A.P; Stoll, D.R.; Carr, P.W. J. Chromatogr. A 2005, 1064, 143-156.
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16. Gradient Method Transfer Calculator
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17. Non-Linear Gradients
Most gradient elution methods use linear gradients as has been shown previously, however, the use
of non-linear gradients may provide modest improvements for some separations. In the early days
of gradient HPLC with online solvent mixing, curved gradient profiles were popular and various
manufacturers offered the possibility of forming gradients of the type shown in Figure 1.
In practice, curved gradients are difficult to reproduce on a consistent basis and are especially
difficult to transfer between different instruments/laboratories, and for that reason they are now
much less popular.
In modern gradient HPLC, the approach is to use multi-segment linear gradients to achieve optimum
resolution for more difficult separations. Generally this involves changes in gradient slope or the
insertion of isocratic segments into the gradient profile. Non-linear gradients can be applied to
separations of homologous or oligomeric samples, which exhibit a decrease in resolution with an
increase in molecular weight.
Separations which produce chromatograms with bunched, overlapping, or widely separated peaks
within different areas of the chromatogram, or where analytes will show alternate selectivity for
gradients of different slopes at varying points in the chromatogram.1
1. Snyder, L. R.; Kirkland, J. J.; Glajch, J. L. Practical HPLC Method Development 2nd Ed. John
Wiley & Sons Inc., 2011.
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18. Segmented Gradients
Segmented gradients can be applied to chromatograms which exhibit critical pairs (or groups) of
peaks that are widely spaced within the initial chromatogram.
The chromatogram shown in Figure 1 (top) has critical peak pairs at points 1 and 2. The issue is that
they are widely spaced in the gradient and a universal reduction in gradient slope will not solve both
problems. Generally, poorly resolved peaks require a change in the slope of the gradient.
Usually this will involve lowering the slope of the gradient profile, however, in some cases a steeper
gradient may force one peak to move through the other and solve the issue. There are no hard and
fast rules regarding the time (or gradient composition) at which the slopes should be altered and the
use of simple chromatographic optimization software can help enormously to visualize gradient
slope changes and their effect on the resulting chromatogram. Segmenting the gradient will enable
independent control over the early and later portions of the chromatogram resulting in the
satisfactory separation of both critical pairs.
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Optimization of Segmented Gradients
From the initial gradient the slope can be segmented in an infinite number of ways. As shown in
Figure 2, starting from the initial gradient and overlaying a grid (4x8 grid in this case) at possible
break points in the gradient can result in 19 possible alternative gradient profiles.1 If a more
complex grid were used then the number of possible gradients would increase. Therefore, it can be
seen that the use of computer software to visualize each of these gradients and the effect on the
chromatography can be invaluable.
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19. Isocratic Hold
When separation problems occur at the midpoint of the chromatogram, an isocratic hold is useful to
help increase resolution. As a rule of thumb, the gradient steepness should be retained from the
original method.
The isocratic composition is calculated by subtracting 10%B from the elution composition of the first
peak in the problematic region of the original chromatogram (52% as indicated in Figure 1). If the
isocratic eluent composition is not satisfactory, the eluent composition should be reduced in steps of
10%B.
The length of the isocratic hold is typically matched to the duration of the problematic region in the
original chromatogram. This of course can be lengthened if required. The gradient steepness after
the isocratic hold should once again be matched to the original method.
Once again, the separation can be empirically optimized and computer optimization can assist
enormously.
Figure 1: Original gradient profile (top) and gradient profile with isocratic hold (bottom).
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Example Calculation
The following equations can be used to calculate the composition of the isocratic hold required to
resolve a critical peak pair in a gradient HPLC method.
Method
Gradient: 5-100 %B
tG = 30.48 minutes
Flow rate = 2 mL/min
VD = 1.7 mL
Critical pair at 9.25 minutes
The rate of change of the mobile phase composition during the gradient can be calculated from
equation 1.
(%Bfinal − %Binitial )
∆%B/min = (1)
tG
(100 − 5)
= = 3.117 %B/min
30.48
Optimization of the %B used in the isocratic hold can be carried out using Equation 2.
VD
Optimized %Biso = (%Binitial + (t i x ∆%B/min)) − ( x ∆%B/min) − 10%B (2)
F
1.7
= (5 + (9.25 x 3.117)) − ( x 3.117) − 10%B
2
= 21.18 %B
Therefore, the isocratic hold would be added into the method at 21%B and held until the peak pair
was resolved before continuing the gradient at the same gradient steepness as the original method.
Where:
%Binitial = initial starting %B from scouting gradient
ti = elution time of the initial peak
Δ%B/min = rate if change of the mobile phase composition (Equation 1)
VD = dwell volume
F = flow rate
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20. Peak Capacity
The separation efficiency of columns under isocratic elution conditions is measured in theoretical
plates. Peak capacity is used to describe the separation efficiency for gradient elution. Peak capacity
describes the maximum theoretical number of components that can be successfully separated with a
given column and set of analytical conditions with Rs =1 (Figure 1 and Equation 1).1-2
Equation 1 can be used to give an approximation of the number of components that can be
separated under a specific set of conditions, if this number is lower than the number of components
in a sample then the method will not produce a chromatogram with resolved peaks.
tG
P≈1+ (1)
w
Where:
tG = gradient time
w = average peak width at 4σ (13.4% of peak height)
Note: This is an approximation but a good guide. The average peak width can be calculated by
adding the peak widths of the first and last peaks and dividing by 2.
Peak capacity is a function of gradient time, flow rate, column length, and particle size. Increasing
column length while keeping particle size and gradient time constant results in a maximum value of
peak capacity being reached, and in fact, for longer columns the value of peak capacity may
decrease (Figure 2).
Improving peak capacity using particle size seems to give more promising results, with the decrease
in particle size giving higher peak capacity values. Increasing the gradient duration will increase the
peak capacity; however, for longer gradients the increase in peak capacity with time becomes small
as a maximum will be reached.
Peak capacity can be optimized using the flow rate at a fixed gradient time (tG). Peak capacity will
increase proportionally to the square root of column efficiency (Equation 2); therefore, doubling
column efficiency will increase peak capacity, but only by 40%.
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√N B∆C
P=1+ (2)
4 B∆C t 0 + 1
tG
Where:
N = efficiency
B = slope of the function ln k (k is the retention factor) versus solvent composition C
ΔC = change in solvent composition
t0 = breakthrough time
tG = gradient time
Figure 2: Plot of tG/t0 vs. P giving the optimal peak capacity for a separation.
1. Gilar, M.; Daly, A. E.; Kele, M.; Neue, U. D.; Gebler, J. C. J. Chromatogr. A 2004, 1061, 183-
192.
2. Wang, X.; Stoll, D. R.; Schellinger, A. P.; Carr, P. W. Anal. Chem. 2006, 78, 3406–3416.
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21. Estimating Gradient Parameters
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22. Gradient Performance Checks
To proportion and mix a reliable gradient the components of the HPLC pump must be functioning
correctly. Carrying out regular gradient performance checks on equipment can be used to:
Test the solvent delivery system (highlight malfunctioning check valves, leaks etc.)
Test gradient performance (indicate failures with low pressure valves, electronic control of
pumps etc.)
Test proper mixing of high salt buffer gradients used in biochemical applications
Test the linearity and influence of stray light on detectors
Gradient performance checks can be carried out using a simple generic step gradient, or under the
analytical conditions which are to be used. For biological applications where systems are required to
mix high salt buffers using the analytical conditions can be advantageous to ensure proper gradient
formation.1
A gradient performance check (on high or low pressure systems) can be carried out as follows:
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Gradient proportioning valves on low pressure mixing systems can also be tested as follows:2
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