Thursday, 17 December 2009

How was 2009 for you? What do you need to work on in 2010?

Training know-how applied to laboratory science

Now that 2009 is coming to an end it is a good time to review how your professional year went and consider what you want to do next year. This may be performed as part of a companywide performance review programme where you will use proscribed templates to identify what you have achieved and what your targets will be for next year. All too often these procedures can feel like a paperwork exercise which is simply done so that a tick can be put in the right box. However, if you want to develop your career further it is a good idea to use a performance review as an opportunity to assess what you can do well and where you want to be in the future.

A training plan is a key component in developing your skills. If you do not have support for formal training then you need to find ways to learn about your chosen subject in an informal way. I am going to concentrate on the technical skills related to working in a pharmaceuticals analytical laboratory but you can probably apply a similar approach to the other workplace skills you require such as so called ‘soft skills’, which includes communication, time management, people management, project planning etc.

What do you currently do well? To figure out what you want to improve you first need to assess your current knowledge and skills. In a laboratory setting this will usually translate into knowledge and skills related to particular analytical tasks. Write out all the tasks which you have used over the past year and try to determine your level of proficiency. It may be convenient to use the following categories:

Analytical chemistry laboratory skills & knowledge
Examples include: Using a balance; Using volumetric glassware; pH measurement; Analytical method validation; Analytical method transfer,etc.

Laboratory techniques
Examples include: High Performance Liquid Chromatography (HPLC); Infra-red spectroscopy; Water determination by Karl Fischer; Titrations, etc.

Laboratory procedures
Examples include: Use of laboratory documentation; Knowledge of standard operating procedures (SOPs); Recording data; Equipment calibration; Deviations and out of specification results, etc.

Pharmaceutical science
Examples include: Forced degradation studies; Stability studies, etc.

This is quite a difficult task. It is advisable to seek help from others. This may mean talking to your line manager but it may also help to talk to an experienced colleague or your peers to give you a range of opinions. You may encounter bias if you only seek out one person’s opinion. The aim is not to come out as high as possible for each identified task but to determine your level of competency as accurately as possible. At the same time as discussing what your current level is you also need to figure out what you need to do next and prioritise which tasks are likely to be most important in your day to day work and most beneficial to furthering your career. The next stage is to convert the information you have obtained into a realistic training plan for 2010.

If you discover that you already have a broad range of knowledge and skills at a highly proficient level (you probably already knew this but it’s nice to have it confirmed!) then you may wish to develop an area of expertise. This is a great way to raise your profile.

Two things to consider:

  1. Pick a topic which you find interesting and if possible, very interesting. It is much easier to develop expertise in a subject area that you are passionate about.
  2. Pick a subject in which you have a realistic opportunity to gain experience and which is a valuable asset in your career plans.
To summarise, figure out what you are able to do well and what you need to be able to do well to move on to the next step on your career ladder.

This advice is based on the approach used by Mourne Training Services for performing training needs analysis. We carry out job analysis for the roles in your laboratory and define competence based standards for the work activities identified. We then assess current capabilities and identify the learning needs for which training solutions are suggested. Contact us if you are interested in our training consultancy services for training needs analysis.

Wednesday, 16 December 2009

Feedback from free HPLC training video

In June of this year Mourne Training Services released a free training video on YouTube: ‘A Brief Guide to HPLC instruments’. The video has been extremely popular with over 9000 views in the last six months from all around the world. It has a 5 star rating and has been selected as a favourite by YouTube users many times. An example of a particularly gratifying comment is this one: ‘great video; thank you very much for a simple to understand component breakdown of the process!’ This type of comment assures us that we have achieved our aim with this video.

It was intended to provide a useful resource which explained how all the bits in a HPLC system come together to enable HPLC analysis but it was also an opportunity to try out our new concept for online training. Stretching our minds back to university days we remembered that being at a lecture and having something explained to you was much better in terms of retaining the information than trying to make sense of the notes later, even if the notes were very good. A one hour lecture could take as much as 3 or more hours to get to grips with on your own. We tested this theory a little further by using a poll on the MTS blog where we asked:

Which of the following two methods of learning do you prefer?
1. Reading well written notes on the topic
2. Watching a video which explains the topic

The response was overwhelmingly for option 2; watching a video. Hardly scientific research but still adding to the overall theory that having something explained to you verbally is preferable, even without opportunities for questions.

Our new online training solution, UTrain, consists of training videos which are similar to ‘A Brief Guide to HPLC instruments’ but contain further information. The videos are supported by exercises which can be undertaken by an individual or as part of a group. Fully completed solutions for these exercises is provided. The training is finished off with an e-learning review/assessment which tests the learning. On successful completion of the assessment a certificate is awarded which is recognised by the Royal Society of Chemistry for the purposes of continuing professional development.

UTrain is available as a subscription service which can be purchased by your laboratory. It consists of a series of modules which are available separately, thus you can choose the training that is needed in your lab. The first four modules are available now on the topic of basic HPLC.

Contact us for more information, or, if you would like to arrange a free trial of UTrain so that you can try it out for yourself.

Tuesday, 1 December 2009

HPLC calculator for pressure conversion

A resource for chromatographers

Last month we gave away a HPLC calculator for working out column equilibration times. This month we are pleased to announce that the calculator has been updated to include converting pressure values into different units. Click here to access the calculator; it will open as an Excel document. The directions for how to use the calculator are provided on the spreadsheet.

Monday, 30 November 2009

Help on: Difference in retention times using Agilent and Waters HPLC systems


Do you have any problems relating to analytical chemistry for pharmaceuticals or training? Send your questions to the MTS helpdesk using our contact form.

"I have a question... I want to know why there is a difference in the retention times observed when using Agilent systems and Waters systems?”

“To answer this question we must first look closely at what exactly is a retention time? It is measured as the time from the injection of the sample to the time the separated component of interest is observed by the HPLC detector. Therefore the total retention time will depend not only on the time taken for the component to travel through the HPLC column but also the time taken to travel through the tubing both before the column and after the column. This time, which is in addition to that spent on the column, will depend on the overall volume of tubing, we refer to this as the ‘extra column volume’.

The amount of extra column volume for different brands of HPLC instruments will be slightly different since they are not all made in the same way. Additionally, the user of the instrument can alter the extra column volume by a variety of actions such as replacing lengths of tubing, cutting off the end of the tubing when removing stainless steel ferrules, installing column switching valves, etc. These factors result in small differences in the retention time observed for different brands of instrumentation.

A feature of the chromatogram which tells us about the extent of the extra column volume is the void volume, t0. This is usually the first disturbance in the baseline and corresponds to the solvent in which your sample was injected. This solvent is unretained and so corresponds to the time required for an unretained component to travel through the column to the detector. If you calculate the capacity factor, k’ you can remove the effects of extra column volume for each peak, this value should be the same for different HPLC systems (assuming all other variables are constant, i.e. same column, same mobile phase, same method etc.).

Capacity factor, k’ = (t – t0)/t0

where t = retention time and t0 = the time taken for non-retained components to elute. The capacity factor is a measure of where the peak of interest is located with respect to the void volume (the elution time of unretained components).

Another reason for a difference in retention times which can lead to larger differences relates to gradient methods. When running these types of method the changes in mobile phase composition are controlled by the gradient proportioning valve (GPV) in the pump (for low pressure mixing HPLC systems, the most common type in use). The consequence of this is that there is a delay between changing the gradient and that effect being experienced at the column. This has to take into account the extra volume between the GPV and the injection port. The total volume from the GPV to the column is called the dwell volume and this is another reason why you may experience different retention times using Instruments from different manufacturers. In my experience I have noticed very little difference when measuring the dwell volumes of Agilent and Waters systems.

To calculate the dwell volume for a particular system, refer to a previous post on interpretation of HPLC methods (Wednesday, 14th October 2009).

In practice we tolerate small differences in retention time on different systems provided there is no negative effect on the observed chromatography but a larger difference may need further investigation.”

Do you have anything to add? Feel free to leave a comment.

Friday, 27 November 2009

Thoughts on applying QbD to analytical methods

The MTS blog post on applying Quality by Design to analytical methods (Wednesday 11th November 2009) has been published as a short article on the Pharma QbD website.

Wednesday, 11 November 2009

Quality by Design for analytical methods


Quality by Design (QbD) is the name given to the principle of fully understanding a process and the effect of the various characteristics which influence the process, rather than just testing the resulting product at the end to check if the process has performed as expected. This concept was adopted by the FDA in 2004 as detailed in ‘Pharmaceutical CGMPs for the 21st century – A Risk Based Approach’ [1] and is included in ICH Q8 'Pharmaceutical Development’ [2] and ICH Q9 ‘Quality Risk Management’ [3].

QbD has been applied to manufacturing processes but can it be applied to analytical methods? When we develop analytical methods we typically select suitable method parameters based on experience and knowledge and then check to ensure that we can achieve the desired results by applying analytical method validation characteristics. Thus we can ensure that the method can quantify at the levels required, that the results are always the same and that they give the true value, etc. This approach does not result in a full understanding of how the parameters of the method can affect the results.

Validation guidelines such as ICH Q2 [4] list the validation characteristics which should be investigated when validating your analytical method. These characteristics include intermediate precision, reproducibility and robustness. These three can provide understanding of the effects of method parameters.

Intermediate precision tests how the method performs when carried out by different analysts, on different analytical systems, on different days, etc. Reproducibility is required when the method needs to transferred to another laboratory and adds to the previous variables investigated for intermediate precision that of carrying out the method in a different laboratory. Robustness testing investigates the effect of slight changes to the method parameters. For example, in a HPLC method the effect of the flow rate, buffer strength and composition of the mobile phase might be investigated.

If performed thoroughly and correctly, the combination of these three validation characteristics can provide a good understanding of how a method performs and yet often this is not the case. Why?

One of the problems is that these characteristics are usually investigated at the end of a validation study due to the effort involved; they are time consuming, require different analysts, systems etc and thus are expensive to perform. For this reason they may only ever be investigated for projects which are in a late stage of development and even then often only the bare minimum of testing is performed. Also, there is a tendency to treat validation studies as a ‘tick-list’ exercise. It is regarded as a separate task which may even be performed by a different set of operators to those routinely using the method, thus valuable knowledge and experience is not gathered together. Another issue is the statistical knowledge required to interpret the results, particularly relating to robustness studies which are best performed using multivariate analysis techniques, such as design of experiments (DoE).

Applying QbD would involve moving these studies which provide an understanding of the method to the beginning of the method development process instead of performing them at the end of method validation. This would mean that the method parameters would be chosen on the basis of these experiments and would be within a design space of the method. The use of automation in these experiments would be desirable to reduce the effort involved and analytical chemists would benefit from a good understanding of the necessary statistics.

. US Food and Drug Administration, Pharmaceutical CGMPs for the 21st Century – A Risk Based Approach, 2004.
2. The International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use, Quality Guideline Q8 Pharmaceutical development, 2006.
3. The International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use, Quality Guideline Q9 Quality Risk Management, 2006.
4. The International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use, Quality Guideline Q2(R1) Validation of Analytical procedures: Text and Methodology, 2005.

Tuesday, 10 November 2009

Tips for trainers - Icebreakers

Training know-how applied to laboratory science

Previously in Learning at Work we looked at the training cycle and how to set learning objectives. In this issue we are going to take a quick look at the use of icebreakers. Love them or hate them, if done well they can get your training session off to a great start. The main purposes of an icebreaker are to get your audience’s attention and to familiarise the training delegates with each other, thereby creating a relaxed learning environment. It may also be used as an opportunity to introduce the content of the training.

The interaction of the members of a group may be an integral part of training. Soft skills training courses, such as management training, often involve role playing exercises. Delegates can find these types of exercises quite challenging and a good rapport with the other members in the group will make them more successful. In technical training, such as using a piece of laboratory instrumentation, the group interaction is less critical but this does not mean that it is unimportant. Group learning situations where the learners can discuss the topic of the training and contribute questions are often preferred to learning as an individual. It can be both a good learning experience and fun.

In my experience the games beloved of some training events involving balloons, juggling balls and singing songs, to give just a few examples, do not go down well in a laboratory training environment. Much preferred is something short and fun, and if it touches on the purpose of the training, it must be relevant. Taking people out of their comfort zone so that you can then give them information relating to laboratory science is not usually productive, the best results for this type of training are obtained when the delegates are relaxed, comfortable and ready to learn.

So what should you use as an icebreaker? One of the factors to be considered is whether you already know the people you are training and if they know each other. Knowing people’s names is one method of providing a relaxed environment. If you do not know your training groups names or they do not know each other I suggest that your icebreaker should be some sort of naming exercise. This can be as simple as getting your group to introduce themselves individually, or putting them into pairs where they first have to find out about their partner and then introduce each other.

I have found that if you ask your learners to include a quick interesting fact about themselves it can lighten the mood and help you to remember who is who. You should always go first to make sure that they know it does not have to be a very personal piece of information. Quick facts that have come up in my training sessions include: It’s my birthday; I play in a band and we have our first gig tonight; I am getting married this summer; my favourite pastime is shopping. As a facilitator you should try to encourage some chat around the response given by each learner.

If you and your group all already know each other then you might want to use an icebreaker which introduces the content of the training in some way. This may involve a discussion of your delegates learning expectations. If you capture these on a flip chart you can revisit it at the end of the training. This activity also has the benefit of making the learners think about what they want and why, thus shifting the emphasis from your responsibility of telling them about the topic to their responsibility of learning about the topic. This can help get their buy in to the training.

Another icebreaker that I sometimes use for a group where we all know each other is to see if they can draw an ampersand. I show the group a printed ampersand, ‘&’, and ask if they know what it is. Everyone usually gets it straightaway. Having hidden the printed ampersand from view I then ask the members of the group to have a go at drawing it. In general, most people find it difficult especially since they are not allowed to look at an example. This activity usually goes down quite well and stimulates discussion among the group. The point that you can make relating to training is that even if you think you know about something it doesn’t always mean you can do it.

Thursday, 5 November 2009

Help on: Mobile phase composition in HPLC


Do you have any problems relating to analytical chemistry for pharmaceuticals or training? Send your questions to the MTS helpdesk using our contact form.

"I wish to have a very "elementary" and concise knowledge for reverse and normal phase column chemistry. Articles that I have read so far are very confusing.
Non-polar stationary phase (RP) requires polar mobile phase hence polar compounds come out first (shorter RT) while the non polar compounds come out later...Which component in the MP affects what? e.g. ACN : H2O ...65:35 ...Which one to increase or decrease to effect the chromatogram and in what order would the peaks elute? "

“When we use the term reversed phase HPLC we are usually referring to the most common type of HPLC, namely partition. In this technique a non-polar stationary phase, typically an alkyl chain such as C8 or C18, is used together with a polar stationary phase, typically a mixture of water with an organic solvent, such as acetonitrile or methanol.

Consider an analysis in which we wish to analyse a mixture of different organic components which all have some polar characteristics due to the functional groups present. The mixture is injected into the mobile phase and thus introduced to the stationary phase. The organic components in the mixture are attracted to both the non-polar stationary phase and also the mobile phase in which they are dissolved, largely due to hydrophobic forces. When the component is in the mobile phase it moves through the column and when it is in the stationary phase it does not. The equilibrium experienced by each component of interest is a partition between the bonded stationary phase and the liquid mobile phase. The mixture can be separated if the extent of this partition is different for each component.

The relatively more polar components will elute from the column first with the shortest retention times because their partition favours the polar mobile phase. The relatively less polar components will elute from the column last with the longest retention times because their partition favours the non-polar stationary phase and thus they spend more time there during the analysis.

Now to move on to the question of the effect of the composition of the mobile phase, the polarity of the mobile phase can be adjusted by changing the proportions of the aqueous and organic solvents present. This has the effect of either increasing or decreasing the retention times for all the components in the mixture. As the organic portion of the mobile phase is increased the retention times will decrease. This is because the organic solvent attracts the components into the mobile phase and thus they spend less time on the stationary phase. This is why we steadily increase the proportion of the organic solvent in gradient HPLC. There is a rule that you can use to predict the effect of changing the composition of the mobile phase which applies to most small molecules in reversed phase conditions:

‘An increase of the organic solvent component in the mobile phase of approximately 10% will result in the retention time being reduced by a factor of 3.’

So for your example, if we change the proportions of the mobile phase ACN: H2O, 65:35 to ACN:H2O, 75:25 we would expect the retention times of your components to be decreased by a factor of 3.

If you change the proportions of the mobile phase for an isocratic method (as in the example above) then the elution order of your peaks should remain the same. However if you change the mobile phase proportions during an injection (a gradient analysis) you may see changes in the order of elution of the peaks, an effect that is used in the method development process.

Normal phase partition HPLC works in much the same way as described above except that the stationary phase is polar and the mobile phase is non-polar and therefore the elution order is reversed. However, the rule of 3 does not apply. It is important to realise that another type of normal phase HPLC, adsorption HPLC (in which the stationary phase is typically bare silica), does not work on the partition principle but rather the analytes and the mobile phase compete for adsorption sites on the surface of the stationary phase. Normal phase partition HPLC (using polar bonded phases) is most suitable for polar components whereas normal phase adsorption HPLC is most suitable for non-polar components.”

Do you have anything to add? Feel free to leave a comment.

Wednesday, 4 November 2009

HPLC calculator for column equilibration

A resource for chromatographers

HPLC column equilibration was discussed in a previous blog post and was subsequently provided in a printer friendly version as a resource in the MTS website free training resources area. We are now giving away a free calculator to make these calculations as simple as possible for you.
Click here to access the calculator; it will open as an Excel spreadsheet. The directions for how to use the calculator are provided on the spreadsheet.

Monday, 19 October 2009

Help on: Retention time shift in HPLC analysis


Do you have any problems relating to analytical chemistry for pharmaceuticals or training? Send your questions to the MTS helpdesk using our contact form.

"I am using 0.1 % TFA in water + 100 % Acetonitrile as mobile phase in gradient RP-HPLC. But since last month I have problem of RT shift. I am not able to rectify it. Please suggest solution for the same. "

"Some of the most common problems which can cause a shift in retention time are temperature, mobile phase composition and equilibration of the column. I shall discuss each of these first and then move on to other potential causes of your problem.
Is your column in a column oven and maintained at a constant temperature throughout your analysis? Temperature is a variable in HPLC analytical methods (we often use it to adjust retention during the method development process) and changes in temperature can result in shifts in retention time. A method that runs at ‘ambient’ temperature can be subject to large variations in the room temperature of a laboratory. So even if the method says ambient it is best to control the temperature.
Mobile phase composition:
How do you create the gradient for your method? If you premix your mobile phases of 0.1% TFA in water and 100% acetonitrile to result in one reservoir which corresponds to the method starting conditions and another which corresponds to the end conditions then it is possible that changes in the volatile components of these mixtures due to evaporation could lead to shifting retention times. However, if you use online mixing and simply have 0.1% TFA in one bottle and acetonitrile in the other then this cannot be the source of the problem.
Column equilibration:
Generally, when using RP-HPLC methods the column can be equilibrated relatively quickly. Check that you have allowed enough time at the end of each injection before the next injection. Typically 10 column volumes will be sufficient (refer to the previous post on column equilibration). An exception to this is when using ion-pairing reagents, these can take a significantly longer time to equilibrate due to the adsorption of the ion-pairing reagent on the surface of the stationary phase. Although TFA can act as an ion pairing reagent I have never had an issue with equilibration at the concentration you are using.

If you can rule out these common causes then we need to look more closely at both the HPLC method that you are using and the problem that you are experiencing. Some questions to consider:
You say your problem started last month, was the method working well before this, and for how long?
If the method has been trouble free for a significant length of time then it indicates that the problem is more likely to be due to a problem with the HPLC system or the column. Consider carefully, are you certain that you haven’t changed any of the method parameters? Review the method thoroughly. Run the method on a different column (preferably new) and see if the drifting retention time is observed.
If the method hasn’t been proven to work then the problem could be due to the method parameters, e.g. sample preparation could be inadequate leading to progressive contamination of the column, or the column and analytes may not be completely compatible.
Are all the peaks in the chromatogram moving (including the solvent front) or is it restricted to certain peaks only?
If all the peaks, including the solvent front, are shifting then the problem may be due to system leaks, or air bubbles in the pump. If the solvent front does not move then the change may be linked to the mobile phase and method conditions. If the shift in retention time is only observed for certain peaks then it may be due to a change in the column which only effect certain types of functional groups , e.g. subtle pH change.
What is the nature of the retention time shift, changing within a run, changing between runs, increasing with time, decreasing with time, no pattern in the drift?
The way in which the retention time is shifting will provide extra information on potential causes and may support a diagnosis based on the discussion above.
A note regarding your mobile phase. TFA does not actually have much buffering capacity, it is very convenient for acidifying the mobile phase when you need a volatile buffer. Depending on the parameters of your gradient it may be that there is very little acid at the end of your gradient. You can add TFA to the organic portion of your mobile phase, i.e. 0.1% TFA in acetonitrile and thus the amount of TFA present throughout the gradient remains constant. This may result in some improvement in your chromatography, however I don’t think that this would explain the shift in retention time that you are experiencing.
Please use the comments if you would like to continue this dialogue and provide some more information which may help to diagnose your problem.”

Do you have anything to add? Feel free to leave a comment.

Help on: Measurement of urinary citrate levels by HPLC


Do you have any problems relating to analytical chemistry for pharmaceuticals or training? Send your questions to the MTS helpdesk using our contact form.

Question:"We would like to measure 24 hrs urinary citrate levels in humans for our upcoming project. How can HPLC help us?"

"To utilise HPLC for the measurement of urinary citrate you need to consider the nature of the analyte, in this case the citrate molecule. The relatively polar nature of this molecule makes it suitable for analysis by either ion exchange HPLC [1] or reversed phase partition HPLC. Reversed phase partition HPLC is usually preferred due to its availability in most laboratories.

Examples in the literature report the use of C18 columns for the analysis of citrate [2,3]. A low pH aqueous mobile phase was used combined with a column preconditioning step consisting of 100% methanol, this organic solvent rinse was repeated between injections to ensure that the chromatography was acceptable. The reason for using a 100% aqueous mobile phase is to obtain satisfactory retention of the relatively polar citrate molecule.

I suggest that you make use of an aqua column which can perform in 100% aqueous conditions, and should adequately retain the citrate molecule. The low pH of the mobile phase means that citrate is in its unionised form and maximises the retention on a RP column. It is important to control the pH adequately since citrate has 3 pKa values at 3.1, 4.7 and 5.4, changes in pH around these values may have an effect on the retention of the analyte and thus the reproducibility of the chromatography.

The citrate molecule contains a weak chromophore which will enable the use of UV detection. A low wavelength such as 210 nm will be suitable. LC-MS may also be used.

The other issue to consider when analysing samples of urine is the matrix and how much sample preparation is required. Khaskhali et al. [2] found that deproteinisation of the urine sample was adequate."

References:1. Kristina L. Penniston, Stephen Y. Nakada, Ross P. Holmes, Dean G. Assimos. Journal of Endourology. March 2008, 22(3): 567-570. ‘Quantitative Assessment of Citric Acid in Lemon Juice, Lime Juice, and Commercially-Available Fruit Juice Products’
2. M. Hassan Khaskhali, M. Iqbal Bhanger and F. D. Khand Journal of Chromatography B Volume 675, Issue 1, 12 January 1996, Pages 147-151. ‘Simultaneous determination of oxalic and citric acids in urine by high-performance liquid chromatography ‘
3. Keevil B. G. ; Owen L. ; Thorton S. ; Kavanagh J. ; Annals of clinical biochemistry 2005, vol. 42 (5), pp. 357-363 ‘Measurement of citrate in urine using liquid chromatography tandem mass spectrometry : comparison with an enzymatic method'

Do you have anything to add? Feel free to leave a comment.

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

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.

Tuesday, 25 August 2009

Help on: GPV testing


Do you have any problems relating to analytical chemistry for pharmaceuticals or training? Send your questions to the MTS helpdesk using our contact form.
"can we use ipa(isopropyl alcohol) in gpv test if yes then why."
"To answer your question about using IPA for a GPV test I am going to make a few assumptions about what you are trying to achieve. I take it that you want to perform a gradient proportioning valve test for a low pressure mixing HPLC system, the intent being to test if the proportions of each solvent that you programme during your HPLC analysis are actually being performed accurately by the system. The normal method to perform this test is use two solvents: The first is water, and the second is water containing a small amount of acetone (approximately 0.1%v/v). You can also use methanol in place of water. It is best to use just one solvent since you want to test the proportioning of the valve only, and don’t want the effects of differing compression of solvents included in the results.

Some manufacturers sell GPV test solvents, e.g. Waters provide propyl paraben in methanol for their test. The purpose of the acetone (or propyl paraben) is to introduce a component into one of the solvents which will have a corresponding UV response, so obviously this approach is used for systems which have a UV detector installed. The response for the solvent containing the UV absorbing compound will change as the proportion of this solvent is introduced by the GPV and therefore it allows you to measure how well the GPV is mixing the solvent lines.

Since your question was about the solvents and not actually how to do the test I will assume that you already know about how to do it. If you want to use IPA for your GPV test then it would only make sense if you regularly use IPA as a mobile phase component. You want to test how well the GPV works in its typical use. The UV cutoff for IPA is at about 210nm so it will work fine with a UV absorbing component added to one portion of IPA and just IPA in the other solvent. However if you routinely use aqueous solvents then water may be a better choice."
Do you have anything to add? Feel free to leave a comment.

Friday, 10 July 2009

Learning objectives: Jargon or a trainer's best friend?

Training know-how applied to laboratory science

Last month we looked at the 4 stages of the training cycle and it was observed that the outcome from the first stage, identification of learning needs, should be a set of learning objectives for the training event being planned. The purpose of these objectives is to state the goals which should be achieved by the learner by the end of the training. Taking the time to get these objectives right can make a huge difference to the success of your training event. A good set of objectives will make it easier to plan and design how the training will be delivered. During delivery they help to focus the trainer and the learner on what they need to achieve. After the training they allow a measure of whether the training has worked by investigating whether the objectives have been met.

Sounds good so far but where do you start? Basically you need to think about what you want your learners to be able to do after the training. Then you need to condense these requirements into a series of statements - the objectives. If you have set objectives before for other tasks then you may have encountered the SMART model, this simple technique can help you to make sure that your objectives are optimised.

S is for Specific
M is for Measurable
A is for Achievable
R is for Relevant
T is for Time-bound

The objective should specify exactly what is required from the learner rather than a vague high level statement. For example, rather than saying ‘learn about HPLC’, you should specify the aspects of HPLC which will be covered in the training whether it is separation theory, column chemistry, setting up a HPLC system, etc.

If the objective can be measured you have an easy way to ensure that the training is working. A careful use of language is required for a measurable objective.

Examples of measurable verbs are: Adjust; analyse; approve; calculate; carry out; complete; decide; describe; explain; implement; obtain; perform; solve; select; state; update; use; develop; identify; list.

Use of some verbs may be difficult to measure, for example: Hear; be aware of; listen to; attend; now; enjoy; grasp the meaning of.

The learner needs to be able to achieve the objective by the end of the training session so a realistic objective needs to be designed.

The objective should be relevant to the required outcome of the training. This helps to keep the training focussed on the learning outcomes without unnecessary distractions.

Typically the objectives need to be achieved by the end of the training event but in some circumstances further practice may be required and a time limit for this should be set.

There are some variations on what each letter in SMART stands for, an example is R which is sometimes quoted as being for ‘Realistic’. It doesn’t really matter providing the objectives are sensible. For the purposes of training the most important feature is that they are measurable, this enables evaluation of how well the training has achieved its aims.

An example of a SMART objective is as follows;
By the end of the training the learner will be able to prepare mobile phase for HPLC using different types of solvents, buffers and additives and understand the effect these have on the chromatographic separation.’

The objective is specific; it relates only to the preparation of mobile phase and defines the required reagents as solvents, buffers and additives. It is measurable; the learner can demonstrate their learning by preparing mobile phase or detailing the steps required to do so, and by explaining the chromatographic effects of the mobile phase. It is achievable; this is a realistic amount of learning to include in a training session. It is relevant; the training is an introduction to HPLC and preparation of mobile phase is a key requirement. It is time-bound, the objective should be completed by the end of the training.

Setting suitable learning objectives for training on analytical chemistry techniques is usually a straightforward task due to the practical nature of the training. If you need to be able to use an analytical instrument or perform a particular analysis then the competent performance of these tasks indicates that the training has been successful. Choose suitable measurable verbs such as ‘perform’, or ‘carry out’. If the training has included background theory then you can consider verbs such as ‘explain’ and ‘describe’. The number of learning objectives which you should set will depend on the actual training required.

Setting objectives is one of those tasks that is often included in a range of training events such as time management and project planning. It can end up feeling like management-speak jargon, especially if it is not explained adequately. The effort involved in setting learning objectives for training events is well worthwhile. They are the basis for the design, delivery and evaluation of the training and a good set of learning objectives will make these stages of the training cycle simple to implement.

Wednesday, 8 July 2009

Free training resources on the MTS website

The MTS website has had a facelift! Last month the new look website was launched. The new layout makes it more user-friendly and enables you to find the information you’re after with the minimum number of clicks. Take a look at the new format and find out more about the services (training courses delivered at your site or via web-based training and expert advice on your in-house training programmes), products (pre-prepared training courses for you to deliver in-house and training books) and resources available from Mourne Training Services.

A new feature of the website is a FREE training resources area. Here you will find articles on topics relating to the chemical analysis of pharmaceuticals including HPLC related subjects and tips for delivering training (these articles may have started life as blog posts!). You will also find links to back issues of Analyse This and links to freebies from MTS such as posters and videos. New resources will be added regularly. If you have any comments, suggestions or requests be sure to let us know.

Monday, 29 June 2009

Dealing with outliers in analytical method validation


Occasionally in validation studies, as in any analysis, outliers may be observed in data sets of results. This is where a value is present in the data set which differs considerably from the majority of the other results. For example, the following data set was obtained for a precision study:

25.4, 25.3, 27.5, 24.5, 24.7, 25.6

The value 27.5 is much higher than the one nearest to it at 25.6 and it is suspected that it may be an outlier. The mean and standard deviation calculated for the data set including and excluding the suspected outlier are presented in Table 1. The mean does not appear to be effected substantially by the suspected outlier but if the suspect value is included the %RSD does not comply with the acceptance criterion of %RSD ≤ 2% for precision. If it is excluded the precision complies with the acceptance criterion.

Table 1

Statistical tests can be performed which will provide confidence in the characterisation of a data point as an outlier. However a decision still has to be made about whether to exclude the data point from the results. Some statisticians object to the rejection of any data from small size data sample, unless it is known that something went wrong during the measurement of that data. Rejection of data during validation studies must be very carefully considered, statistical evidence alone may not be enough to justify the rejection of data.

The most popular statistical test applied to detect suspect outliers in the results from chemical analysis is Dixon’s Q-test. One (and only one) observation from a small set of replicate observations (typically 3 to 10) can be examined. The test assumes a normal distribution of the data. The null hypothesis for this test is that there is no significant difference between the suspect value and the rest of the values, any differences must be attributed to random errors.

The test is applied as follows:

1. The values comprising the data set are arranged in ascending order:
X1, X2, X3, ........ Xn

e.g. 24.5, 24.7, 25.3, 25.4, 25.6, 27.5

2. The experimental Q-value is calculated, defined as the ratio of the difference between the suspect value and the nearest value to it, to the range of the data.

If the suspected outlier is a low value:

If the suspected outlier is a high value:

e.g. Qexp = (27.5 – 25.6)/(27.5 – 24.5) = 0.633

3. The value of Qexp is compared to a critical Q-value (Qcrit). Refer to Table 2 for critical values of Q for Dixon’s test, from work by Rorabacher[1]. The value of Qcrit corresponding to the confidence level required for the test is selected, usually 95%.

e.g. Qcrit = 0.625 (95% confidence level)

4. If Qexp > Qcrit, then the suspect value can be characterised as an outlier.

e.g. Qexp > Qcrit, therefore data point 27.5 can be characterised as an outlier.

Table 2

The problems associated with the Q-test are that it may be misleading if more than one outlier is present, and also if an outlier is detected, a decision then has to be made about whether to exclude the data point from further statistical calculations. Methods which are more robust than the Q-test, such as the Huber method (described in the AMC technical brief, no. 6[2]), are becoming increasingly favoured for treatment of outliers since they consider all data present in the set, and not only three data points as in the Q-test. Robust statistics utilise approaches such as the use of the median and median absolute difference to estimate the mean and standard deviation respectively. In this way the outlying data has no effect and the does not have to be rejected. Any approach used to deal with outliers will have to be justified fully in the validation report.

1. D. B. Rorabacher, Anal. Chem, 63, 139-146, 1991, ‘A Statistical treatment for rejection of deviant values: critical values of Dixon's "Q" parameter and related subrange ratios at the 95% confidence level’.
2. Analytical Methods Committee, AMC Technical Brief, No. 6, 2007, ‘Robust statistics: a method of coping with outliers’ (available on RSC website,

This blog post is an excerpt from 'Validation of Analytical Methods for Pharmaceutical Analysis' by Oona McPolin, available to purchase through the MTS website.

Wednesday, 10 June 2009

Free HPLC training video

This HPLC training video describes the different parts of a HPLC instrument. A picture of how the whole system operates is built up gradually by introducing each part and defining the role that it plays.

You are welcome to use this video in your HPLC training programmes. It is an example of the resources included in UTrain, an MTS product which enables you to easily deliver your training in-house. A series of videos are used to deliver the knowledge part of the course. These are accompanied by a workbook for each learner which details exercises and practical experiments that give learners an opportunity to apply what they have learned. Then e-Learning modules are used to review the learning and administer an assessment to test whether the learning has been absorbed. The videos and e-Learning modules are accessed through e-MTS, the virtual environment for learning provided by MTS.

The first UTrain course deals with basic HPLC and is due to be released later this year. It is based on the MTS course, An Introduction to HPLC for Pharmaceutical Analysis, recognised by the Royal Society of Chemistry for the purposes of continuing professional development. If you have any queries about UTrain then please let us know.

Monday, 8 June 2009

The training cycle explained

Training know-how applied to laboratory science

Previously, it was established that most people who work in a scientific laboratory environment have some responsibility for providing training (see blog post dated 8th May 2009, I’m a trainer? But that’s not in my job description. Is it?). Also the training cycle was introduced. In this article we will look at the training cycle in a bit more detail. The four steps are as shown.

Step 1: Identifying the learning needs
Identification of learning needs may also be referred to as training needs analysis, or TNA. The remit of this step can be rather broad since it can apply to all the training required by an individual or a group, even a whole company. If you are responsible for a particular type of training provision then typically it will already have been determined that the individuals who come to you for training have been identified as needing the training. Therefore the scope of this step narrows considerably. You need to consider what the learners need to be able to do at the end of the training. For example if you are providing training on an analytical instrument do they need to be able to:
  • Use the instrument
  • Clean the instrument
  • Calibrate the instrument
  • Service the instrument
  • Troubleshoot the instrument?
The complexity of the technique will determine whether all or some of these tasks need to be incorporated into the training that you are planning. You may need to develop training which can be delivered over time as the learner gains experience with the technique.
The outcome of step 1 is a set of learning objectives for the training that specify what the outcome should be.

Step 2: Design the training
When you come to design your training you need to consider how you are going to achieve the learning objectives that you have set. It may be that a practical session where the learner gets hands on experience of using an instrument or piece of equipment is most suitable. In some tasks a theory session where the concepts relating to the task can be fully understood is appropriate.
When designing the training you need to consider the way in which people learn and be aware of the differences in learning styles. In general adults learn well if the training is relevant to what they will do in the workplace so use case studies and realistic exercises in your training. Methods of training that you can consider are: lectures and presentations, demonstrations, exercises, case studies, practical sessions, question and answer sessions, discussion groups and e-learning.
Make sure to consider the time and other resources that you have available for the training. You may need to prepare visual aids such as PowerPoint presentations (much maligned but valuable if used well), and handouts to accompany the training. Try to keep it simple and stick to the point.

Step 3: Deliver the training
The delivery of the training is the step that some people can find daunting, particularly if presenting to groups of learners is required. Some keys things to remember when delivering training are:
  • Speak clearly and make sure that all learners are able to understand what you are saying.
  • Ensure that the learner’s expectations are addressed early on in the training.
  • Explain the structure of the training at the start.
  • Review regularly to ensure that the material covered has been understood.
  • Try to deal with questions as they arise but if you don’t know the answer, don’t be afraid to say you will get back to the learner later.
  • Give useful and constructive feedback to learners.
  • Check your timing and have a contingency plan in case some parts of the training take longer than you expected.
  • Deliver the training consistently so that all learners receive the same training.
Step 4: Evaluate the training
When you have gone to the trouble of designing and delivering training then you will want to be sure that it is working. This is the purpose of training evaluation. A common way to evaluate training is to get the learners to complete a form at the end of the training where they give feedback on whether they thought it was useful, what they thought of the facilities and the trainer, etc. This information is very useful and may be used to improve the training in the future but it does not measure what was actually learned by the delegates. To do this some type of assessment is usually administered. This may be a written test, or the learner may have to analyse a sample by implementing the training that they have received. You need to decide what is most appropriate for your training.
To assess long term implementation of learning is more difficult. A number of different approaches are possible but all assess how successfully the learner is completing the task in question. The opinion of the learner, colleagues and managers may be canvassed to obtain a balanced view of how well the training has worked.

In summary the training cycle provides a structured way for you to approach your training responsibilities. The core of all successful training programmes are good learning objectives, sometimes known as learning outcomes. In the next instalment of Learning at Work the process of setting realistic and appropriate learning objectives will be discussed.

Thursday, 4 June 2009

Analytical method validation by phase of development


An analytical method which is used in pharmaceutical analysis should always be validated to ensure that the results generated are trustworthy. These results may be used to make critical decisions during the drug development process relating to issues such as safety of the drug, the synthetic route and the manufacturing process. However performing formal validation studies as per ICH guidelines requires considerable resources. In the early stages of the development of a drug the investment required for formal validation may not be desirable due to a number of reasons including:

  • The development and optimisation of the analytical methods is ongoing.
  • The development of the synthetic route for the drug substance is ongoing.
  • The development of the formulation of the drug product is ongoing.
  • The drug may not progress into later stages of development.

The ICH guidelines apply to ‘validation of the analytical procedures included as part of registration applications submitted within the EC, Japan and USA’ [1], and thus do not formally apply to the early phases of drug development. The guidelines from the FDA regarding INDs for phase 2 and phase 3 studies [2] require ‘appropriate’ validation data for methods which are not from a pharmacopoeia or official reference standard.

The consequence of this is that it is common for pharmaceutical companies to use a phased approach to analytical method validation studies. Validation performed for early phase drugs tends to be less extensive than that performed for late stage drugs. However the objective of validation still applies, i.e. to demonstrate that a method is suitable for its intended purpose.

The following list provides suggestions for validation of early phase drugs:

  • A formal validation protocol is not yet mandatory. Internal guidelines, or a standard operating procedure (SOP), may be used to summarise the general validation requirements. This may be referenced rather than producing time consuming documentation.
  • The extent of testing and the number of replications may be reduced.
  • Wider acceptance criteria may be adequate in early phases of development.
  • Specificity and evaluation of the quantitation limit are the primary characteristics to ensure that assay and impurity methods meet their intended purposes of potency and safety.
  • A second method to evaluate accuracy is unlikely to exist in the early phase of development, therefore accuracy may be inferred from the results of precision, linearity and specificity.
  • For precision testing synthetic mixtures of drug substance and placebo may be used, rather than authentic samples, thus enabling the combination of accuracy, precision and potentially linearity at the same time.
  • Formal intermediate precision experiments are not yet needed but if different laboratories need to operate the method then the handover will require suitable validation.
  • The evaluation of the detection limit for impurity methods may be delayed.
  • Formal robustness testing is not yet required. Robustness studies associated with method development are likely to be ongoing at this stage.
  • The validation report may be presented in a simplified tabular format, together with the conclusions. This type of summary report fulfils the expectations of the regulatory authorities, e.g. phase 2 and phase 3 INDs.

Further information regarding validation of analytical methods by phase of development is available from Bloch in ‘‘Method Validation in Pharmaceutical Analysis. A guide to Best Practice’ [3] and also from Boudreau et al. in a paper developed from a PhRMA 2003 workshop [4].

1. International Conference on Harmonisation (ICH) of Technical Requirements for Registration of Pharmaceuticals for Human Use, Topic Q2 (R1): Validation of Analytical Procedures: Text and Methodology, 2005,
2. Guidance for Industry: INDs for Phase 2 and Phase 3 Studies, Chemistry, Manufacturing, and Controls information, US Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research (CDER), 2003.
3. M. Bloch, ‘Validation During Drug Product Development – Considerations as a Function of the Stage of Drug Development’, in ‘Method Validation in Pharmaceutical Analysis, a Guide to Best Practice’, Eds. J. Ermer, J. H. Miller, Wiley, 2005, p243-264.
4. S. P. Boudreau, J. S. McElvain, L. D. Martin, T. Dowling, S. M. Fields, Pharm. Technol., 28 (11), 54-66, 2004, ‘Method Validation by Phase of Development – An Acceptable Analytical Practice’.

This blog post is an excerpt from 'Validation of Analytical Methods for Pharmaceutical Analysis' by Oona McPolin, available to purchase through the MTS website.

Tuesday, 2 June 2009

Priming injections

A resource for chromatographers

For most HPLC analyses a priming injection is required prior to the analysis. This injection will usually result in a slightly different chromatogram to subsequent injections of the same solution.

The reason for this is not fully understood but is probably due to the presence of (at least) two types of active sites on the column for interaction with the analyte molecules. One of these sites equilibrates much more slowly than the other and is gradually saturated with the analyte in the first few injections thus stabilising the response for further injections.

For this reason it is common practice to perform one or more ‘test injections’ before starting the analysis injection sequence. The SST solution or the calibration standard is commonly used for the test injection. The test injections may be programmed into the injection sequence if the run will be unattended, e.g. it is started at the end of a working day.

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

Friday, 8 May 2009

"I'm a trainer? But that's not in my job description. Is it?"

Training know-how applied to laboratory science

Are you a trainer?
If you work in a science laboratory environment then you probably are. In most working environments it is normal practice for an experienced member of staff to show new and less experienced staff how to perform common work related tasks. This practice is very suited to a laboratory setting due to the large range of tasks that need to be learned by a new starter. These include learning about: the range of procedures to be followed; the ways of working; the operation and calibration of equipment and instrumentation; computer software applications used in the laboratory, etc. Sometimes this type of training is provided within a formal framework, where individuals are assigned to maintain and train others on a specific piece of equipment or analytical technique, but sometimes it is simply a case of “will you show John how to use the Karl Fischer?” An initial training session may be followed up with support for the learner as they gain competence in the task in question.

On-the-job training has clear advantages for the laboratory if it is well implemented; it is cost effective, flexible, and the learner is doing the job while they learn. It is also good for the learner; they are trained by someone with a good working knowledge of the task and implementation of the learning is usually possible immediately. But what about the trainer? Is training others a welcome and enjoyable responsibility, or an unenviable extra duty to try to fit into a busy work schedule? This depends on how the individual feels about being a trainer. Some people enjoy it more than others and some find the whole concept very daunting .

Acting as a trainer in the workplace has a number of benefits, the most important being that of improving your career prospects. The ability to train others is a valuable addition to your CV, particularly important in the current economic climate. Delivering training in a particular area hones your own skills and knowledge in that area since explaining it to others requires full understanding on your part. This maximises your assets and can lead to recognition of your expertise, an important consideration in promotion prospects. The interaction with the staff members that you train can increase your exposure in the workplace and thus enhance your reputation and standing. Additionally, the skills involved in training can be used in a managerial role. If these advantages do not appeal then there may be another incentive to get involved in training. It is likely that a responsibility for providing training is written into your job description, particularly if you have one of the fairly broad, generic job descriptions much favoured in recent years.

Of course the benefits listed above only apply when the training being delivered is successful and effective. One of the problems when using in-house staff as trainers is that the quality of the training can be variable and the individuals involved are not always given guidance on how to plan and deliver training. The purpose of this series of articles is to provide advice and tips on the best way to approach a training event. The word ‘event’ is used here to signify any training programme, large or small, which needs to be implemented in your laboratory.

The implementation of training is usually described using the training cycle shown below. There are four main stages:

1. Identify the learning needs
2. Design the training
3. Deliver the training
4. Evaluate the training

Next time we will look at each of these stages in more detail to give you an overview of what you need to do to implement your training event.

Wednesday, 6 May 2009

Free HPLC calculator from Thermo


You can find numerous useful resources for pharmaceutical analysis on the internet which don’t cost anything. Suppliers and manufacturers of analytical equipment and instrumentation need to position themselves as experts so that they can gain your trust in their products. They do this by providing a range of useful resources relating to their product area; often these resources are available online.

An example of a useful resource for HPLC is the method development calculator from Thermo, available at:

This calculator allows you to transfer a HPLC method to a different set of column dimensions. You enter the current method conditions in the left hand column and the new column dimensions in the right hand column. Then the calculator will work out the required flow rate, injection volume, predicted system pressure, gradient conditions and suggested equilibration time. You can also specify a flow rate and get adjusted values for the parameters listed above. There are two calculators, one for gradient and one for isocratic methods. The application is aimed at transferring standard HPLC methods to ultra high pressure conditions but the calculations hold for whatever transfer you require.

I like this calculator because it is well laid out and easy to use. I also like the fact that an explanation of all the equations used is provided. I think that this could be a useful tool to save time in your HPLC method development.

Friday, 1 May 2009

Error and uncertainty in analytical measurement - Part I


When analysing pharmaceuticals the objective of our efforts is to produce a result, an analytical measurement, which gives important information about the sample of interest. Examples are: the determination of impurities in an active pharmaceutical ingredient; and the assay of the amount of the active in a dosage form. In these examples the information obtained may be used to ensure that the product is safe and of high quality. It is vital that the analytical results obtained are reliable since they are used to make decisions which can have an impact on human health.

A reliable result does not mean that the analytical measurement is exactly equal to the actual value of the parameter being measured (the true value). We accept that the result is an estimation of the true value and that the difference is due to error and uncertainty introduced during the measurement process. However, for the result to be deemed reliable, we need to determine the magnitude of this difference between the analytical measurement and the true value. This requires a quantification of the error and/or uncertainty associated with the analytical method.

So what are error and uncertainty, and what is the difference between them?

Error is defined by ISO [1] as “the result of a measurement minus a true value of the measurand”, (where the measurand is the “particular quantity subject to measurement”). In Figure 1 the true value of an analytical measurand is shown by a, the centre of the target. An analytical measurement is shown by b. The difference between the true value and the measured value is given by x. Therefore the error for the measurement is equal to x. Error is a single value for each analytical measurement. In theory a known value for error can be used to correct the result.

Error in analytical measurement is regarded as being made up of two different types: random error and systematic error. Random error is caused by the many uncontrollable variables that are an inevitable part of every chemical or physical measurement. This type of error can usually be reduced by multiple measurements. Systematic errors have a definite cause and are of the same value for a number of replicates analysed in the same way. They are independent of the number of measurements and therefore cannot be reduced by increasing the number of measurements. A further type of error is a spurious error, or blunder, which invalidates a measurement. This can occur when the person performing the analysis makes a mistake or an instrument fails.

Uncertainty is defined by ISO [1] as a “parameter associated with the result of a measurement, that characterises the dispersion of the values that could reasonably be attributed to the measurand.” This parameter may take the form of some multiple of the standard deviation, or confidence limits relating to an analytical measurement. It is usually expressed as a range within which the true value is known to fall. The uncertainty may be determined for a particular analytical method or procedure and thus applied to all results obtained for the method, provided that it is applied in the same way each time.

In Figure 2 the true value of an analytical measurand is again shown by a, the centre of the target and an analytical measurement is again shown by b. The uncertainty of the analytical measurement b is shown by the red shading. The true value is contained within this area, or range of values.

Figure 3 illustrates the difference between error and uncertainty. As previously, the true value of an analytical measurand is shown by a at the centre of the target. This time an analytical measurement, c, is shown. This result has been determined using the same analytical method as in Figure 2 and thus has an associated uncertainty shown by the red shading. The difference between a and c is very small and thus the error for this measurement is very small. The analyst knows that the true value lies within the area of red shading but has no way of knowing just how close the measurement is to the true value. In practice it is usually possible to quantify the uncertainty for an analytical procedure, and thus a range within which the true value lies, but quantification of the error for each analytical measurement is not usually possible.

Now that we have defined error and uncertainty, and the distinction between them, we can move on to the sources of error and uncertainty in pharmaceutical analysis and how they may be quantified. This will be discussed in the next issue of Analytical Chemistry Revision Notes, Error and uncertainty in analytical measurement - Part II.

1. International Vocabulary of basic and general terms in Metrology. ISO, Geneva, (1993). (ISBN 92-67-10175-1).
These definitions are reproduced in the publication: S.L.R. Ellison, M. Rösslein, A. Williams, (Eds.), EURACHEM/CITAC Guide, Quantifying Uncertainty in Analytical Measurement, second ed., 2000. This publication has the advantage of being freely available online.