No thanks! I would like to know more about CHROMacademy

 Over 3000 E-Learning topics / 5000 Articles & Applications
 

You Need a Fancy System to Run Fast HPLC – or do you?

column lengthFast HPLC is where we obtain the same (or better) separation, but with a shorter runtime. This is obtained simply by exploiting certain parameters (the column particle size, internal diameter, length and particle morphology) to increase the efficiency of the separation. If the column and conditions are more efficient we can do the same job in less time.

If we require an efficiency of c.12,500 to allow us to separate our peaks,  the following table illustrates what mix of parameters we can use to obtain the same efficiency. »

What do these three parameters do?

Particle size – smaller particle size increases efficiency – simple as that, however it also causes the back pressure to increase. We have to find a balance between what efficiency we’d like and what our system can handle. Although we may not be able to use the ultra efficient sub 2u particles, we can go a long way with particle sizes in the 3u range or Superficially Porous Particles [core-shell or solid core] .

More from CHROMacademy on Fast HPLC

You can learn more from our Fast HPLC eLearning module

You should also check out the Fast HPLC webcast and our comprehensive Fast HPLC tutorial



Superficially Porous Particles

Superficially porous silica technology has been available since the mid-70's and consists of a solid silica core surrounded by a relatively shallow layer of porous silica material. The inner core is chromatographically inert whilst the porous outer layer is used for the separation of analytes.

These materials were originally designed for the separation of macromolecules and consisted of silica with a wide pore diameter (typically 300Å). Modern advances in superficially porous materials allow the materials to be produced with 90 - 120Å pores which is much more amenable to the separation of small molecules. The core of these materials is typically 1.5 - 2.0μm in diameter with a 0.5μm outer layer making the whole particle 2.5 - 3.0 mm diameter. The main advantage of these materials is that they typically generate one third of the back pressure seen with sub 2μm materials, whilst achieving comparable efficiencies.

Like their reduced diameter counterparts, superficially porous particles gain efficiency through the reduction in band broadening due to Mass Transfer. However, very recently it has been concluded that this process is insignificant for analyte molecules below about 600Da. Current thinking explains the increase in efficiency as being due to the very homogenous packed beds which result from superficially porous materials. Very narrow particle size distributions can be achieved when engineering these particles and as such the quality of the resulting packed beds increases and a significant reduction in eddy diffusion results in increased efficiency.

There were some initial concerns regarding the capacity of these materials as they theoretically have limited silica surface area. This issue is explored further in this module.

 

CHROMacademy Forum

Content on this page requires a newer version of Adobe Flash Player.

Get Adobe Flash player


Column length – increasing column length increases efficiency, but also increases runtime, back-pressure and reduces sensitivity as the peaks become broader. If we double column length we double the runtime, but only increase efficiency by a factor of 1.4.

Column Internal diameter – for each internal diameter there is an optimal flowrate. As we reduce internal diameter we use lower flowrates – reducing solvent use or allowing us to interface directly to an MS without needing a flow splitter.

One important thing to consider with gradient methods is that as we reduce the flowrate, this increases the re-equilibration time between injections. This is because of the system dwell volume – the volume the gradient mixture has to travel (e.g. through the pump, tubing etc.) before reaching the column. In older HPLCs, particularly pumps mixing mobile phases in a proportionation unit before entering the pump heads, this can be several mls and at 0.3ml/min will take several minutes longer to equilibrate. This can offset any reduction in runtime obtained from modifying the other parameters. However, smaller particle sizes are more efficient over a larger range of flowrates, so it is possible to increase flows (backpressure permitting) to obtain a faster equilibration time.

Typical flowrates are :

4.6mm ID – 1.0ml/min
3mm ID – 0.6ml/min
2.1mm ID – 0.3m/min

Injection volume – if we reduce the particle size, column length and internal velocity we gain the advantages of shorter runtime and better sensitivity. One important point to remember is that all these reductions reduce the amount of stationary phase material meaning we have to reduce the injection volume in order to avoid overloading the column. Any loss of sensitivity we get by reducing the injection volume is more than offset by the increase in sensitivity achieved by the other changes.

We have a lot of parameters to optimise to allow us to find the right solution for the equipment we have available


Getting it all together:

HPLC Method Conversion

There are a number of simple equations which can assist with translating HPLC methods to faster equivalents and some of these are explained alongside the simple method translator below.

Content on this page requires a newer version of Adobe Flash Player.

Get Adobe Flash player


To summarise :

To reduce runtime – reducing particle size increases efficiency, this allows us to reduce column length. If using a 150 x 4.6 x 5u, we can obtain similar efficiencies on 100mm x 4.6u x 3.5u or a 75 x 4.6 x 2.6u solid core technology.

loading data
loading data
loading data
loading data
loading data

group  subsCHROMacademy can deliver to corporate clients on a multi-user subscription basis.
Served up from secure servers to the corporate intranet or individual desktops.

  • Microsite - your own learning site powered by CHROMacademy
  • Your Landing Pages -with your logo and branding
  • Customized Assessments - Based on content agreed upon Certificate of Completion
  • Certification Programs - Offer your learners a goal to strive towards
  • LMS : Connect - Our Learning Management System is S.C.O.R.M. compliant and will connect to your system
  • Engagement Package - E-newsletter stimulation program derived from your content and ours
  • Full archive of Essential Guide webcasts & tutorials
  • 1000’s of eLearning topics - HPLC / GC / Sample Prep / Mass Spec
  • Ask the Expert - our experts will answer your chromatography questions within 24 hrs.
  • Assessments - test your knowledge
  • Application notes & LCGC articles
  • Troubleshooting and virtual lab tools

Request a quote

 

 Home | About UsContact Us | SubscribeTerms and Conditions | Advertise | Privacy Policy 

loading data

loading data

loading data

 

loading data


loading data