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The CHROMacademy Essential Guide
-
Basics of Preparative HPLC

 

  sponsored by Shimadzu

The Essential Guide from LCGC’s CHROMacademy presents an educational webcast on the Basics of Preparative HPLC.  In this session, Dr. Charles White (YMC Europe GmbH, UK Operations) and Tony Taylor (Technical Director, Crawford Scientific), present an introductory guide to the theory, instrumentation and applications of Preparative HPLC.  The session will consider all the relevant aspects of theory and practical implementation and will consider the requirements imposed at analytical; scale when considering scale-up to prep.  We will also consider logical approaches to scaling an analytical separation with target recoveries in mind as well as highlighting some of the major pitfalls of the technique and practical tips to overcome them. Throughput and method robustness will also be considered and are factors that are often overlooked by the novice preparative HPLC user.

A must see for anyone using or about to use preparative HPLC.

Tony Taylor
Technical Director
Crawford Scientific

Charles A White
YMC Europe GmbH,
UK Operations

 

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Topics include:

  • Prep HPLCUses of preparative chromatography
  • Column Considerations
  • Preparative Separation Goals
  • Analytical separations designed for preparative applications
  • Method development
  • Optimizing loading at analytical scale
  • Volume & Concentration Overloading
  • Sample Solvents
  • Methods of Scale Up
  • Scalable and non-scalable factors
  • Productivity and throughput
  • Robustness and method verification
     

Who Should Attend:

  • Anyone using or considering using preparative HPLC to recover analyte materials to milligram quantities

Key Learning Objectives:

  • Understand when to use preparative HPLC and have realistic expectations regarding yield
  • Understand the impact of designing analytical HPLC methods for ultimate us in preparative scale separations
  • Highlighting selectivity as the key to unlock higher yield per injection
  • Adopting logical strategies and using key tools and equations to scale from analytical to prep.
  • Appreciate the issues encountered with scaling up, including solubility and loadability
  • Understand the equipment required for preparative chromatography and matching column dimension to required yield
  • Appreciate the need to consider method robustness and throughput as important variables in preparative chromatography
   

 

Analytical and preparative scale High Performance LC separations have many similarities but a number of differences. The prime object of an analytical scale separation is to produce a “work of art” in a chromatogram which has sharp, well resolved, symmetrical peaks. The aim of preparative scale HPLC is to produce a quantity of pure compound as easily as possible in the most economical way.

The aim of this Essential Guide is to outline the ways to progress from analytical separations to preparative purification as easily as possible, to remove some of the “fear” of increased scale and to avoid a number of potential pitfalls Preparative chromatography can be performed using analytical equipment with an analytical column to produce a few µg of material for infra-red, NMR or other off-line analytical methods for structural elucidation of a minor component for example. At the other end of the scale is the fully automated process method providing ton quantities of, for example, active intermediates for the pharmaceutical industry.

The larger the scale of operation, the further the technique is removed from analytical chromatography, both in terms of scale and ideology; the bigger the scale, the more non-chromatographic parameters have to be considered. We hope that this guide gives a balanced consideration to all scales of operation.

 

Figure 1.  Analytical (top) versus preparative (bottom) HPLC chromatograms.

 
 

Animation 1. Analytical vs. preparative HPLC systems.

 

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It is not possible to draw a clear distinction between preparative and analytical HPLC. Since its origins, liquid chromatography has been a preparative technique to recover analytes at both large and small scale.[1]

The term “preparative chromatography” may bring to mind large columns and high eluent flow rates, but it is not the size of the column, instrumentation or the volume of mobile phase used that determines if a separation is classed as ‘preparative’ or ‘analytical’. A preparative separation is the isolation and purification of a component from a sample. In other words, in preparative chromatography, the sample goes from detector into a sample collector while in analytical chromatography the sample goes from detector into waste.

Depending on the goal of the separation, the scale of operation can range from analytical scale columns to massive production scale units. Table 1 summarises the scale of operation for commonly used column sizes.

 

Table 1. Definition and scales of operation.

Scale Column I.D. (mm) Quality of Product Typical Column Length (mm) Purpose
Analytical 4.6 1 - 40mg 250 Biological materials for activity testing
Semi-Prep 10 - 30 100mg - 3g 250 Reference compounds
Prep 50 - 70 5 - 10g 250 - 1000 Intermediates for lab synthesis
Pilot 100 - 300 20g - 5kg 300 - 1000 Pharmaceutical development
Process >300 kg - tons 500 - 1000 Large scale production

Table 1 is in no way comprehensive as special circumstances can dictate very specific system configurations. Figure 2 shows a general set up for large pilot scale separations up to 50kg product; whilst Figure 3 shows a very specific customer column (note the size of the operative in the foreground).

 
 

Figure 2.  1000 mm x 400 mm i.d. columns and pre-columns.
(Courtesy of YMC Co Ltd, Japan)

 

 
   

Figure 3.  4000 mm x 1600 mm i.d. column.
(Courtesy of NovaSep, France)

 
Many preparative separations are achieved on analytical scale columns and systems so that a few mg of material can be isolated for structural or characterisation studies. (Table 1 and 2)
 
 
 

Initial Considerations

Before any preparative separation can be undertaken, the choice of column must be made. This may sound obvious, but you have to consider if you are to perform the separation many times over a period of time, or are simply looking to do a one-off separation. We will revisit the various approaches to sample recover later in this tutorial.

Important considerations:

  1. size of the column – will you want to do the separation in one step or are you happy with multiple injections? This will affect eluent flow rates and pump requirements
  2. type of column – will you want to reduce costs by packing the column yourself or will pre-packed columns be more appropriate?
  3. do you have the required pumps, etc to work with the chosen column?

Pre-packed columns are commercially available from many column manufacturers and come in a wide range of sizes as shown in Table 2. Whilst these can be more expensive than packing your own columns for long-term projects, they offer a guaranteed performance ’out of the box’.

 
 

Table 2. Typical commercially available pre-packed column sizes

Internal Diameter (mm) Column Lengths (mm) Guard Column Length (mm)
10
20
25
30
50
70
100
150
200
50 100 150 250      
50 100 150 250      
  100 150 250      
    150 250      
      250 300 500 1000
      250 300 500 1000
      250 300 500 1000
        300 500 1000
          500 1000
10 or 20
10 or 20
10 or 20
20
50 or 100
100
100
200
200
 

Packing your own columns is generally only economically feasible for those who will be frequently using preparative chromatography on a preparative/pilot/process scale. The cost of the bulk packing material is lower but this has to be off-set against the initial hardware costs.

The hardware may appear externally similar to pre-packed columns, but internally there are many differences to allow for the chromatographic bed to be compressed continually so that voids do not occur in the packing. These so-called SAC (static axial compression) or DAC (dynamic axial compression) columns are available from a number of suppliers and range in diameter from 25mm i.d to over 600mm i.d. as shown in Figures 4 and 5.

 
 
  Glass Pilot column – courtesy of YMC Europe GmbH
 
  BPG™ Columns – courtesy of GE Healthcare Life Sciences

Figure 4.  Types of axial compression columns for lower pressure and bio separations

 
 
  Self-packer modules – courtesy of Agilent Technologies
 
  Large-scale steel DAC columns – courtesy of YMC Co Ltd, Japan

Figure 5.  Types of axial compression columns for higher pressure applications

 
 

Preparative Columns

Preparative HPLC columns consist of a tube (made of stainless steel, glass or synthetic polymers) filled with micro-particulate porous silica. The performance of the separation is influenced by the packing material (particle size, shape and particle size distribution) as well as the quality of the packed bed.

 

Particle Size and Shape

The world of preparative HPLC columns is continuously evolving. Historically, preparative HPLC has been dominated by columns packed with irregular particles of large size and broad size distribution. Nowadays, columns packed with spherical particles of small size and narrow size distribution are the standard for preparative HPLC separations. In addition, modern preparative columns are well known for their good mechanical stability and their high loadability.

As a rule of thumb, the smaller particle sizes result in higher plate numbers (N) and greater analytical efficiency. In preparative HPLC, particle size is important; however, since most times, the column may be overloaded, the smaller particles currently available for analytical HPLC (sub 2μm) are not used in large scale applications. As a matter of fact, most industrial applications use columns packed with particles of relatively large size (between 10 and 30μm).

 

Stationary Phases

Preparative HPLC columns packed with different support materials have been developed, the most important include:

  • Silica
  • Organic polymers
  • Chiral stationary phases
  • Monolithic stationary phases

Silica based are the most widely used of all stationary phases for preparative HPLC. In the early stages of preparative HPLC, silica based columns were packed with large and irregularly shaped particles for which axial dispersion was not uncommon. Nowadays, carefully engineered spherical particles with a narrow particle size distribution had considerably reduced this problem. Underivatized silica and C18 reversed-phase materials are available in packed column as well as bulk quantities and they dominate the preparative HPLC world.

Organic polymers are also a very important group of support materials currently used in preparative HPLC. Compared with silica-based columns, organic polymers show a much higher stability at extreme conditions of pH. Polystyrene-divinylbenzene (PS-DVB) is one of such materials and its representation is shown below.

 
 

Figure 6.
Polystyrene-divinylbenzene (PS-DVB) representation.

 

Chiral stationary phases are becoming more and more important in the preparative HPLC world as, these columns permit the direct separation of enantiomeric compounds. The most important types of chiral stationary phases include:

  • Naturally occurring polymers such as cellulose and amylose
  • Synthetic chiral polymers such as poly(meth)acrylamides
  • Chirally modified silica gels

Monolithic stationary phases can be considered as a single large "particle" of porous material that fills entirely the column volume without any interparticular voids typical of packed columns. Because of that, all the mobile phase must flow through the stationary phase. Monoliths do not need to be packed into a column since they can be prepared in situ by polymerization.

Most polymeric monolithic stationary phases, are known to swell in organic solvents. This frequently leads to a lack of stability. Furthermore, the preparation of polymeric monoliths usually leads to micropores, which negatively affect the efficiency and peak symmetry of the column. Therefore, it is not easy to obtain high efficiency for small molecules. The low column capacity may be another significant disadvantage of monolithic packed columns; this limitation may be attributed to the low specific surface area of monoliths compared to traditional packings.

In order to optimise the recovery per injection of a preparative separation the selectivity must be optimised, allowing the highest loadability. More on this later in this Essential Guide. Most companies now offer their stationary phase materials in both analytical and preparative scale, but it is worth checking with your manufacturer ahead of time to avoid disappointment when scaling from analytical to preparative scale later on.

 
 
 
The main steps in preparative HPLC method development are outline in Figure 7. As HPLC can be regarded as a preparative technique, all considerations made for analytical HPLC method development are valid when developing preparative HPLC methods, although their relative emphases can and will vary.
 

Figure 7. Strategy for preparative HPLC method development.

 
 

Preparative Separation Goals

With any preparative separation there is always balance between the costs of the separation and subsequent downstream processing, time to develop and perform the separation and available systems to operate the process.

Once the scale of the separation has been defined and the timeframe over which the application will be required (and any GMP implications agreed), initial investigative separations may be performed. Irrespective of the scale of operation or the application area involved, the key aspects of preparative chromatography are the same.

If the separation is restricted to a single laboratory, with no GMP implications, the selection of chromatographic supports is more open than if the project involves multi-site production over several years. This is because the latter will require guaranteed long term, possibly world-wide supply of the chromatographic phase with consistent and reproducible performance.

The aim of scale-up to preparative separations is to provide the simplest, most cost-effective but most robust method which can be performed without loss or product degradation within an acceptable timeframe.

 

Analytical Separations Designed For Preparative Applications

Many preparative separations start life as an analytical method, but thought should be given to the whole process, especially if the method is to require larger scales of operation than lab-based systems.

  • An analytical method which requires a complex gradient may prove difficult to replicate on the large scale
  • A method which used large amounts of buffer salts and modifiers may give problems at a later stage where removal of such additives requires a complex process
  • The use of complex eluents may have severe cost implications
  • Exotic phases may not be available for larger scale applications
  • Organic solvents may introduce problems of explosion-proofing equipment
  • Removal of large volumes of water is more difficult than similar volumes of less expensive solvents (which can be re-used to further reduce costs)
  • Elevated temperatures may prove costly to reproduce on a large scale
  • Non-bonded silica is more economical than bonded phases
 

Definition Separation Requirements

The first step in preparative method development is to identify the problem and challenges associated. You should bear in mind the following aspects:

  • Sample information
  • Analyte(s) of interest (type, number, concentration, required level of purity, etc.)
  • Other separation strategies suitable for your sample
  • Detection
  • Amount of material to be isolated
  • Required degree of accuracy, precision, etc
  • Method verification
  • Costs

It is important to note that the ultimate value of the product and the costs of preparative HPLC versus the costs of implementing alternative separation methods will determine whether or not preparative HPLC should be implemented. The following situations are practical examples where HPLC should be selected over other separation techniques:

  • For the separation of peptides there is no single technique that competes with reversed phase HPLC
  • The separation of oligonucleotides is dominated by a combination of ion-exchange and reversed phase techniques
  • The separation of racemic mixtures is, no doubt, dominated by chiral HPLC
  • The separation of thermally labile compounds of biological/biochemical origin (such as those commonly found in toxicology and pharmaceutical analysis) is efficiently achieved by HPLC means
 

Select the Correct HPLC Mode

In principle the same separation mechanisms that are used with analytical scale chromatography are also available with preparative HPLC; however, the preparative HPLC universe is dominated by reversed and normal phase applications, this can be explained in terms of the high prices of mobile phase and additives as well as the limited number of preparative columns and their price.

The increasing pressure to separate large amounts of valuable products by liquid chromatography is creating new opportunities for other forms of preparative HPLC such as ion exchange, size exclusion and chiral chromatography.

The following factors should be considered when selecting the appropriate HPLC mode for your separation.

  • Solubility
  • Molecular weight
  • Functional groups
  • Sample matrix
  • Detectability
  • Other separation alternatives

For more information on HPLC method selection, please visit the HPLC department from CHROMacademy.[2].

 

The following table will help with your preparative HPLC mode selection:

Table 3. Preparative HPLC mode selection

HPLC Mode Typical Analyte Eluent System Temperature
Normal Phase Lipophylic analytes such as: oils, fats, lipids, etc. Organic solvents (heptanes, alcohols, chloroform, etc.) Usually performed at elevated temperatures
Reversed Phase
  • Neutral compounds with molecular weights < 2000
  • Weak acids and bases
  • Strong acids and bases (ion pair)
  • Homologs and benzologs
  • Proteins and peptides
Aqueous mixtures of methanol, acetonitrile and additives. Usually performed at elevated temperatures
Ion Exchange
  • Inorganic ions
  • Acids and bases
Aqueous buffers, ionic solutions Usually performed at elevated temperatures
Chiral Enantiomers Aqueous or organic solvents Usually performed at lower temperatures than other forms of preparative HPLC; however, high temperature applications are also common.
Size-Exclusion Polymers, proteins, nucleic acids Aqueous buffers or organic solvents Usually performed at elevated temperatures
 

As with any form of chromatography, once the correct HPLC method is selected, then method optimization should be performed.

 
 
 

So, before selecting the analytical method of choice, you need to consider if normal phase might be more appropriate than reversed phase or if you need a high purity bonded phase to avoid the use of buffer salts and modifiers, etc.

For more information on analytical HPLC method development please visit the HPLC Department from CHROMacademy.[2]

Important: You must consider the downstream requirements as this can dictate the separation mechanisms which can be used.

Once you have chosen the desired technique and chromatographic phase which gives the desired separation of the principle compound(s) from other products, such as starting materials or side products, the method should be optimised. A good starting point is to use analytical scale columns, typically 150 x 4.6mm i.d., packed with 5 µm material as this can lead to a simple, empirical route for up scaling, but there are more mathematical approaches to be considered.

 
 

Method Optimisation

It should be born in mind that that the chromatographic column or more exactly, the stationary phase used in scale-up, MUST be commercially available in both analytical and preparative columns (or as a bulk preparative material) and that the materials are ‘scalable’ to prevent further optimisation work during the scale up process.

Chromatographic resolution (Rs) is at the heart of all preparative separations, the greater the resolution, the greater sample components are separated. Baseline separation should be the aim of any initial method development. Using the definition in Figure 8,[3] the greater the resolution between the peaks, the greater the sample loading and throughput will be.

 
 

Figure 8.
Resolution between two peaks.

 

Resolution is a function of three other parameters, the retention factor (k), the selectivity (α) (separation of the peak apices) and the efficiency (N) (width) of the peaks.

 

Important: the overloading process (that will be covered later in this session) will result in a loss of resolution (Rs). Therefore, method development should aim to maximise resolution, in such a way that separation targets are not compromised even if resolution is lower than expected.

 
 

The efficiency (N) of a chromatographic peak: is a measure of the dispersion of the analyte band as it travelled through the HPLC system and column. In an ideal world, chromatographic peaks would be pencil thin lines – however, due to dispersion effects the peaks take on their familiar ‘Guassian’ shape.

 

The retention (or capacity) factor: is a means of measuring the retention of an analyte on the chromatographic column.

 

Figure 9.
Definition of retention factor.

 
 
 

The selectivity (or separation factor) (α): is the ability of the chromatographic system to ‘chemically’ distinguish between sample components (see Figure 10).

 

Figure 10.
Definition of selectivity.

 

Selectivity can be optimised by screening different analytical columns.

For chiral separations, it is highly recommended that you contact your column manufacturer as, during the overloading process (that will be covered in the pages to follow) selectivity can be adversely affected.

Maximizing selectivity factor will significantly impact:

  • Throughput (recovery per injection)
  • Particle size of packing material
  • Dimensions of preparative column
  • Solvent choice

Important: you must bear in mind that "equivalent" stationary phases from different manufacturers can have different separation results. This situation is always a factor to consider, especially when dealing with chiral HPLC. Sometimes, even the "same" stationary phase from different manufacturers will render different separations.

 
 

Temperature

Temperature plays an important role in HPLC; this is because both the kinetics and thermodynamics of the chromatographic process are temperature dependent.

In nearly all reversed phase separations, an increase in temperature will reduce analyte retention. Additionally solvent viscosity is reduced at elevated temperature which in turn means lower backpressure.

At elevated temperature the solute transfer from the mobile phase to the stationary phase is more efficient. This leads to a flatter van Deemter curve (extended working range) at higher linear velocity or higher flow rate. For more information on flow rate and band broadening considerations, please visit the "Band Broadening" module from the CHROMacademy HPLC Department. [4]

By increasing the temperature, the amount of organic solvent in the mobile phase can be reduced to maintain retention. In some cases a small increase in temperature (of only a few degrees Celsius) produces a similar effect on retention as changing mobile phase composition. [5]

Increasing temperature will speed up the HPLC analysis for several reasons. First, an increase in column temperature reduces the viscosity of the mobile phase and therefore the column backpressure is reduced, permitting faster flow rates [4]. An increase in column temperature enhances analyte mass transfer (increasing efficiency). The use of high temperatures is limited by the boiling point of the mobile phase, and by thermal stability of the analyte and stationary phase.

NOTE: When transferring methods from traditional HPLC, it should be noted that altering the column temperature may also affect the selectivity of the separation, especially when there are ionisable compounds within the sample.

Whilst elevated temperature control of small scale preparative separations is common, increasing scale can cause problems. With increasing inner diameter the heat transfer from the air surrounding the column to the whole cross section of the column (filled with the ambient temperature mobile phase) becomes more and more difficult. This may result in poor peak shape, as the radial temperature gradient causes different separation conditions for every part of the sample depending on its distance relative to the column wall (non-laminar flow effects). This can be illustrated by the separation of some flavonoids from a Gingko extract on a 20 mm i.d. column (see Figure 10).

The chromatogram at the bottom shows the separation at 40°C with direct introduction of the mobile phase onto the heated column. The separation shown in the middle shows the effect of pre-heating the mobile phase (this measure is often successful in analytical scale), by extending the inlet capillary in the oven. When the eluent in the reservoir bottle was also preheated to 40°C (top chromatogram) the separation was achieved with excellent peak shapes.

Conclusion: Not only the column, but the whole system has to be in a stable and defined state. Preheating of the eluent is therefore an essential step to improving the separation results.

Important: Have you considered the cost of heating eluent flows of 1 L/min or greater or heating the mass of the steel column; what effects will varying the sample volume loaded have if the sample is not at the same temperature as the eluent and/or column?

 
 
 

Figure 11.
The effect of column and system heating on the separation achieved

 
 

Particle Size

Column efficiency is dictated by the particle size of the packing material; the smaller the particle, the more efficient the separation, but at a higher cost and system pressure.

However, larger particles will reduce the column back-pressure and allow lower specification pumps to be used but at the expense of column efficiency. Table 4 shows the relationship between these parameters for a C18 material packed in a 250 x 4.6mm column eluted with methanol:water (50:50).

Table 4. Influence of particle size on column efficiency and back pressure

Particle Size (μm) Column Efficiency (N) Back Pressure
(psi) (bar)
5 20,000 3,300 228
7 14,300 1,680 116
10 10,000 830 57
15 6,700 370 26
20 4,500 250 17
40 2,300 60 4
50 2,200 35 2.4
 

For economic reasons, preparative separations very rarely use particle sizes less than 5μm whilst larger particle versions of the media with truly seamless scalability will only affect the efficiency of the separation and not the elution time. Therefore initial work on a 5μm particle can be very helpful in gaining an in-sight into the optimum particle size whilst allowing selection of the most appropriate stationary phase. It is possible to scale from smaller particle sizes to preparative particle dimensions, however it is important that the support materials, stationary phase and bonding technology remain consistent, otherwise changes in selectivity may occur which may require further optimisation of separation conditions.

 

Figure 12. The effect of particle size on the elution profile of a C18 column 250 x 6.0mm i.d. eluted with acetonitrile:water (50:50) when using a scalable stationary phase.

 
 

Pore Size

In addition to particle size, pore size has to be considered. For example, whilst it is generally accepted that pore sizes of 30nm and above are required for proteins, etc, the use of 20nm porosity particles can result in greater loadability due to the larger surface areas of smaller porosity media. There is some overlap in the optimal ranges of the different porosity materials, rather than a strict cut-off, and there are gains to be made in loadability by exploring the lowest possible porosity material which can be used. Reference to manufacturer’s specifications can also help as the difference in surface area of apparently similar phases can be quite marked. The higher the surface area; the greater the sample loadability.

 
 

Figure 13. The influence of pore size on target analyte molecular weight in analytical HPLC.

Remember you are not looking to produce a master piece of a chromatogram; all you are looking for is the maximum separation of the target material from the rest of the sample content. Closely eluting peaks will reduce loadability, purity and yields.

 
 

Solvent Selection

If alternative solvents can be used, always consider the cost, viscosity and any associated hazards. There will always be a trade-off between the cost of the solvent, the lowest viscosity and the safest solvent which can also be removed easily after separation to allow economic recovery of the pure product. The key issues in selecting solvents are:

  • Selectivity
  • Viscosity and backpressure
  • Cost efficiency
  • Impurities
  • Recycling (possibility of)
  • Toxicity & flammability
  • Cost of waste disposal
  • Cost of storage
 

Table 5. Common solvents for preparative chromatography.

High Viscosity Comments Consider for
Water Need high purity to avoid rogue peaks, most expensive to recover target materials  
Acetonitrile Low viscosity, decreased UV adsorption, expensive, toxic difficult separations
Methanol High viscosity, low price, good solubility for salts non-critical separations
2-Propanol Less toxic than methanol, higher viscosity  
Tetrahydrofuran High viscosity, good miscibility, high solubility for salts special selectivity
Hexane Flammable, low cost, low viscosity normal phase
Ethly Acetate Flammable, low viscosity normal phase
 
 
 

Preparative HPLC provides two ways of achieving purification of large sample amounts: scale-up of the analytical system and column overloading.

Scale-Up of the Analytical System

The use of a chromatographic system capable of dealing with increased amounts of sample. This well known form of preparative HPLC uses:

  • larger column diameters
  • higher eluent flow rates
  • larger sample volumes (constant concentration)

With this approach peaks can remain sharp with good shape; however, large columns and high eluent consumption are needed to separate relatively small amounts of sample.

 

Column Overloading

Injecting increased amounts of sample to overload the column is usually the method of choice as it permits preparative HPLC even with analytical columns (separation of milligrams of sample). Larger amounts of sample would require scaling-up the system. As expected, under column overloading conditions, peak shape is usually compromised.

In analytical chromatography, the assumption is made that the adsorption isotherm is linear which leads to the simple relation between retention factor, k, and the adsorption constant:

k = F x K

where:
F = column phase ratio
k = retention factor
K = adsorption constant

However, the linearity of the adsorption isotherm is limited to a certain very low concentration range where there is less than a monolayer of sample on the stationary phase. At higher concentrations the adsorption isotherm becomes non-linear and often has a convex shape. This non-linearity can be described as a Langmuir adsorption isotherm by mathematical expression:

where:
Cs = column phase ratio
Cm = retention factor
K = adsorption constant
qs = adsorption constant

In chromatography, when the concentration of the solute is such that the adsorption isotherm is non-linear, the column is said to be over-loaded. In order to obtain the maximum loading on the column, both the volume injected and the concentration of sample need to be maximised.

There are two ways of performing column overloading: concentration and volume overloading. Both of them lead to a decrease in chromatographic resolution. [6]

Concentration Overloading

When the sample of interest has good solubility in the mobile phase, then concentration overload is the technique of choice. Here, sample concentration is increased while the injected sample volume remains constant.

When performing concentration overloading, the capacity factor is reduced and peak shapes tend to be triangular rather than Gaussian.

 
 
 

Figure 14. Analytical HPLC run versus preparative HPLC concentration overloading.

 

Advantages of concentration overloading:

  • Availability of the analytical equipment
  • Good stability of the column packing
  • Low cost
  • Less solvent use, less environmental pollution
  • Increasing the concentration of the sample, whilst retaining a constant injection volume, results in concentration overloading. Due to the saturation of column interactive sites, the breakthrough point of the sample peak and the peak maximum is earlier than for an analytical loading as shown in Figure 15. The peak shape becomes more saw-tooth in shape with the final elution time close to that for the analytical separation. Column efficiency (as dictated by particle size) has little effect on such overloading.

     
     
     

    Figure 15. The effect of increasing sample concentration on a 50 x 4.6mm id column packed with C18 material and eluted at 1ml/min with acetonitrile:water (60:40).

     

    Concentration overloading is particularly useful for those samples which have good solubility in the mobile phase.

    In preparative HPLC, peak splitting and shouldering can occur due to poor sample solubility. Typically co-solvents, such DMSO or DMF, can be used to aid sample solubility and decrease the deleterious peak effects.

     
     

    Volume Overloading

    When the sample of interest has good solubility in the mobile phase, then concentration overload is the technique of choice. Here, sample concentration is increased while the injected sample volume remains constant.

    When performing volume overloading, peaks tend to be broad and rectangular rather than Gaussian. After a certain volume, further increasing sample volume injected, does not lead to an increase in peak height, and indeed peak height (and area) may begin to reduce.

     
     

    Figure 16. Analytical HPLC run versus preparative HPLC volume overloading.

     

    Concentration overloading can separate larger amounts of sample than volume overloading and is usually the technique of choice (if solubility permits it). Due to solubility issues, is not uncommon to use both techniques in combination.

     
     
     

    Figure 17. Analytical HPLC run versus preparative HPLC volume overloading.

     

    The effect of increasing injection volume whilst maintaining sample concentration on a 50 x 4.6mm id column packed with C18 material and eluted at 1ml/min with acetonitrile:water (60:40).

    Increasing the volume of sample loaded onto the column, without changing any other parameters, including sample concentration, causes the peak to broaden and tail until the peak shape becomes almost rectangular due to the large volume injected. The peak also elutes at a later time as shown in Figure 17 (above). Column efficiency and therefore smaller particle sizes will help to improve peak shape and so allow larger volumes to be loaded. This technique is particularly useful when a compound has poor solubility in the mobile phase.

     
     

    Sample Solvent Considerations

    To achieve maximum loading, the solvent used to dissolve the sample has to be considered. If the solvent is more highly eluotropic than the mobile phase (for example if DMSO or DMF have been added), it will tend to elute the compounds as if they were in a stronger solvent and so elution can be earlier than expected. However, as the sample solvent dilutes within the mobile phase, the elution power decreases and material is retained on the column. In extreme cases this can result in split or shouldered peaks for the same compound. There is also a risk that as the sample is diluted by the mobile phase, precipitation of sample can result in column blockage. To this end, when using a co-solvent, the proportion of co-solvent used and the volume injected have to be carefully balanced.

    Important: For maximum overloading, it is always better to try to use a solvent that is weaker than the mobile phase.

    Figure 18. Optimisation of the analytical method.

     
     

    Which Overload Method?

    Table 6. Summary of column overloading parameters.

    Volume overloading   Concentration overloading
    Determined by injection volume   Determined by solubility of compound in mobile phase
    Appropriate when sample has poor solubility   Appropriate when sample has good solubility
    Throughput determined by column diameter   Throughput determined by selectivity
    Linear (analytical) area of adsorption isotherm   Non-linear (preparative) area of adsorption isotherm
    Small particle sizes improve loadability   Particle size has very little influence on concentration which can be loaded
     

    Whilst concentration overloading is favoured over volume overloading for preparative chromatography because of the higher amount of sample that can be separated, in practice a combination of both techniques is often required to maximise the amount of sample that can be purified at any one time. The aim is to maximise the loading until there is virtually no base line between the target peak and closely eluting peaks, as shown in Figure 18.

     

    Detector Overloading

    Detector overloading, where the signal response of the detector is no longer a linear relationship with analyte concentration, may be observed as flat-topped peaks with lower heights than expected. This may not be a problem as the key parts of the chromatogram are the bottom of the peaks and the separation between them, from which fraction diversion valves are typically controlled. These are in the linear detector response region and give the appropriate response. Detector overloading will not allow peak integration to be used for compound quantity assessment, but this is not normally required, particularly for the larger scale preparative separations.

     
     

    Figure 19. UV detector overload in preparative HPLC.

     

     
     
     

    Preparative HPLC leads to separations with reduced chromatographic resolution. There are, however, resolution requirements that must be met for the separation to be practical.

    It is well known that selectivity is adversely affected when performing column overloading. However, the higher the selectivity the larger the amount of sample that can be separated in a single analysis, prior to resolution being compromised.

    When performing method development and optimization for preparative HPLC, the following steps should be followed:

    1. Selection of the appropriate mode of chromatography (normal phase, reversed phase, etc.)
    2. Optimization of the separation (stationary phase, mobile phase, temperature, etc.)
    3. Optimization of the throughput (sample amount, column overloading)
    4. Scaling-up
     

    Methods of Scale Up

    Development work carried out at analytical scale will establish the separation conditions required for the scale up to preparative conditions and retention and selectivity optimised. Some basic calculations and relationships can be used to scale the analytical conditions to ensure maximum recovery at preparative scale.

    As the maximum loadability of a sample is directly related to the amount of chromatographic support, we can now calculate the Scale Up Factor. If, for example, we need 100 times more packing material to obtain the desired yield from the preparative separation, the scale up can be expressed as:

     

     

     

     

     

     

     

    Figure 20. Column scale up.

     
     

    NOTE:

    • Doubling the column length doubles the loading (and the column back pressure)
    • Doubling the column diameter quadruples the loading (and does not affect back pressure)
     
       

    Figure 21. Linear relationship between column length and loadability.

       
     
     

    Table 7. Column loadability.

      Column Size (mm) Load (mg) Flow (mL/min.)
    20x 250 x 20 200 9.5
    5x 250 x 10 50 2.4
    1x 250 x 4.6 10 0.5
    a 250 x 4.6 1 0.5

    Figure 22. Effect of column diameter on loadability.

       
     
     

    Scaling up a method is not just a case of bigger is better. The usual approach to scale-up separations from analytical to preparative HPLC is based upon the following assumptions: [7]

    • Both columns (analytical and preparative) should be packed with the same stationary phase and no meaningful differences in terms of chemistry, particle size or morphology should be found
    • Plate count is kept constant upon scale-up and therefore, the injected amount of sample and the column volume should be increased proportionally
     

    Linear Scale Up

    In order to achieve an accurate scale up which applies across a wide range of column sizes, removal of as many variables from the equation as possible is necessary. If we assume that the column length and retention times remain unchanged, that is to say we use constant linear flow of eluent within the column, the scale up becomes easier as a number of parameters are adjusted by the same ‘Scale Up‘ whilst other parameters remain unchanged, allowing us to select other columns with only simple calculations.

     

    Figure 23. Linear scale up.

     

    The use of linear scale up has the following advantages:

    • Reduced effort in method development
    • Minimisation of sample volume
    • Low costs for method development
    • Easy to use method

    For those who wish to change column length there are other methods of scaling up, including on-line scale up calculators which allow for changes in column diameter, length, flow rate, etc in a single operation

     

    Scalable and Non-Scalable Factors

    With linear scale up the following parameters are scalable and should be scaled up in order to maintain the separation developed at the analytical scale.

    • Flow rate
    • Column internal diameter
    • Sample volume loaded
    • Solvent consumption will be scalable
    • Total fraction volume
    • Yield
    • In theory all connecting tubing should also be scaled up. However this is not always practical and the effect, relative to the volume of the column, is negligible
     
     

    Simple Calculations

    1. Linear velocity should be kept constant in order to "reproduce" chromatographic conditions between columns of different size.

    Where:

    D: Column Internal Diameter
    PC: Preparative Column
    AC: Analytical Column

    Example: It was found that for a C18 30cm×3.0mm, 5μm the optimum eluent flow rate was 0.6 mL/min and the amount of sample loaded was 15μL. Calculate the equivalent flow rate in a C18 30cm×20.00mm, 5μm column

    Note that the flow rate will dictate the size of the preparative column required for the separation.[8]

    Figure 24. HPLC column classification.

     
     

    Where particle size is changed between analytical and preparative analyses, the following equation can be used:

    As optimum flow rate is inversely proportional to the particle size, it is possible to calculate the optimum flow rate:

    Where:

    dp: refers to the particle size
    D: Column Internal Diameter
    PC: Preparative Column
    AC: Analytical Column

    Example: It was found that for a C18 30cm×3.0mm, 5μm the optimum eluent flow rate was 0.6 mL/min and the amount of sample loaded was 15μL. Calculate the equivalent flow rate and sample load in a C18 30cm×20.0mm, 10μm column

    Note that the flow rate will dictate the size of the preparative column required for the separation.[8]

     
     

    2. Scale up of sample load (maintain the overloading status)

    Where:

    D: Column Internal Diameter
    L: Column Length
    PC: Preparative Column
    AC: Analytical Column

    Example: It was found that for a C18 30cm×3.0mm, 5μm the optimum eluent flow rate was 0.6 mL/min and the amount of sample loaded was 15μL. Calculate the sample load in a C18 30cm×20.0mm, 5μm column

     
     
     

    Overview

    Controlling gradients in large scale separations is more difficult than in the analytical lab. In fact, process control is always easier at lab scale than at large preparative or industrial scale, the main reasons being:-

    • When two or more mobile phase components are mixed, air bubbles are formed. Traditional lab degassing strategies are usually unpractical in large scale HPLC separations
    • The proportioning of mobile phase in gradient operation must be precisely controlled
    • Large scale gradient instrumentation has large hold-up volume
    • Scaling up gradients is not easy or reproducible, and always requires optimisation at the final scale

    Where possible, isocratic separations should be considered as this will alleviate the need for time-wasting re-equilibration of the column and expensive gradient formation/mixing systems. Also it can be possible to inject a repeat sample before the previous run has finished, provided that no cross contamination can be proved.

     
     

    Generic Gradients

    To a first approach, when developing a generic preparative HPLC gradient process, the separation conditions (temperature, packing material, additives, etc.) should be set as in the analytical separation. In addition the following rules should be followed. [9, 10]

    First rule of thumb: under column overload conditions the main component usually elute at approximately two-thirds of the concentration of polar modifier observed in the analytical separation.

    Second rule of thumb: elution should be started at a concentration that corresponds to the predicted concentration separation minus 10%. Then apply a change in concentration corresponding to 5% over 10 minutes.

    Example: the results of a gradient HPLC separation (gradient conditions) show that a valuable analyte elutes at a concentration of approximately 30% of the polar modifier. Design an equivalent large scale gradient separation under column overload conditions.

    First rule of thumb, under column overload conditions it will elute at approximately 2/3 × 30% = 20%.

    Second rule of thumb, the analysis should be started 10% earlier than the previously calculated concentration, this is approximately 20% - 10% = 10%; then a gradient with a rate of change or organic modifier corresponding to 5% in10 minutes should be applied up to the initial elution concentration (30% organic. Under these conditions the analyte will elute in the region shown in Figure 25.

     

    Figure 25. Generic gradient method development for preparative HPLC.

     

    When considering preparative gradient analysis is reversed phase mode one should bear in mind that the higher the aqueous content of the mobile phase in which the analyte is collected, the more time and energy required to recover the material with an increased possibility of analyte degradation through the use of increased temperatures for solvent evaporation.

     
     
     

    Productivity Considerations

    Defined as the total amount of output (service, product, profit, etc.) per unit input (labour, equipment and capital), productivity is a measure of the effectiveness of productive effort. There are many ways of defining and calculating productivity, however, in preparative HPLC, productivity might be measured based upon the revenue generated by the price of the purified analyte divided by the total separation costs (initial investment and running costs).

    The lack of productivity will make sank any project however good. The following pointers should be considered when performing preparative HPLC method development:

    • There are situations where HPLC cannot be replaced by any other single separation technique (separation of peptides, racemic mixtures, etc.)
    • Column load is a decisive productivity factor as, low column load capacities will indicate that several injections need to be undertaken in order to achieve the required amount of product
    • Sometimes it may be better to purify a valuable product in smaller portions rather than compromising separation targets
    • The initial investment (time, money, human resources) to establish a pilot plant is vast compared to performing analytical development. The situation gets even worst with large scale industrial applications
     

    Illustration: The HPLC separation of certain polypeptide can be achieved by using either reversed or normal phase. The following table looks at issues that will be reflected in higher running/investment, which in turn will be reflected in lower productivity.

     

    Table 8. Reversed phase vs. normal phase productivity considerations.

    Technique Comparison
    Stationary Phase Stationary phases for reversed phase are usually more expensive (usually twice as much) than the ones for normal phase, however columns tend to be cheaper.
    Loadability Smaller loadability of peptide samples in reversed phase compared with normal phase columns
    Column Resolving Power Higher in reversed phase than in normal phase
    Eluent Systems Eluent systems for reversed phase are predominantly aqueous and are normally less expensive than the ones used for normal phase (which are predominantly organic)
     
     

    Are We Ready to Go Ahead?

    Now we have established the scale of operation for the preparative scale, are we ready to go-ahead with the practical work?

    No! We must consider the financial implications and instrument requirements. If we have chosen a long thin column for our separation, what is the likely back pressure? Should we have chosen a shorter, wider column as these will:

    • generate lower back pressures, requiring less expensive pumping systems
    • reduce the solvent requirements and provide more concentrated fractions, further lowering downstream costs

    Have we chosen to overestimate the column size by opting for a single injection? If we consider the application below in table 9 and consider the cost implications of multiple injections on smaller columns compared with a single injection on a large column to provide, for example, 500g of product.

     

    Table 9. Effect of column size on production costs.

      Column ID
      4.6 20 50 150
    Scale Up Factor 1 18.9 118 1060
    Sample purity (%) 50 50 50 50
    Product yield (%) 98 98 98 98
    Solvent consumption/run (ml) 20 378 2360 21200
    Sample consumption/run (mg) 3 56.7 354 3180
    Product yield (mg) 0.147 2.78 17.35 155.8
    Injections per 500 mg 340 18 3 1
    Time consumption (min) 6800 360 60 20
    Solvent consumption (litre) 6.8 6.8 7.1 21.1
     

    Have we chosen to overestimate the column size by opting for a single injection? If we consider the application below in Table 9 and consider the cost implications of multiple injections on smaller columns compared with a single injection on a large column to provide, for example, 500g of product.

    Again No! We have to consider the costs of materials. The amount of solvent used by the 4.6, 20 and 50mm i.d. columns is very similar and so costs of obtaining and disposal of the solvent will be similar but significantly different to that for the 150mm i.d. column as shown in Table 9.

    Once the cost of manpower and the cost of materials for the overall operation are factored in, the situation becomes somewhat clearer. It is better to use the 50mm i.d. column, in this case, to perform 3 injections rather than have the massive overproduction potential of the single injection on the 150mm i.d. column. However should the potential of this particular application increase to requiring production of 1500g, then the economics move in favour of the single injection on the 150mm i.d. column.

    Table 10. Economic factors affecting preparative separations.

    Column Dimension Flow Rate (mL/min) Volume / 500g Costs Yield / Run
    4.6 mm 1 6.8 L 400.00 € 0.147 g
    50 mm 118 7.1 L 420.00 € 17.4 g
    150 mm 1060 21.1 L 1,250.00 € 156 g
             
        4.6 mm ID 50 mm ID 150 mm ID
    Solvent   400.00 € 420.00 € 1,250.00 €
    Waste Disposal   40.00 € 40.00 € 120.00 €
    Personnel (200 €/h)   22,700.00 € 1,200.00 € 70.00 €
    Materials   800.00 € 2,500.00 € 15,000.00 €
    Total   23,940.00 € 4,160.00 € 16,440.00 €
             
    50g Product sales (1000 €/g)   50,000.00 € 50,000.00 € 50,000.00 €
     

    Robustness and Method Verification

    Hopefully your work at the analytical scale proved that you had a robust method, but it is always essential to verify this at the final preparative scale because occasionally minor issues arise due to:

    • Changes in the grade of the solvent
    • User-packed columns as not as efficient (or reproducible) as you hoped
    • You have changed a non-scalable factor

    Once the method has been verified as producing the required product in the expected yield and purity and in a reproducible manner, you have reached your goal of a preparative separation.

     
     
     

    Overview

    As was previously stated, sample exiting the detector is diverted into a fraction collector. This is usually achieved by using a diverter valve that is switched based upon certain triggering events such as detector signal or time programming.

     
     

    Figure 26. Preparative HPLC fraction collection principle.

     

    For the collection of fractions vials, test tubes or well-plates are commercially available; most fraction collectors can handle all those fraction containers. For very large scale applications bigger containers can be used.

    Fraction collectors are available at different sizes and designs. Some of them combine the autosampler and the fraction collector on a single device either with a single needle and valve for both operations (injection and fraction collection) or with two independent devices, one for injection and the other one for fraction collection.

     

    Figure 27. HPLC fraction collector suitable for analytical to preparative applications.

     

    Fraction collectors are available at different sizes and designs. Some of them combine the autosampler and the fraction collector on a single device either with a single needle and valve for both operations (injection and fraction collection) or with two independent devices, one for injection and the other one for fraction collection.

     

    Figure 28. HPLC fraction collector classification.

     
     

    Sample Collection

    As the major difference between analytical and preparative separations is that in the later the sample has to be collected and isolated rather than simply pumped through a detector, it would be an omission to exclude some discussion on fraction collection.

    The first point is to consider the detection system used. If a destructive method such as ELSD or mass spec is employed then, in order to collect intact product, the detection system must be configured to work off-line with a splitter. In order to maximise yields, the split ration must be as high as possible, commensurate with good detection.

     

    Figure 29. The layout of a splitter to allow destructive detectors
    to be used for preparative separations.

    If refractive index is used, there are limitations on the flow rate which detector cells can accommodate and again off-line working with a splitter is recommended.

    Note: for maximum yields sample splitters are best avoided with the entire sample passing via the detector to the collector.

    Depending on the scale of operation various collection methods are available:-

    • Manual collection
    • Time based collection
    • Detector based collection
     
     

    Manual collection means that the user either triggers the switch to collect a fraction (or simply inserts a collection vessel after the detector) based on observation from the chromatogram. Whilst this offers flexibility due to operator interpretation of the chromatogram, it has the drawback of requiring full-time operators and is subject to a lack of reproducibility. It is therefore only recommended for low throughput applications and very valuable samples.

    Time based collection relies on a timer to operate the fraction collection system and therefore has a degree of automation. However this method is subject to errors due to variations in elution times.

    Detector based collection relies on the detector signal to operate the collection system. Such systems allow full automation, but are only as good as the set up of parameters used to determine if a signal is due to a compound eluting or an artefact. A number of parameters are built into automated systems and include:

    • Threshold operation
    • Peak gradient
    • Data point duration

    Threshold operation, where the detector triggers collection when the signal rises above (to start collection) or falls below (to end collection) a certain level, is the simplest method. However such a system does not account for changes in baseline or unresolved peaks.

    Peak gradient is used to determine the rate of increase of a signal. This is always a maximum at the start and end of a peak and low for baseline changes which therefore go undetected..

    Data point duration allows for rapid changes in the slope to be acted upon if the duration of change is more than a number of data points. This will allow distinction between spikes due to air bubbles, etc and genuine sample peaks

    In practice, the more sophisticated the collection system, the more these parameters can be selected and used in conjunction with each other to ensure that only the require peaks are collected. Some systems will even allow for “cuts” to be made in a peak such that the early and late eluting fractions, which are more likely to be contaminated by other, closely eluting materials, can be removed for subsequent re-chromatography. This is an excellent method for increasing yield of the main component in the initial separation, whilst allowing recovery of an additional quantity of purer material on re-chromatography of the “target-rich” fractions.

    Important: It is very important to incorporate time delays into the collection system to allow for the time it takes for the sample to leave the detector (at the start trigger point) and arrive at the sample collector and also (at the end trigger point) to arrive at the sample tube or selection valve. These delays can easily be calculated from the volume of the connecting tubing and the eluent flow rate.

    Important: Changes in flow rate will change these time delays

     
     

    Triggering Events

    The following are some of the most commonly used triggering events used to decide whether a fraction must be collected or not:

    Manual fraction collection: the diverter valve, used to collect eluent fractions, is manually triggered by the user (pressing a button or using the control software). This approach is usually based upon a signal plot on the instrument display or in the software. The major drawbacks are the lack of automation and the low throughput that can be achieved with this method. Manual collection can be used in the following situations:

    • low-throughput applications
    • valuable samples
    • as a safety feature to manually interrupt a run

    Peak-based fraction collection: collection is based upon detector response (some approaches utilize peak shape). The easiest way to perform peak-based fraction collection is to trigger on signal threshold i.e. as soon as the signal exceeds a predefined limit then the diverter valve is switched to collect sample. When the signal falls back below the predefined limit, then the diverter valve is switched to its original position and the collection of sample is terminated. A more reliable method uses peak shape to act the diverter valve. See figure below.

     
     

    Figure 30. Selected peak-base fraction collection strategies.

     

     

    Warning: threshold must not be set too high because otherwise some peaks could be missed.

    Time-based fraction collection:
    only fractions eluting at certain predetermined time intervals are collected.

     
     

    Fraction Delay Time

    Once the triggering event prompted the sample being collected, the valve position is switched accordingly. However, due to the spacial separation between the detector and the diverter valve, there is a finite period (flow rate dependent) that is required for molecules exiting the detector to reach the valve and from the diverter valve to the sample collector.

     

    Where:

    t1: time at which the detector finds the peak of interest

    t2: time at which the end of the peak is measured by the detector

    tA: time required for the eluent system to travel from the detector to the diverter valve

    tB: time required for the eluent system to travel from the diverter valve to the sample collector (to reach the tip of needle)

    Figure 31. Fraction delay illustration.

    By looking at the previous figure, the following information can be deduced:

    Table 11. Fraction delay relationships.

    Fraction Collection Sample Diverted to Collector Relationship
    Start When start of peak arrives at diverter valve t1+ tA
    End When end of peak arrives at needle tip t2+ tA+ tB
     

    The delay volume, defined as the whole empty volume from the detector to the end of the tip from the sample collector, should be used in conjunction with the delay time, otherwise, the system should be recalibrated again and again. The delay volume requires to measurements:

    • the empty volume between the detector and the diverter valve (VA)
    • the empty volume between the diverter valve and the capillary tip of the sample collector (VB)

    The total delay volume (VD) can be calculated as:

    VD = VA + VB

    There are many ways to calibrate your product recovery system. The classic approach uses dyes as calibration standards. In essence, once the dye is detected, the diverter valve is switched to the sampling position, and a timer is started; once the dye can be seen coming out of the fraction the timer is stopped. This approach considers tA and tB. There are other procedures but the best solution is to follow your HPLC manufacturer’s recommendations.

     
     

    Types of Collector

    There are numerous fraction collectors available, but they essentially fall into 3 categories:

    • Manual systems
    • Fraction collectors
    • Valve-based collection systems
     

    Figure 35. Various types of fraction collection systems.

     

    The manual system is generally only suitable for low throughput and very rare samples where total operator supervision is required.

    Fraction collectors are very suitable for lab-based systems where the tube volumes that are available is between 1mL and several hundred ml. With the higher flow rate systems it should be noted that a waste/sample valve should be incorporated (as shown in Figure 36) so that solvent and sample do not spray the unit whilst moving between tubes.

    Valve-based units are most suited to large scale operations where sample collection vessels can range in volume from about 20mL up to several litres (or greater). These units are fully automated and can be set up to pool fractions from repeated runs or to collect the main fraction in successive vessels for repeated injections. By using a waste/sample valve in addition to the sample selection valve, the latter does not have to switch back to a waste position and so only moves forward 1 position every time. This prevents cross contamination of collected sample as the result of eluent flow passing the previous collection positions as it moves to the waste position. Depending on flow rate and system pressures the number of collections per valves can be between 4 and 12 but some systems allow for additional valves to be incorporated into the final valve position to extend the number of collection vessels which can be used.

     
     
     
    1. Yuri Kazakevich and Rosario LoBruto. “HPLC for Pharmaceutical Scientists” ISBN-13: 978-0-471-68162-5. PP 937 – 980. John Wiley & Sons. 2007.
    2. HPLC Department from CHROMacademy.
    3. “Chromatographic Parameters” from “The Theory of HPLC”, HPLC Department from CHROMacademy.
    4. “Band Broadening” from the “HPLC Channel”.
    5. Bowermaster J, McNair HM (1984) Journal of Chromatography Science 22:165–170.
    6. Udo Huber and Ronald E. Majors. “Principles in preparative HPLC” Agilent Technologies Inc. Publication Number 5989-6639EN. Printed in Germany. April 2007.
    7. Anurag S. Rathore and Ajoy Velayudhan. “Scale-Up and Optimization in Preparative Chromatography” Chapters 2 and 3. Marcel Dekker, Inc. 2003.
    8. Lin Yuan. “Analytical vs. Preparative HPLC” Shimadzu application note.
    9. Yung-Huoy Truei, Tingyue Gu, Gow-Jen Tsai and George T. Tsao. “Large-Scale Gradient Elution Chromatography” Advances in Biochemical Engineering/Biotechnology. Vol 47. Pp 1-44. 1992.
    10. Donald A. Wellings. “A Practical Handbook of Preparative HPLC” ISBN 13: 978-1-8-56-17466-4. Chapters 1 – 6. Elsevier. Italy 2006.
     
     
     
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