Tuesday, 8 September 2009

PocketLab: HPLC data for your lab coat pocket

This useful little booklet is made using a single sheet of paper. It contains information on the following:

  • Common buffers used for RP-HPLC with pKa and UV cutoff data.
  • Common solvents used for HPLC with polarity index, water solubility and UV cutoff data
  • Pressure conversion table for converting between bar, psi, atm and MPa (useful if you have more than one type of HPLC system in your lab)

The links below open as pdf documents (available for A4 and Letter sized paper) which contains clear instructions on how to fold your PocketLab including advice on suggested printer settings.

Click here to download the A4 PocketLab for useful HPLC data

Click here to download the Letter PocketLab for useful HPLC data

Monday, 7 September 2009

Lab Tech Guy on white powder on the balance

And the winner is...

Congratulations to Marjorie from Boston in the United States who has won an iPod Shuffle for submitting this month's question for Ask Lab Tech Guy. Marjorie enjoys listening to the Dixie Chicks and Hootie & the Blowfish. What will you listen to on your iPod Shuffle? Send any contributions for Analyse This to at@mournetrainingservices.co.uk. You need to be subscribed to Analyse This to be eligible.

Friday, 4 September 2009

UTrain: the definitive in-house training solution for basic HPLC

The free HPLC training video featured in the June edition of Analyse This introduced the new, online, in-house training solution from MTS; UTrain. The first UTrain training course will be available soon. It provides a solution for in-house training on basic HPLC. Based on our popular course, ‘An Introduction to HPLC for Pharmaceutical Analysis’, it is recognised by the Royal Society of Chemistry for the purposes of Continuing Professional Development. The course is split into four modules which may be purchased separately.

UTrain is a comprehensive online, in-house training package which practically delivers itself! It is made up of 3 parts which are accessed through our virtual environment for learning, e-MTS.

The first part is knowledge transfer. This is accomplished by the use of training videos which explain the topic fully. These videos are available to all learners and may be watched again as required.

Which of the following two methods of learning do you prefer?

  • Reading well written notes on the topic
  • Watching a video which explains the topic

Please take part in our poll on the top right hand side of this page.

(Note: Poll is now closed, 31st October 2009)

Next the learning from the video is applied by following a set of exercises. These are provided as pdf documents which can be printed out and completed either in groups or by individuals. They include practical tasks where appropriate. The solutions which are provided for the exercises are very detailed, these are the handouts for the course.

A review of the learning is combined with an assessment in a specially designed e-learning module which provides a method to validate and evaluate the training.

Contact us if you have any queries about UTrain. Detailed information will be posted on the MTS website and on this blog in the coming months.

Wednesday, 2 September 2009

What is HPLC column efficiency?

A resource for chromatographers
Column efficiency, also known as plate count, is a measure of the dispersion of a peak. Narrow peaks take up less space in the chromatogram and thus allow more peaks to be separated. They are also easier to integrate since they give better resolution and less overlapping. Efficiency is usually explained using the concept of theoretical plates. This model supposes that the column contains a large number of separate layers. Separate equilibrations of the sample between the stationary and mobile phase occur in these plates. The analyte moves down the column by transfer of equilibrated mobile phase from one plate to the next. It is important to remember that the plates do not actually exist, they are a means to help understand the process at work in the column. They also give a measure of column efficiency by stating the number of theoretical plates in a column, N. A high value for efficiency indicates that more peaks can be separated. The number of plates will increase with the length of the column. The calculation for efficiency is related to the peak width and is as follows:

where t is the retention time of the peak of interest and W is the peak width at the base (as shown in Figure 1).
Figure 1
Similar to the measurement for resolution, the measurement for efficiency may also be performed using the peak width at half height:

where Wh/2 is the peak width as half height. It can be seen that N is related to the analyte peak and thus the column behaves as if there are different numbers of plates for each solute in a mixture. When the peak width increases resulting in broad peaks, it is due to band broadening. There are a number of different reasons for band broadening:

  1. The path taken by the analyte molecules through the column varies due to chance. Some molecules will travel in a fairly straight line whereas others will undergo several diversions. The effect of this is that not all the molecules will elute at the end of the column at exactly the same time.
  2. Sample molecules in a solvent will spread out without any external influence due to molecular diffusion.
  3. Analyte molecules travel from the moving mobile phase to the surface of the particle, through stagnant mobile phase in the pores to the internal surface on the packing. It interacts with the stationary phase and then is transported back to the moving mobile phase. This process is referred to as mass transfer and not all molecules will experience mass transfer in an identical way therefore band broadening will occur.
  4. The mobile phase travels in a laminar flow between the stationary phase particles, the flow being faster in the centre than near a particle. Thus some molecules travel more quickly than others. This flow distribution is reduced by ensuring that the particles in the packing have a narrow particle size distribution.
  5. The tubing in the HPLC instrumentation contributes to band broadening, this is known as the extra-column effect.

HPLC columns which contain packing of smaller particle sizes give better efficiency because the diffusion paths are shorter allowing solutes to transfer in and out of the particle more quickly and thus reducing band broadening.

A typical acceptance criterion for efficiency would be > 2000. Although the value for new columns would usually be very much higher than this (values in the tens of thousands) the system suitability acceptance should be based on a value which indicates that the efficiency is no longer sufficient for the separation. These calculations only apply to isocratic separations. For gradient methods the peak width remains fairly constant throughout the run due to the changing mobile phase composition and therefore the value for N would appear to increase with retention time. A more useful measure of the column efficiency would be the peak width at half height for the analyte. Monitoring this value could provide a measure of when the column efficiency is no longer sufficient for the separation. Resolution depends indirectly on efficiency and therefore if resolution is a parameter in the system suitability test then a measure of efficiency is already included.

The calculation for efficiency using the peak width at half height is common to the USP, EP and JP although the terminology and notations used are not identical. However, due to a slight difference in rounding, the constant in the equation is 5.54 in the USP and EP but is 5.55 in the JP.

This blog post is an excerpt from 'An Introduction to HPLC for Pharmaceutical Analysis' by Oona McPolin, available to purchase through the MTS website.