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Troubleshooting GC SeparationsThe CHROMacademy Essential guide to
Translating and Transferring HPLC Method


The Essential Guide from LCGC’s CHROMacademy presents an informative session on method translation and transfer in HPLC. In this session, Tony Taylor (Technical Director,Crawford Scientific) and Scott Fletcher (Business Development Manager, Crawford Scientific), present a definitive guide on everything that one would need to consider for translating and transferring HPLC methods.

The session includes in-depth consideration of the hardware and data acquisition requirements for simple method transfer or translation to different column / particle size and morphology. We present many worked examples of calculations, approaches and pitfalls associated with method scaling and transfer and include up to date concepts such as frictional heating and pressure based selectivity to ensure you are fully up to date in this subject area. A must see for everyone translating or transferring HPLC methods.

Topics include

  • Instrument components important for method transfer
  • Characterizing your system for simple method transfer or translation to faster methods
  • Geometric scaling of method parameters (flow rate / loading etc.) for different columns / particle sizes
  • Translating gradients – pitfalls and tricks
  • UHPLC to HPLC – because sometimes we have to!
  • Pressure and Temperature effects on Selectivity

Find out more about this Month's Essential Guide Webcast »

 

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Q & A from the CHROMacademy Essential guide to "Translating and Transferring HPLC Methods"

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The CHROMacademy Essential guide to
Translating and Transferring HPLC Method

A definitive guide on everything that one would need to consider for translating and transferring HPLC methods.

The session includes in-depth consideration of the hardware and data acquisition requirements for simple method transfer or translation to different column / particle size and morphology.

We present many worked examples of calculations, approaches and pitfalls associated with method scaling and transfer and include up to date concepts such as frictional heating and pressure based selectivity to ensure you are fully up to date in this subject area. A must see for everyone translating or transferring HPLC methods.

Key Learning Objectives

  • Understand method transfer variables
  • Understand hardware requirements for running Fast HPLC methods
  • Investigate ‘geometrical scaling’ to convert flow rates and other method parameters between columns and particles of different sizes
  • Learn to convert gradient HPLC methods to maintain equivalent selectivity
  • Consider important factors in reverse scaling from UHPLC to HPLC methods
  • Investigate important aspects of temperature and pressure when scaling methods including Fractional Heating and Pressure Induced Selectivity changes
  • Build a portfolio of useful equations and rules of thumb for method scaling and transfer

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Introduction to Method Translation

Very often we have a need to transfer or translate an existing HPLC method to a different instrument or to gain speed by altering our column geometry, stationary phase particle size or particle morphology.

In order to do this successfully, we need to understand the underlying principles and important instrument aspects which govern this translation or transfer.  This Essential Guides provides information on the basic relationships which allow us to ‘geometrically scale’ our method parameters.  No matter if you are transferring, for example,  from a 150mm x 4.6mm column with 5μm stationary phase particles to a 100mm x 2.1 column with 3μm particles, or with 2.7μm superficially porous particles or to sub 2μm particles, certain relationships hold which can help us to estimate the optimum eluent flow, pressure, sample volume or the expected efficiency we might obtain.

Further, there are ways to transfer the gradient profile in order to maintain selectivity as well as constraints and considerations regarding the hardware we chose for the new analysis, governed by the geometry of the column, packing material and the ultimate speed (and hence the backpressure) of the newmethod.

There may also be times when we need to transfer an existing method from one instrument to another, without any change other than the actual instrument used, the instrument manufacturer or the laboratory in which it is situated.  Many of the considerations highlighted below will be pertinent also to this situation also.

NOTE:  The following guidelines are intended to provide a good ‘starting point’ for the translated method – further method optimization may be required.

Readers should also note that this Essential Guide does not fully consider column stationary phase selection.  Our starting point is that you already have an HPLC method which produces satisfactory resolution (selectivity) and the method needs to be transferred to another HPLC system or a decrease in analysis time is required.

 

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Q & A from the CHROMacademy Essential guide to "Translating and Transferring HPLC Methods" Forum / Essential Guides

 
 

Instrument Parameters

Under isocratic conditions, method translation may be carried out under the assumption that the stationary phase selectivity holds at different flow rates, column geometry or temperature conditions.  This means that selectivity will remain approximately constant after method translation. 

There are certain caveats to the presumption that selectivity will remain unaltered.  Frictional heating effects, pressure induced selectivity changes and changes in manufacturing (packing) / bonding for particles of different diameter or morphology all need to be considered.  These issues will be further explained in subsequent sections of this Essential Guide.

The mobile phase linear velocity should also remain constant after method translation and this last condition is fundamental for translating methods where the column geometry changes.

For more information on HPLC method development, please visit the links below:

Developing Better Methods for Reversed Phase HPLC (Part 2) webcast & tutorial » - available to Premier Members only

Reverse Phase (Partition) Chromatography e-learning module » - available to Premier Members only

Normal Phase (Absorption) Chromatography e-learning module » - available to Premier Members only

Mobile Phase Considerations e-learning module » - available to Premier Members only

 

The model that we are going to explain, is just the first step in your isocratic method translation. Once you have calculated the new experimental conditions the architecture of your HPLC instrument needs to be considered, and the aspects which require attention include: [1-3, 4-9]

  • System Dwell volume (including gradient mixing and pulse dampening components)
  • Extra column volume (volume of connecting tubing, fittings and the detector flow cell where appropriate

More on these aspects after the gradient method translation part of this webcast and in the accompanying Essential Guide.

The optimum flow rate (F) when translating methods to columns of different geometry is proportional to the square of the column internal diameter (D) and inversely proportional to the particle size (d).  The equation below will ensure that the linear velocity of the eluent remains the same in the translated method.

For columns that are packed with the same stationary phase particle diameter, the previous expression reduces to the well known relationship:

Figure 1 shows simulated chromatograms for a translated separation of 6 neutral test compounds when column geometry is altered.

fig 1

 

 

« Figure 1.  Illustrative example of geometric scaling of eluent flow to a column of reduced diameter and particle size, using DryLab® software simulation.  Separation of 6 neutral test compounds of varying polarity.  Eluent: 56% acetonitrile : 44% water.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

This Essential Guide will consider in a subsequent section the extra column volume considerations which may affect the efficiency of the separation carried out using the narrow bore HPLC column.

Note that this translation gives us an equivalent separation, and does not result in a reduction in analysis time at this stage.  Also however note the inherent increase in efficiency of the separation, evidenced by the sharper peaks in the lower chromatogram.  This phenomenon can be exploited to achieve faster separations as we will see in the next section.

The optimum injection volume (V) under changed HPLC conditions is proportional to the square of the column internal diameter (D) and to the column length (L) and can be calculated using the following relationship:

The efficiency (N) of a column is proportional to its length and inversely proportional to the packing material’s particle size.  So when transferring a method, the efficiency of the new column can be approximated by:

The original and transferred pressures can be estimated using the following expression:

Some column manufacturers have developed their own strategies for pressure translation. One of them uses backpressure factors (γ):

Where γ is a parameter inherent to the column.  For selected values of γ please refer to table 1.

 

Table 1.  Backpressure factor (γ) for selected columns

Particle Diameter (µm) Backpressure Factor (γ)*
1.7 8.7
1.8 7.7
2.2 5.2
3.0 2.8
3.5 2.0
5.0 1.0

 

 
Method Translation Example 1:

A separation using a C18 25cm×3.0mm×5μm column has an optimum eluent flow rate of 0.5 mL/min and the amount of sample loaded was 15μL, yielding an efficiency of 11000 theoretical plates.  Calculate the equivalent flow rate and sample load in a C18 10cm×2.1mm×1.8μm column.  Calculate the relative pressure increase when using the fast format HPLC column if the original method generates a backpressure of 250 bar.

The table below lists all required information to solve this problem. 

Table 2.  Tabulated information

Parameter Original Method (1) Translated Method (2)
F (mL/min) 0.5 ?
D (mm) 3 2.1
L (cm) 25 10
d (μm) 5 1.8
V (μL) 15 ?
P (bar) 150 ?
N (theoretical plates) 18,133 ?

 

Flow Rate:

 

Injection Volume:

 

 

Efficiency:

 

 

Pressure:

 

The example above indicates a useful feature of method translation.  If one assumes that equivalent efficiencies will produce equivalent separations (a big leap of faith but one worth taking sometimes!), then we can look to use smaller columns and particles to give an equivalent separation in a shorter time than before – so called ‘Fast’ HPLC.

The following illustrative example should help in visualising this:

 

 

fig 02« Figure 2.  Separation of 6 neutral test compounds of varying polarity.  Eluent: 56% acetonitrile : 44% water

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

You should notice that the retention time of the last peak, in this isocratic separation, was 23.95 mins in our original separation, but 3.31 minutes in the translated method – no separation quality was lost.  The downside of course is the accompanying rise in system back pressure, which may require special HPLC equipment.

Example 2 outlines some considerations when translating methods from Fast HPLC systems back to ‘standard’ column / system geometries.  This may seem somewhat counter intuitive, but increasingly, laboratories without upgraded pressure equipment need to translate methods from literature or method development colleagues with somewhat greater capital purchase budgets!

 
 
Example 2:

It was found that for a C18 5cm×1.0mm×1.7μm column, the optimum eluent flow rate was 0.4 mL/min and the amount of sample loaded was 5μL.  Calculate the equivalent flow rate and sample load when using a C18 10cm×2.1mm×3.5μm column.  Calculate the ‘analytical’ scale pressure if the fast HPLC column resulted in a pressure of 770 bar.

The table below lists all required information to solve this problem. 

Parameter HPLC (2) Fast HPLC (1)
F (mL/min) ? 0.6
D (mm) 4.6 2.1
L (cm) 10 5
d (μm) 3.5 1.7
V (μL) ? 5
P (bar) ? 770

Table 3.  Tabulated information

Flow Rate:

 

Injection Volume:

 

Pressure:

 

This model is just the first step in your isocratic method translation. Once you have calculated the new experimental conditions the architecture of your HPLC instrument needs to be considered, and the aspects which require attention include:

  • System Dwell volume (including gradient mixing and pulse dampening components)
  • Extra column volume (volume of connecting tubing, fittings and the detector flow cell where appropriate)

 

These aspects will be considered later in this essential guide.

 

fig 03

Figure 3.  Separation of 6 neutral test compounds of varying polarity.  Eluent: 56% acetonitrile : 44% water

 
 

Volume Loading Considerations

As a rule of thumb, the injection volume should be kept between 1% and 5% of the total column dead volume.  For example, for a C18 10cm×3.0mm×5μm with a void volume fraction Φ = 68%, the injection volume should be calculated as follows:

Therefore, the injection volume V, should be something between 1% and 5% of VColumn, so in this case it should be something between 5 and 25 µL, which matches well with our calculated injection volume above

 
 

Mass Loading Considerations

The best source of information for mass loading is your column manufacturer.  However, a brief description of the type of calculations made in order to establish the correct amount of sample to be loaded without compromising peak shape is provided below.  Broadly speaking, this method considers a reduction in peak efficiency when increasing the amount of sample.  The details of the derivation can be found elsewhere. [10-13]

An approximate expression for the thermodynamic contribution to column efficiency is:

Where:

Nth is the efficiency of the peak measured using the peak width
ko is the retention factor for small sample mass,

The loading factor (Lf is the sample mass (Wx) divided by the column saturation capacity (Ws), and is given by:

The column HETP can be assumed to be the sum of two terms:

From which:

But

And Nth was already defined as:

So the following expression can be deduced:

Where to is the void volume of the column and W the peak width at base of the overloaded peak.

The previous expression can be used to calculate the mass loading capacity.  The figure below illustrates one way to achieve such end:

fig 04

Figure 4.  Mass loading capacity determination.  For different amounts of loaded sample, different values of retention factor (ko), efficiency (No) and peak width (W) are obtained.

So, measurements of peak width (W) and retention factor (Ko) for a given sample mass (Wx) will permit the determine the column’s loading capacity (Ws).  Of course there are more precise methods to determine column capacity, but this is out of the scope of this Essential Guide, and interested readers are invited to explore relevant literature.

 
 

Translating Gradient Conditions

Under gradient conditions, method translation should be carried out under the assumption that the mobile phase velocity remains constant for both the original and converted separation in order that the relationships used for translation of the gradient profile are accurate. 

If the gradient profile with time changes (%organic within the column per unit time) between the original and translated separation, then the separation selectivity and resolution may be drastically altered.  The gradient must therefore be ‘scaled’ according to some empirical relationships in order to preserve the separation selectivity.  This is further explained below.

For more background information on HPLC gradient operation, interested readers are invited to visit the links below:

Understanding HPLC Column Characterization and Selection webcast & tutorial » - available to Premier Members only

Gradient HPLC e-learning module » - available to Premier Members only

 
 

Gradient Profile Translation

If the laws that govern method translation under isocratic conditions are valid with gradient elution (this assumption is not unreasonable), then similar relationships might be applied. [1-3, 4-9]

For the mobile phase flow rate (F) and the gradient time (tg), the following expressions hold:

 

Where,tg1 and tg2 are the original and new gradient segment times and V01 and V02 are the column void volume of columns 1 and 2 respectively.  There are different ways of calculating these two parameters.  A general model considers a void volume (i.e. which is the interstitial volume within and between the silica particles) of approximately 68% of the total column volume. This porosity figure is generally applicable to fully porous silica particles typically used by column manufacturers.  For absolutely accuracy within the calculations, or where fully porous silica is not being used, this number should be adjusted after consulting your specific column manufacturer.  If we assume 68% porosity, then the following expressions hold:

 

In general, if both columns have a similar void volume, the following expression holds:

A separate, but closely related, approach to gradient segment translation scales the gradient segment volume in proportion to the column void volume, whilst maintaining the same initial and final eluent composition.  A general relationship for this shown below as a re-arrangement of the well known equation for determining average retention behaviour in gradient operation:

 

S is a constant determined by the solute (usually between 2 and 5)
tg is the gradient time
Vm is the column void volume, approximately 68% of the column volume (0.68πr2L)
ΔΦ is the change in volume fraction of organic (final %B – initial %B)

 

Where:

 
Example 3: 

It was found that for a C18 15cm×4.6mm×5μm the optimum eluent flow rate was 1.0 mL/min, a gradient of 20% to 60% of B in 15 minutes and the amount of injected sample was 15μL.  Calculate the equivalent flow rate, sample load and gradient conditions in a fast C18 10cm×2.1mm×1.8μm column.  Note the particles used in both columns were of the same porosity.

The table below lists all required information to solve this problem.  The sub-indices 1 and 2 can be assigned as we wish.

Table 4. 

Parameter HPLC (1) Fast HPLC (2)
F (mL/min) 1.0 ?
D (mm) 4.6 2.1
L (cm) 15 10
d (μm) 5 18
V (μL) 15 ?
tg(min) 15 ?

 

 

For columns with similar void volume:

 

Calculating the new gradient segment time using the equation for average retention in gradient HPLC:

 

 

For separation 1:

 

For separation 2:

 

But  should be the same for both methods, so:

 

 

The calculated new gradient segment time shows close agreement between the two approaches. 
Using these methods will help to ensure that the selectivity and hence the resolution, of the method remains unchanged.

 

 

« Figure 5.  Gradient separation of polynuclear
aromatic hydrocarbon test mix

 

Dwell Volume Considerations

One important aspect to consider when transferring gradient separations, is the effect of the system dwell volume (Vd) – assuming that the translated method is run on a different HPLC system. [3, 14, 15]

Defined as the system volume between the point of mixing in the pump and the top of the column, the delay volume is responsible for a composition delay between the pump and the column (under gradient conditions).

The dwell volume (Vd) and the flow rate (F) will determine the time lapse in the gradient reaching the column, also known as the dwell time (Td).  In essence, once the composition of the mobile phase is formed in the pump, a finite time interval is required for this new composition to reach the column.  This time interval or dwell time (Td) is given by:

 

fig 06

 

 

 

 

 

 

 

« Figure 6.  The dwell volume imposes a time difference between the moment at which the mobile phase composition was changed (at the pumping system in this case) and the moment it reaches the HPLC column.

 

 

Gradient dwell volume is the total system volume in a gradient system between the point where the gradient is formed and the inlet of the column. In a high-pressure-mixing system, this includes the mixing chamber, connecting tubing, and injector. In a low pressure mixing system this ALSO includes the volumes of the pump head, pulse damper and other connective tubing.

The dwell volume can be converted into a dwell time by multiplying by the eluent flow rate. The dwell volume has a significant impact when transferring methods between different HPLC systems. It is important to account for differences in dwell volume (time) between systems in order to accurately reproduce a separation. This is usually achieved by having an ‘isocratic hold’ section at the beginning of the gradient profile which can lengthen or shorten according to the differences in dwell time between the two HPLC systems. [15, 16]

Calculating Dwell Volumes

Replace column zero dead volume connector
Run gradient 100%A (aq) to 100%B (aq + 0.1% Acetone)
Calculate dwell time;   Td = t1/2 – (tG/2)
Convert to dwell volume; Vm = Td x F

 

Dwell volume can be easily measured using your UV detector to plot the gradient profile generated by your system. [17]

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« Figure 7.  Calculating Dwell Volume.

 

For more in dwell volume and time, please visit the links below:

 


 
 

Consider a method that was developed on a system with small dwell volume.  If this method is to be transferred to an instrument with larger dwell volume, a systematic increase in retention time (and possibly selectivity) will be observed.  As an illustration: when working at 1 mL/min, a system with 1mL dwell volume will add 1 min gradient delay.

Differences in dwell volume, column dead volume and flow rate will cause noticeable differences between the observed and programmed gradient profile.  Retention time issues (usually a general shift for all peaks) and altered selectivity for early eluting peaks are common symptoms of such problem.

 

fig 07

« Figure 8.  Changes in dwell volume can cause changes in retention time and resolution (DryLab simulation).

 

In method translation, what it really matters, is the “effective” dwell volume, which is defined as the total volume of starting eluent delivered to the column after sample injection. 

If the dwell volume of the system used in the original method (Vm1) is larger than the second HPLC system (Vm2), then you should compensate by adding an isocratic hold such that:

Hold + Vm2 = Vm1

In the opposite case, delay injection after the gradient is started, in order to simulate the lower dwell volume.  This function is available on most modern HPLC systems.

Vm2 – Delay = Vm1

 

 

fig 09

 

Figure 9.  Gradient profile with delayed injection.  The blue dashed line represents the moment when the gradient conditions were imposed, the red line represents the actual moment when the gradient reaches the column.  Some instrument manufacturers offer software to co-ordinate sample injection at a defined time post gradient start.

 

fig 10

  50µl mixer 425µl mixer
Peak Name Ret. Time RRT Ret. Time(min) RRT
Acetophenone 0.353 0.51 0.376 0.38
Valerophenone 0.688 1 0.996 1
Caprylophenone 0.976 1.42 1.414 1.42

Figure 10.  Dwell volume effect on mixers of different dead volume.

 

3. Instrument Considerations

Once the theoretical method translation is done, you must consider whether or not your HPLC system can cope with the conditions imposed by the new method. 

The instrumentation used in fast HPLC have been developed to deal with high pressures (sometimes exceeding 1000bar), while traditional HPLC systems typically do not exceed 400 bar.

In general terms the working principles of high and standard pressure HPLC systems are very similar because the critical elements required for a good chromatographic separation remain the same (accurate and reproducible gradient missing, ripple free continuous delivery of mobile phase, fast introduction of sample plug, low dead volume, efficient column, lowest volume detector flow cell to achieve required sensitivity, enough data points to effectively the model the peaks eluted etc.).

For more in fast HPLC Instrumentation, please visit the link below:

Fast HPLC e-learning module » - available to Premier Members only

 
 

fig 11Tubing and Accessories

Stainless steel tubing is required for very high pressure applications where other types of tubing may fail due to bursting.  The tubing and accessories currently used with fast HPLC instrumentation can be made from any appropriate HPLC material (usually stainless steel, Teflon, Delrin, PEE, fsc, etc).  In essence, most polymeric HPLC tubing and accessories can hold between 250 and 400 bar and are resistant to attack by many solvents; however, this is not enough when dealing with fast HPLC separations where pressures in excess of 1000 bar are not uncommon.

Whilst PEEK fittings can handle up to 1000 bar pressure, PEEK tubing is normally designed to operate at pressures not exceeding 680 bar, likewise, PEEK ferrules should only be used below 500 bar.  Some fittings, manufactured from proprietary blends of PEEK can cope with pressures exceeding 1500 bar.  Consult your vendor to check the pressure rating of your tubing and fittings.

The tubing length must be minimized before the column so that the delay time for the gradient to reach the column is as short as possible.  Larger internal diameter tubing should be used before the column to prevent unnecessary system back pressure. 

 

» Figure 11. Schematic showing the effect of incorrectly matching fittings

 

 

 
 

Fittings are used to connect the various components of an HPLC system together while maintaining a leak free flow-path.  Using the correct fitting eliminates dead volume that can contribute to peak broadening, decreased resolution, split peaks, and sample carry-over.  No matter how sophisticated the instrumentation, it cannot compensate for poor fittings.

Unfortunately HPLC manufacturers have not standardized their fitting sizes and this is always a source of trouble as using fittings from different manufacturers may lead to an excessively large dead volume and band broadening.

In order to achieve high quality separation, extra-column dead volume should be minimised.[21]  The use of specially designed low dead volume fittings and eluent filtering devices cannot be overemphasised.  Figure 12 illustrates how separation quality is adversely affected by using large extra-column dead volumes.  

 

fig 12

 

Figure 12. Extra-column dead volume effect.  Courtesy of Agilent Technologies.

 
 

fig 13 « Figure 13. High pressure fitting with a front and back ferrule

 

Column end fittings provide a means to connecting an HPLC column to the tubing.  The choice of end fitting should be based on compatibility with the pressure required.  There are some basic rules you should consider always:

  • Stainless steel fittings should not be used on PEEK tubing
  • Fittings should not be over tightened
  • Fittings from different manufacturers usually do not match
  • Ensure the fitting is made to ensure low dead volume as shown in Figure 13 above
 
 

fig 14


Figure 14.
Fittings from selected manufacturers.

 
fig 15

Figure 15. End fitting geometries (dead volume highlighted in red colour) from selected manufacturers.

 
 

Frit: A major cause of column deterioration is the buildup of particulate and chemical contamination at the head of the column. This can lead to increased back pressure and anomalous chromatographic results.  HPLC Columns normally contain stainless steel inlet and outlet frits (acting as filters) which retain the column packing and allow the passage of the mobile phase.  The pore size of the frit must be smaller than the particle diameter of the packing, e.g., a 0.5 μm frit for 1.8 μm packing.  Sample depositing on the column inlet frit can lead to peak shouldering as demonstrated below. 

fig 18 « Figure 18. Column inlet frits.
 
 

Mobile Phase Considerations

When translating methods to fast HPLC, bear in mind that extreme conditions of pressure are used, and any blockages will increase the system back pressure dramatically and therefore should be avoided as carefully as possible.  In general you should:

  • Use only high grade organic solvents and water
  • Use high quality reactants and salts to prepare any required buffer or solution
  • Implement ultra-filtration (0.22 µm)
  • Avoid microbial growth, use freshly prepared mobile phases
  • When preparing your buffer, add the organic to the aqueous component
 
 

Inejctor

Development of fast HPLC has been directly related to availability of suitable hardware (columns, pumps, inlet systems, low dead volume fittings, etc.) that allow precise flow control under the analysis conditions, as well as the ability to manufacture a wide variety of column packing materials.  As with all fast HPLC hardware, low dead volume is of overriding importance.

Modern fast HPLC injectors implement temperature control to guarantee that mobile phase, sample loop and column remain at the same temperature, thus providing maximum retention time reproducibility while minimising thermal diffusion effects.  As a rule of thumb, the injector should be placed as close as possible to the column in order to minimise dead volume, decrease diffusion problems and minimise temperature changes, so sample reaches the column at the correct temperature.

When injection valves are rotated between the load and injection positions, a momentary stop in flow occurs.  Pump pressure rises and column pressure decreases, therefore, a pressure surge occurs as the rotation is completed.  Such pressure surges can damage column packing materials. 

 

fig 19« Figure 19. Pressure surge with traditional six-port HPLC valves.  Once the flow is re-established, the column will be exposed to an increased pressure that could adversely affect its integrity.

fig 20

 

« Figure 20. The implementation of a bypass will guarantee a constant flow of mobile phase, so no pressure surges will be experienced.  The dashed arrow on the bypass indicates a continuous flow (which varies with valve position) exists during operation.

 
 

The resistance to flow to through the bypass and the back pressure across the normal flow path will determine the split flow ratio (within the valve).  Broadly speaking, a small percent of the total flow will be diverted through the bypass during normal operation (inject or load positions); however during the intermediate rotational state of the valve (neither injecting, nor loading positions), the entire flow will be diverted through the bypass.[22]

A widely used approach to eliminate pressure surges and flow interruption is the Rheodyne Make Before Break (MBB) valve.  This device implements a bypass only during transition from load to inject positions.  MBB valves can be found with most modern high pressure HPLC instruments.[23]

fig 21

Figure 21. Make Before Break (MBB) valve.  A continuos flow of mobile phase to the column is provided during the entire rotation of the valve by the use of connecting passages in the rotor seal.  These connecting passages are made in such a way that they operate when the valve is neither at the load nor at the inject positions.  Courtesy of Rheodyne

 
 

Pump

For fast HPLC it is necessary to use a variety of devices which have to endure extreme conditions during the analysis, this is particularly true for pumps, which have to be able to deliver a continuous pulse-free flow of mobile phase at extreme pressure conditions (sometimes exceeding 1000 bar) while dealing with corrosive (acidic/basic systems) and/or aggressive (presence of electrolytes, additives, etc) eluent systems.

Due to the high operating pressures the compressibility of the liquids sometimes becomes noticeable.  Close control of the rotational speed of the motor through the reciprocating cycle of the pump and compressibility compensation will improve the smoothness of the delivered flow. These characteristics are implemented with most commercially available fast HPLC pump designs.

Broadly speaking, traditional and fast HPLC pumps have a similar architecture; however, in order to deliver the required flow rate at increased pressure, high pressure HPLC pumps require some improvements.  Some of these are outlined below.

One of the most important improvements of high pressure pumps is the capacity to alter pump efficiency during operation.  This is usually achieved by considering changes in mobile phase compressibility (pressure readings) and altering the rotational speed of the motor accordingly (this usually requires a calibration curve, which is mobile phase dependent).  This drastically reduces the need for pulse dampers, and some modern instruments do not use pulse dampers at all, which dramatically reduces the system dwell volume and facilitates the use of ‘ballistic’ gradients (a large change in eluent composition over a short time).

New materials have been developed in order to produce high pressure pumps with higher mechanical stability and lower dead volume.  Pump pistons, piston seals and check valves are the object of continuous research, so the quest for the “best” pump continues.

Another important improvement is the development of high pressure mixing devices with optimum mixing performance and minimum delay volume.

 

Summary – Pump Adaptations:

  • The traditional pressure limit for conventional HPLC systems is 400bar (~6000psi)
  • UHPLC systems can operate at pressures exceeding1000bar (14,500psi) and some UHPLC systems permit the use of high flow rates at these pressures
  • UHPLC pistons and pistons seals have been redesigned to withstand these pressures, with some systems incorporating silica carbide pistons in place of the traditional sapphire
  • Accurately controlled piston drives are employed in order to negate the need for a pulse damper – reduces Vd
  • Low volume mixers have been designed in order to permit the use of quaternary pumps – reduces Vd and allows ballistic gradient formation
 

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  fig 24

Figure 22. Dual reciprocating fast HPLC pump

 

Figure 23. High pressure binary pump design

 
 

Several fast HPLC pump types have been developed; they implement in-series dual piston designs. 

Binary pump: Integrating two identical pump units (each pump unit presenting a dual piston design) integrated in one housing. Binary pumps are required for applications where very precise flow rates and gradients are required.

Quaternary pumps: Implementing an isocratic pump design, this type of pump uses a multi- valve system for solvent proportioning which allows the user to mix up to four solvents under low pressure.

Notice now in the case of:

  • quaternary pumps, the mixing of solvents takes place before the pump
  • binary pumps, the mixing of solvents takes place after the pump

 

Dwell volume is larger in the case of quaternary pumps versus binary pumps, and the binary pump is capable of mixing very fast (ballistic) gradients, very reproducibly due to the high pressure nature of the gradient formation and the ability to control each of the two pump flow rates very accurately and reproducibly.  The implications of this difference are important, and early UHPLC pumps were predominantly of the binary design.  Only with the advent of very low dead volume instrumentation (mixers, valves and degassers) have quaternary pumps for fast HPLC been introduced. 

 

 

Figure 24 » High pressure quaternary pump design

  fig 24
 

 

4. Columns

The HPLC column's separation performance is highly influenced by the morphological properties of its packing material (porosity, particle size, shape and distribution). Different packing materials have been developed to enable operation at high flow and high efficiency, including:[21, 24]

  • Small (often sub 2μm) fully porous particles
  • Superficially porous spherical particles
  • Non-porous spherical particles
  • Monolithic stationary phases

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« Figure 25. Fast HPLC packing materials

 

As was explained at the beginning of this essential guide, we are not going to cover column selection or characterization.  The previous information is just to give you a flavour of the topic.  A more comprehensive treatment on this can be found in the links below:

 

 
 

Particle Size

fig 26Important to verify the scalability of stationary phase chemistry across the particle size range:

  • Changing particle diameter / morphology may force manufacturers to use different bonding processes or silica substrates
  • It may even require a change in the nature of the bonded phase ligand
  • This may lead to differences in separation selectivity
  • A scalable particle is shown below

Figure 26 »:  Particle size effect on separation.  Courtesy of Agilent Technologies »

 

However – this is not always the case!

 

fig 27

« Figure 27:  Particle size effect on separation Sometimes we don’t get the results we want.  Note the broad peak appearing over 5 mins with the 1.8μm column. 

 
 

Column Protection

Remember to protect your valuable column by implementing best sample preparation practices (in particular appropriate filtration strategies).  Likewise, the use of guard columns is strongly beneficial for challenging situations.

 

fig 28« Figure 28:  High pressure guard column (suitable for pressures up to 1400 bar). 

The use of a guard column is probably the best way to protect your column from fouling.  The guard column is installed to trap particulates and impurities that could adversely affect your column.  The ideal guard column should have no effect on separation and will have very low dead volume.  There are many interesting commercially available products that will comply with such demanding requirements.

 
 

Extra Column Volume

“The total volume contributed by all system components and capillary tubing from sample injection to detection that are not directly involved with the separation process”

The analyte band will diffuse in the mobile phase as it passes through the system – van Deemter B-Term

Extra columns volume is the predominant factor in loss of efficiency when using narrow internal diameter columns

Peak dispersion can be calculated by the Aris-Taylor equation;

 

Where:

σOBS is the observed peak dispersion
DM is the diffusion coefficient
F is the flow rate
L is the column length
id is the column internal diameter

Note that the peak dispersion is proportional to L (effectively the length of tubing used) but is also proportional to the tubing internal diameter (id) raised to the fourth power – thus highlighting the importance of choosing tubing with the appropriate internal diameter!

 

fig 29

Figure 29:  Extra column volume effect

 

 

5. Temperature Control

Temperature impacts separation efficiency; it is imperative to accurately and reproducibly control the column temperature.  During the fast HPLC process, the temperature of the mobile phase could be larger than its intended value, for the following reasons : [25-31]

  • First - the mechanical work provided to the fluid by the pumping system can be converted into heat.  This problem can be solved by implementing temperature control before the column
  • Second - the adsorption of analyte molecules on the stationary phase (as in any adsorptive process) is characterized by its heat of adsorption (energy released during the adsorptive process)
  • Third - certain detectors are affected by the temperature of the column’s effluent (for example refractive index detectors).  Post-column temperature control should be implemented in these cases

 

While flowing through the column, the eluent system experiences frictional forces that increase its temperature (frictional heating). These frictional forces are related to the speed of the eluent system, its viscosity and by the particle size of the column’s packing material.

The particle size of the column’s packing material is crucial not only in determining the column back-pressure but it is the dominant factor responsible for frictional heating.  In essence, if the particle size of the packing material is reduced, then frictional heating will be increased.  This phenomenon could cause selectivity issues as well as a reduction in the overall separation efficiency.

Due to the heat transfer properties of stainless steel columns, power dissipation and temperature control are more efficient with small I.D. columns, so in order to minimize the effects of frictional heating, the use of smaller diameter columns (1 – 2.1 mm) in conjunction with temperature control is recommended.  A typical frictional heating profile within a column is shown below.

When using columns packed with particles of smaller diameter, frictional heating effects will increase.  Therefore, when translating from HPLC to fast HPLC methods, the temperature of the fast HPLC method could be smaller than in the one in the original method.  If this is not taken into consideration, the targeted selectivity or efficiency would not be achieved.

 

fig 30

« Figure 30.  Oversimplified temperature profile in a HPLC column with no temperature control.  The temperature increases in the flow direction (left to right); each temperature zone is identified with a different colour.

Frictional Heating is proportional to the pressure and flow rate

 
 

Table 5.  Frictional heating effects at constant pressure.

L (cm) F(mL/min) ΔP(psi) Tinlet (C) Toutlet (C) ΔT (C)
5.0 1.07 13290 22.3 31.3 9
15 0.36 13250 21.4 25.5 4.1
45 0.12 14150 21 21.7 0.7

 

fig 31

 

Figure 31:  Frictional heating effects at constant flow.

 
 

When using columns packed with particles of larger diameter, frictional heating effects will decrease.  Therefore, when translating from fast HPLC to HPLC methods, the temperature of the HPLC method could be higher than the one in the original fast HPLC method.  If this is not taken into consideration, the targeted selectivity or efficiency would not be achieved.

In order to avoid frictional heating associated problems, fast HPLC utilizes temperature control devices.  Under well-thermostatted conditions, a radial temperature profile will develop, with the centre of the column at higher temperature than the wall. Under certain circumstances altering the temperature of the eluent system before entering the column can be used to compensate for this radial gradient, allowing more efficient analysis.

 

fig 32

 

Figure 32.Oversimplified temperature profile in a HPLC column with temperature control.

 
 

In order to reduce frictional heating associated problems, fast HPLC utilises temperature control devices.  Likewise, it has been reported that mobile phase temperature control before the column will improve the quality of the separation.

fig 33

Figure 33.  Effect of mobile phase preheating on separation efficiency under fast HPLC conditions.

 

Accurate column temperature control is essential to reproducibility of retention times and the stability of the detector baseline.  A small temperature variation can lead to errors in quantification or peak identification.  The smaller the column internal diameter, the easier it is to control frictional heating as dissipation of heat from the column walls is more effective.  If selectivity chnages due to frictional heating are suspected – increase the oven temperature to overcome any potential variability.

 

6. Selectivity Considerations

Perhaps the most difficult part of performing method translation is to make predictions about column selectivity.   Factors such as: frictional heating, extreme conditions of pressure and columns of different geometries packed with new materials (with properties that we never imagined before), are just but a few reasons for the difficulties in making selectivity predictions.

Many attempts have been made to make selectivity predictions, and many approaches have been used; one of the most promising ones consist of grouping columns according to a predefined set of experimental parameters that have an impact on selectivity.  Once this is done, a multivariate analysis such as a principal components analysis (PCA) is performed, so facilitating selectivity predictions.  However, much work is still needed.

Figure 34: Spider diagram representing the various characteristics of the stationary phase.

 

fig 34  

« Figure 34: Spider diagram representing the various characteristics of the stationary phase.

 

HR - hydrophobic retention
HS - hydrophobic selectivity
SS - steric selectivity
HBC - hydrogen bonding capacity
BA - base activity
C - chelation
IEX - ion exchange capacity at pH2.6 and 7.6

AI - acid integration

 

For more on stationary phases for HPLC, Please visit the link below:

Understanding HPLC Column Characterization and Selection »
webcast & tutorial » - available to Premier Members only

 

 

Detection

In fast HPLC (as in traditional HPLC), a detector is commonly placed at the far end of the system, so as to monitor the passage of the components as they emerge from the column.[21]

The detectors used in fast HPLC separations are basically the same type as used in traditionally HPLC separations. However, there are a couple of additional requirements:

  • Increased detector frequency
  • Smaller flowcell volume

In fast HPLC the detector is coupled to a column that is always operated much faster than usually. Thus, the detector has to be compatible with very narrow peaks in time, so the detector must have a very high scan rate.  Modern fast HPLC detectors present scanning rates of more than four times the speed of a traditional HPLC detector.

 

Detector Frequency (Hz)
Fast HPLC 80 - 100
Traditional HPLC 15 - 20

Table 6.  Typical separation speeds of HPLC detectors

 

 

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« Figure 35.  Determination of Gamma-Hydroxybutyrate (GHB) and Gamma-Butyrolactone (GBL) by fast HPLC/UV-VIS (215 nm).  Column: C18, 50mm×4.6mm×5.0μm (packed with partially porous particles).  Mobile Phase: 2.5 mL/min 70% methanol in 10mM buffer KH2PO4 (pH=3.0).

 
 
fig 36

 

« Figure 36:  Detector response measured at different acquisition rates.  Courtesy of Agilent Technologies.

Detector flow cells are often the main source of extra column volume in conventional HPLC

In order to minimize this, and in conjunction with the reduction in injection volumes, reduced flow cell volumes are commonly employed

If injection volumes are not correctly scaled then overloading of the detector can be encountered – Flat Topped Peaks!

 
 

fig 37

 

 

« Figure 37.  Cell volume effect on signal response

 

 
 

Modern detection systems for fast HPLC are designed not only with reduced dead volume (which is critical when dealing with reduced amount of samples) but are designed to improve light transmission and some of them implement multi-wavelength operation. 

The detector cell should provide maximum absorbance, so it should be as long as possible.  However, the total cell volume should be small (as previously indicated).  As a rule of thumb, the flow cell volume should not exceed half of the maximum allowed injection volume (which depends on the analyte and the permitted degree of band broadening).

Modern detector cells are designed to improve light transmission.  Different approaches have been used.  Some of them are illustrated below.

fig 38

 

« Figure 38.  This advanced design prevents light from hitting the cell walls.

 
fig 39

 

 

« Figure 39.  This advanced design uses the reflection properties of electromagnetic radiation.

 

 

References

  1. Ronald E. Majors.  “Method Translation in Liquid Chromatography”  LC-GC Europe.  August 1, 2011
  2. Incognito “HPLC Method Translation and Rules of Thumb”.  Pp 11-15.  The Column.  19 March 2012.  Volume 6.  Issue 5.
  3. Adrian Clarke, John Nightingale, Partha Mukherjee and Patrik Petersson.  “Holistic LC strategies, from UHPLC to HPLC and back”  Chromatography Today.  May/June 2012.
  4. Davy Guillarme and Jean-Luc Veuthey.  “Guidelines for the Use of UHPLC Instruments –Requirements for UHPLC Instruments, Method Development in UHPLC and Method Transfer from Regular HPLC to UHPLC”  Technical note 008383_02.  Copyright ©2009.
  5. Method Transfer Calculator – Introduction, Instructions and Help.  Thermo Technical Note
  6. Easy Method Transfer from HPLC to RSLC with the Dionex Method Speed-Up Calculator.  Dionex Technical Note 75
  7. Michael Swartz.  “HPLC to UPLC Method Migration:  An Overview of Key Considerations and Available Tools”  Waters 2007
  8. Systematic Transfer of HPLC Methods to UHPLC.  Restek Technical Note. 2008
  9. Exploiting Particle Size to Reduce Solvent Consumption in Analytical HPLC.  Thermo Technical Note
  10. Morgane M. Fallas, Uwe D. Neue, Mark R. Hadley, David V. McCalley.  “Investigation of the Effect of Pressure on Retention of Small Molecules Using Reversed-Phase Ultra-High-Pressure Liquid Chromatography”  Journal of Chromatography A, 1209 (2008) 195–205
  11. Morgane M. Fallas, Uwe D. Neue, Mark R. Hadley, David V. McCalley.  “Further Investigations of the Effect of Pressure on Retention in Ultra-High-Pressure Liquid Chromatography”  Journal of Chromatography A, 1217 (2010) 276–284
  12. David V. McCalley.  “Some Practical Comparisons of the Efficiency and Overloading Behaviour of sub-2µm Porous and sub-3µm Shell Particles in Reversed-Phase Liquid Chromatography”  Journal of Chromatography A, 1218 (2011) 2887–289
  13. Morgane M. Fallas, Stephan M.C. Buckenmaier, David V. McCalley.  “A Comparison of Overload Behaviour for Some sub 2 µm Totally prous and sub 3 µm Shell Particle Columns With Ionised Solutes”  Journal of Chromatography A, 1235 (2012) 49– 59
  14. Adam P. Schellinger and Peter W. Carr.  “A Practical Approach to Transferring Linear Gradient Elution Methods”  Journal of Chromatography A, 1077 (2005) 110–119
  15. John W. Dolan.  “Dwell Volume Revisited”  LCGC 05/01/2006
  16. Ronald E. Majors.  “Are You Getting the Most Out of Your HPLC Column?”  LCGC North America Volume 21 Number 12 December 2003
  17. “HPLC Pumping Systems” from  “Instrumentation of HPLC” from CHROMacademy
  18. John W. Dolan.  “Obtaining More Consistent Results”  LCGC North America.  01/01/2006
  19. John W. Dolan.  “How Fast Can a Gradient Be Run?”  LCGC North America.  08/01/2011
  20. Michael D. Jones, Peter Alden, Kenneth J. Fountain, Andrew Aubin.  “Implementation of Methods Translation between Liquid Chromatography Instrumentation”  LCGC 10/01/2010
  21. “Fast HPLC” from “The Theory of HPLC”, CHROMacademy
  22. Hermann Hochgraeber.  “Autosampler for High-Performance Liquid Chromatography”  Dionex Softron GmbH.  United States Patent Number US 8196456B2.  June 12, 2012
  23. Rheodyne.  Operating Instructions.  Model 7710 Sample Injector.
  24. John Palmer.  “Making Sub-2 Micron Particle Technology Easy.  LC Columns and Consumables”  Agilent Technologies.  October 12, 2007
  25. André de Villiers, Henk Lauer, Roman Szucs, Stuart Goodall, Pat Sandra.  “Influence of Frictional Heating on Temperature Gradients in Ultra-High-Pressure Liquid Chromatography on 2.1 mm I.D. Columns”  Journal of Chromatography A, 1113 (2006) 84–91
  26. Fabrice Gritti and Georges Guiochon.  “Mass Transfer Equation for Proteins in Very High-Pressure Liquid Chromatography”.  Analytical Chemistry.  2009, 81, 2723–2736
  27. Fabrice Gritti, Michel Martin and Georges Guiochon.  “Influence of Viscous Friction Heating on the Efficiency of Columns Operated under Very High Pressures”  Analytical Chemistry.  2009, 81, 3365–3384
  28. Fabrice Gritti and Georges Guiochon.  “General HETP Equation for the Study of Mass-Transfer Mechanisms in RPLC”  Analytical Chemistry.  2006, 78, 5329-5347
  29. Fabrice Gritti and Georges Guiochon. “Complete Temperature Profiles in Ultra-High-Pressure Liquid Chromatography Columns” Analytical Chemistry.   2008, 80, 5009–5020
  30. R. Ohmacht and B. Boros.  “Effect of Pressure on Solute Capacity Factor in HPLC Using a Non-Porous Stationary Phase”  Chromatographia Supplement, Vol. 51.  PP S-205 - S-210. 2000
  31. A. Felinger, B. Boros and R. Ohmacht.  “Effect of Pressure on Retention Factors in HPLC Using a Non-Porous Stationary Phase”  Chromatographia Supplement Vol. 56.  PP S-61 - S-64.  2002

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Learn chromatography from the experts

Whether you work in a lab or manage a lab, you will benefit from being a member of CHROMacademy.

As a member of CHROMacademy, you will get access to our vast e-Learning archive full of great interactive content and animations.
All our Essential Guide Webcasts and tutorials and LCGCs archive of magazine articles and webcasts from your favourite authors - John Dolan, John Hinshaw, Mike Balough, and Ron Majors. Plus vendor application notes, electronic laboratory tools and calculators and with our 'Ask the Expert' function - help is always at hand.

 

Analytical Chemists

  • I feel empowered to fix things
  • I can troubleshoot effectively
  • I know where to go for help
  • I understand my analyses
  • I know where to get applications
  • I’m up to date
  • I’m more employable
  • My career is progressing
 

Laboratory Managers

  • Improved equipment utilization
  • Faster method development/problem solving
  • Flexible workforce with a common standard
  • Better quality data
  • Get up to speed quicker
  • Lower T&E
  • Less reliant on me
  • I spend less time on training
 

Subscribe for $399 per/year and access:

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The Essential Guide from LCGC’s CHROMacademy presents an informative session on method translation and transfer in HPLC. In this session, Tony Taylor (Technical Director,Crawford Scientific) and Scott Fletcher (Business Development Manager, Crawford Scientific), present a definitive guide on everything that one would need to consider for translating and transferring HPLC methods.

The session includes in-depth consideration of the hardware and data acquisition requirements for simple method transfer or translation to different column / particle size and morphology. We present many worked examples of calculations, approaches and pitfalls associated with method scaling and transfer and include up to date concepts such as frictional heating and pressure based selectivity to ensure you are fully up to date in this subject area. A must see for everyone translating or transferring HPLC methods.

Scott Fletcher
Technical Manager
Crawford Scientific
Tony Taylor
Technical Director
Crawford Scientific

Who Should Attend:

Anyone wishing to:

  • change HPLC methods to use different column geometry or particle size,
  • speed up methods using UHPLC or more highly efficient particle morphologies
  • transfer methods from one instrument to another

Key Learning Objectives:

  • Understand the variables in method transfer between systems, column and particle geometries
  • Appreciate what changes to hardware and instrument acquisition methods may be required when transferring methods between systems, to different column geometry from HPLC to UHPLC and vice versa
  • Investigate the principles of geometrical scaling to convert flow rates and other method parameters between columns and particles of different sizes
  • Discover the important relationships in converting gradient HPLC methods to maintain equivalent selectivity
  • Consider important factors in reverse scaling from UHPLC to HPLC methods
  • Investigate important aspects of temperature and pressure when scaling methods including Fractional Heating and Pressure Induced Selectivity changes
  • Build a portfolio of useful equations and rules of thumb for method scaling and transfer