Mourne Training Services

Ireland HPLC Training Schedule for 2012

2011-12-13T11:17:00.006Z

The dates for the next MTS open enrolment training courses in Ireland in 2012 are now available:

Tuesday 5th June
How to Run HPLC Methods

Wednesday 6th June
How to Troubleshoot HPLC

Thursday 7th June
How to Develop HPLC Methods

Friday 8th June
How to Develop HPLC Methods for Challenging Separations

We also plan to run the course 'Validation of Analytical Methods for Pharmaceutical Analysis' on Monday 29th & Tuesday 30th October.

The location is Dublin; full details will be on the website early next year. Contact us for more information.

MTS Recommends... Measuring pKa using UV/Vis

2011-12-12T11:55:00.011Z

When delivering HPLC method development training, I usually advise that the pKa of an analyte is taken into consideration, if appropriate, when selecting a suitable buffer pH. The problem with this approach is that the pKa value is not always known. A resource which may prove helpful is provided in the Chemistry Resources section of the Chemagination website where there are directions on ‘How to measure pKa by UV-vis spectrophotometry’.

Help on: Acceptable Variation for HPLC Retention Time

2011-12-09T11:16:00.002Z

MTS HELPDESK

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:
“I assess assay results for imported pharmaceutical products performed by a third party lab. The methods we use do not always specify an "allowed" retention time range. What would a reasonable guide be when assessing this – plus or minus 10%? Basically, what I should like to know is when should we ask the laboratory to investigate/explain a retention time shift from one assay to the next? Put another way, when should the laboratory make system adjustments like the temperature of the column, flow rate etc. in order for the retention time to conform? Often the method will say the active elutes at say 10 minutes. Does this mean that 9 to 11 minutes is also OK, or are there justifiable limits that we can impose?”

Answer:
“The reasons for variation of retention time from analysis to analysis includes small differences in the following: the composition of the mobile phase (in terms of the aqueous and organic solvent portions); the pH of the mobile phase (particularly important for ionisable molecules like acids and bases); HPLC columns (column to column variation), and HPLC systems (particularly the system volume). As a result of these variables it is expected that the retention time will vary and usually small variations do not cause any problems. The potential problems associated with retention time variation from run to run (not injection to injection in the same run, that is a more serious problem which may indicate a problem) includes:

The change in runtime means that a peak is not fully collected by the end of the run and thus the analysis has to be repeated.
For analysis of mixtures, such as impurity analysis, peaks may be difficult to assign if the retention time varies.
Automatic integration events may have problems relating to identifying peaks correctly, thus requiring extra time for reprocessing.

To answer your question, it is difficult to set an allowable retention time range without a good understanding of the individual method. Ideally the person who develops the method should investigate its robustness and thus gain information on the range of retention times which might be expected due to normal variation and where the method still performs as required. Of course, it is not an ideal world and often you will be presented with data from methods where this has not been done, or if it has, it is not written into the method. From experience I would say that about 10 to 20% of the time quoted in the method is a reasonable guide but be careful not to apply it too restrictively. If you have an analysis where the retention time is on the limits, the best way to assess if there is a problem is to look at the chromatograms for the analysis and compare to a previous satisfactory analysis (preferably an example chromatogram should be included in the method). If there aren’t any differences except for retention time, and the system suitability testing is all fine, then the results should be satisfactory to use. If the retention time is very different to that expected and well outside of this approximate range then I would suspect that something has been changed in the way the method was applied and would want the analyst to investigate further.

Although pharmacopoeia sometimes include adjustments to temperature, flow rate etc. to make sure that a peak is at a particular retention time these changes are actually deviations to the method and unless you are following a pharmacopoeia method or have validated your method to show that these alterations are valid then it is not a good idea to change the method parameters.”

New Associate @ NSF DBA

2011-12-05T17:21:00.013Z

I am delighted to announce that I am now an associate of NSF DBA. You will probably be familiar with NSF DBA, formerly David Begg Associates, if you work in the pharmaceutical industry since they are Europe’s largest provider of pharmaceutical training, both in-house and external, and also provide pharmaceutical auditing and consulting services. I will be contributing my expertise in the area of analytical chemistry, and in particular the analysis and testing section of Qualified Person (QP) training.

HPLC Training in Helsinki

2011-12-02T11:45:00.003Z

I will be delivering 3 HPLC training courses from the ‘How to...’ series in Helsinki at the end of January 2012 in collaboration with Phenomenex. The courses are:

Tuesday 31st January
How to Troubleshoot HPLC
Wednesday 1st February
How to Develop HPLC Methods
Thursday 2nd February
How to Develop HPLC Methods for Challenging Separations

Contact the Nordic Phenomenex office for more details: nordicinfo@phenomenex.com

USP Chromatographic Columns

2011-11-24T10:43:00.041Z

PEAK SOLUTIONS

A resource for chromatographers

If you have ever followed an HPLC method in the United States Pharmacopeia, then you have probably already encountered the problem of selecting a suitable HPLC column for the analysis. Columns are designated by a letter and number which identifies the stationary phase, e.g. L1 refers to ‘Octadecylsilane chemically bonded to porous silica or ceramic micro-particles, 1.5 to 10 μm in diameter, or a monolithic rod’. Unfortunately there are hundreds of columns that fit this description and due to selectivity differences they may not all give similar results.

The outcome of the USP Working group on HPLC Columns is that you can now look up the chromatographic column which was used to validate the procedure. The free online database provides a cumulative listing of columns referenced in gas- and liquid-chromatographic methods related to revisions made to USP–NF since January 1980. USP Chromatographic Columns can be accessed via the USP website, you will need to register to access the database, then search for the monograph in question. Once you know the actual column which was used for the method you can either use this or an equivalent. The USP Column Database provides a tool which can be used to find equivalent columns.

MTS Recommends... ‘HPLC Columns’ by Uwe Neue

2011-11-11T10:34:00.004Z

‘HPLC Columns - Theory, Technology, and Practice’ by Uwe D. Neue, Wiley- VCH, 1997

Although this book was published some time ago in 1997, it is still a fantastic resource for chromatographers. Neue managed to combine a thorough overview of the theoretical background relating to column technology with a practical guide, all in a very readable style.

Help on: Assessing Peak Purity Data

2011-11-09T10:52:00.005Z

MTS HELPDESK

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:

“I am developing a HPLC indicating assay for a hormonal drug. I am using the peak purity option to analysis the purity of my peak once the sample has been subjected to 20% degradation under various conditions. The purity results show that my peak is not pure although its 5 overlaid spectrums are more or less identical. The resolution of my drug from its degradation products is greater than 2. I need to know how I can accurately interpret my peak purity results in order to ensure that no other degradation product is co-eluting at the same time as my main peak. I would appreciate any suggestions with regards to this matter.”

Answer:

"I assume that the results you are referring to are from the software that you are using for peak purity analysis. The purpose of this software is to compare the spectra obtained at time points across the peak and detect differences which cannot be observed by eye. Therefore it is possible that there are spectral differences which may be explained by the presence of another peak eluting at a similar time as your drug. When a drug is degraded, the degradation products are often very similar in structure leading to similar retention times and UV spectra. This means that the spectral differences observed during peak purity analysis may be very small.

To investigate whether your purity result is actually due to another peak or is just a result of the way the spectra have been compared by the software requires knowledge of how the purity result was generated. I recommend that you read through any available information on how the software compares the spectra.

Things to consider:

What reference spectrum is being used, particularly important if it is a gradient method;
Compare the peak purity plot observed for your forced deg sample with that for a pure reference standard;
Loss of linearity can result in spectral differences which are not due to another peak, try diluting your sample and compare the peak purity plot obtained with the original.

A final note, peak purity can never be proven. You can only gain confidence that no other peaks have been detected. This is because the UV spectrum for similar compounds may be identical, also if the apex of both co-eluting peaks is at the same time then the spectra across the peak are likely to be identical.”

Free HPLC Calculator for Method Development

2011-11-03T13:27:00.011Z

PEAK SOLUTIONS
A resource for chromatographers

The MTS free HPLC calculator now includes a method development tool for HPLC. The tool is based on a scouting gradient approach for determining suitable initial conditions for a mixture of analytes, where a single gradient analysis is used to decide whether isocratic or gradient elution is most suitable, and to select promising mobile phase composition. This will be familiar to delegates on MTS training courses and you should be able to start using the calculator straightaway. I plan to post more details in the new year on how to use the tool for those of you who have not attended one of my courses.

HPLC Training in Athlone

2011-10-31T11:10:00.005Z

MTS hosted three days of HPLC training last week in Athlone, Ireland. Thank you to all delegates who attended for a great event and for all the feedback on the training. Don’t forget that you can upgrade your certificate of attendance to a certificate of training by completing the online training assessment, the directions are in your course handout.

Get in touch if you have any problems with the assessment or indeed any questions which came up when you applied your new skills back at work. If you missed these courses then contact us, we plan to run some more in 2012.

Separation Science Europe 2011

2011-10-13T09:37:00.007+01:00

Earlier this week, I presented "A Comparison of Recommended Strategies for Reversed Phase HPLC Method Development" at the Separation Science Europe 2011 Conference. The venue for the conference was the Royal institute in London. It was amazing to present at the same location as eminent scientists from the past such as Michael Faraday and Humphry Davy. The conference programme included a fascinating mix of topics relating to LC and GC applied to a wide range of analytes in the pharmaceutical, environmental and bioclinical sectors.

The highlight for me was a presentation from Ron Majors about ‘Just Enough’ sample preparation where Ron described how analysis by LC-MS/MS and GC-MS/MS has enabled the use of simplified sample preparation procedures. Four examples were presented: Protein precipitation in blood plasma; QuEChERS for pesticide residues; Dried Blood (Matrix) Spotting; and Pharmaceutical pollutants in river water with almost no sample preparation. The potential of reducing both time and error by reducing sample preparation is very appealing.

Help on: Problem with the first injection from HPLC vials

2011-09-23T12:20:00.003+01:00

MTS HELPDESK
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: “When I use a single vial for a series of standard injections the area for the first injection is often lower than that for the subsequent injections. What could cause this?"

Answer:
“There are a variety of potential reasons for injector repeatability problems but the most likely explanation when the first injection from an individual vial gives a lower response than subsequent injections is that the vial was overfilled. This creates a vacuum when the needle pierces the cap and withdraws the test solution and thus the syringe does not withdraw the correct amount. On the next injection the vacuum effect is no longer present and thus the syringe withdraws the correct amount. To prevent the problem, take care to leave a small headspace when filling a vial rather than completely filling the vial.”

Separation Science Europe 2011

2011-09-22T12:53:00.002+01:00

I will be presenting "A Comparison of Recommended Strategies for Reversed Phase HPLC Method Development" at the Separation Science Europe 2011 Conference, on the 10th October.

Abstract:
The benefits of using a strategic approach for developing HPLC methods are easily apparent. The numerous possible chromatographic parameters in a typical HPLC method make choosing the most suitable ones for a particular separation very daunting. In particular, how to select one column from the hundreds available? There are a number of different strategies which can be applied, these include: trial and error, changing one variable at a time; finding a method in the literature or finding a method in the literature for a similar compound; and sophisticated column screening experiments combined with computer modelling, peak tracking methods, experimental design and column comparison tools. In this presentation, current recommended method development strategies are reviewed and compared to give delegates an appreciation of the types of strategies which may be applied, so that they can identify the one which is most applicable for their method development needs.

How to Troubleshoot HPLC

2011-09-20T16:13:00.002+01:00

MTS will continue to work with Phenomenex in the delivery of HPLC training seminars in the UK with a series of troubleshooting courses in November. The dates and locations are as follows:

8th November - Edinburgh
9th November - Crewe
15th November - London

Course price is £195 + VAT per delegate. The price includes: Full day training (including post training assessment), course literature, technical brochures, lunch and refreshments.

If you are interested in attending, contact Phenomenex:

Tel: UK: 01625 501367 or email: ukinfo@phenomenex.com

HPLC Calculator for Void Volume

2011-08-25T11:40:00.009+01:00

PEAK SOLUTIONS
A resource for chromatographers

The MTS HPLC calculator now includes calculation of void volume. This calculation may be useful when multiples of column volumes are required, e.g. for equilibration, and also in HPLC method development to estimate t0, the time in the chromatogram when unretained compounds, e.g. the solvent front, will elute.

5 Step Strategy for HPLC Method Development

2011-08-23T10:01:00.054+01:00

The 5 step strategy for HPLC method development which is taught in MTS training courses is now available in a summarised format as a free download. Click on the picture to download this useful guide to the important steps in HPLC method development. It is designed as an A5 postcard but will also print well on an A4 sheet.

BARQA Annual Conference 2011

2011-07-22T13:35:00.000+01:00

I have been invited to speak on the topic of analytical method validation at the BARQA Annual Conference as part of the session 'Crossing the Plimsoll Line' on 28th September. The title of the presentation is Analytical method validation: Establishing that test methods are 'fit for purpose'. The full programme for the conference can be accessed on the BARQA website.

How to Become an Expert on HPLC

2011-07-12T12:54:00.040+01:00

HPLC Training Courses from Mourne Training Services

October 2011 - Radisson Blu Hotel, Athlone, Ireland

Mourne Training Services will deliver three training courses on HPLC topics in Ireland during October, which together provide comprehensive instruction on how to develop HPLC methods and how to troubleshoot HPLC problems. These courses are ideal for those who are using HPLC and want to improve their expertise. The cost for each one day course is €275 + VAT per person. Contact us to find out about our academic discount and discounts for group bookings.

How to Develop HPLC Methods
(Click here for more information)
25th October 2011

Learn how to select appropriate method conditions and perform suitable investigative experiments to obtain a set of method parameters which enables the desired separation for mixtures of analytes.

“Excellent course, found it very informative and useful." – Angela Boag
"Very enjoyable & informative, Oona was extremely helpful.” –Keighley Campbell

How to Develop HPLC Methods for Challenging Separations
(Click here for more information)

26th October 2011
Learn how to implement strategies to achieve satisfactory separation for ‘complex’ samples and use computer modelling to develop robust and fit for purpose HPLC methods.

“I enjoyed the exercises as they made you think and apply what we just learnt.” – Julie Cooper
"Very good, learnt a lot. Thank you.” – Sally Housden

How to Troubleshoot HPLC
(Click here for more information)

27th October 2011
Learn how to find solutions for problems encountered when running HPLC analysis by diagnosing symptoms and implementing appropriate preventative measures.

"Excellently presented and questions answered consistently and clearly. Problem solving sessions very useful for building up knowledge learnt on the day.” – Robert Hetterley
“I am very pleased that I learned a lot of new things today and I am more confident in troubleshooting HPLC.” – Anna-Marie Corina Cristea

On-site Training
(Click here for more information)
We also offer all these courses (and others) as on-site training where we visit your company to deliver the training. This may include customisation to meet your unique requirements and ‘hands-on’ sessions in your laboratories. Contact us for more information.

Interpreting a HPLC analytical method

2011-06-08T12:00:00.000+01:00

PEAK SOLUTIONS
A resource for chromatographers

The detail which is included in a HPLC analytical method will dictate how much interpretation is required by the analyst when following the method. For a very detailed method the analyst should be able to follow it easily but if the method does not include certain details the analyst will have to make some decisions about how to perform the analysis.

Local laboratory proceures
Routinely parts of the HPLC analytical method are covered by the local procedures in the analytical laboratory and thus will not be included in the HPLC analytical method. Examples are:
The sequence of the injections to be performed.
The procedure to use when preparing the mobile phase, e.g. the grades of solvents which should be used.
The routine use of guard columns or cartridges.
The implementation of wash programmes for column post analysis.

A good knowledge of the local laboratory procedures is required to ensure that all requirements are met.

HPLC column not defined or unavailable

When following a pharmacopoeia method, the column to use will be defined by the bonded phase type (e.g. octadecyl silane) and the particle size range (e.g. 1.5 to 10µm). This makes it difficult to choose which column to use since these parameters may be used to describe hundreds of columns. It is up to the analyst to select a suitable column. There may be a preference in the laboratory for a particular column to use in these situations. Some method development may be necessary to achieve the correct results following the monograph.

Another problem related to columns which may be encountered is that the column needed for a method is no longer available. In this case an equivalent column needs to be sourced. To find an equivalent column a column classification system is required. There are a number of researchers working in this area and their work has produced data for comparing columns [1,2].

Dwell volume
When using gradient methods the effects of dwell volume can lead to changes in the retention times when methods are transferred between different instruments. Ideally methods are developed to be robust to changes in dwell volume but if the change results in reduced resolution a solution will be required.If the retention times are shorter than expected due to the difference in dwell volume then the current system has a smaller dwell volume than the previous system. A solution is to introduce a ‘hold’ of the initial gradient mobile phase composition at the beginning of the analysis. The length of the hold can be determined by measuring the dwell volume on the two systems and dividing the difference by the flow rate, or, it can be determined experimentally by injecting some test samples with different hold values programmed into the gradient table.If the retention times are longer than expected due to the difference in dwell volume then the current system has a larger dwell volume than the previous system. Unless the injector has a function whereby the injection can be performed after the gradient has started (unlikely in older systems which are more likely to have a large dwell volume) the method cannot be satisfactorily run on the instrument.

The dwell volume of an HPLC system is easy to measure:

Remove the column from the system and use a short length of 0.010″ tubing to connect the injector directly to the detector.
For solvent A, use HPLC grade water; for solvent B, add about 0.1% acetone to water (methanol or acetonitrile can be used instead of water).
Set the detector wavelength to 265nm.
Run a typical gradient from 0 to 100% B (e.g. 0-100% in 10 minutes at 1 mL/min flow).
Record the detector signal during this gradient.
Print out or display the "chromatogram" from the gradient run. It should look like Figure 1. Draw the best straight line fit to the flat portion at the beginning of the plot. Draw the best straight line fit to the linear ramp of the gradient. The time at which these two lines intersect is the dwell time (tD). The dwell volume is the product of the dwell time and the flow rate used for the test.

Figure 1

References:
1. D. Visky, E. Haghedooren, P. Dehouck, Zs. Kovács, K. Kóczián, B. Noszál, J. Hoogmartens, E. Adams, , J. Chromatogr. A, 1101, 103-114, 2006, ‘Facilitated column selection in pharmaceutical analyses using a simple column classification system’.(This research group provide their column classification data on this website: www.pharm.kuleuven.be/pharmchem.)

2. M.R. Euerby, P. Petersson, J. Chromatogr. A, 994, 13-36, 2003, ‘Chromatographic classification and comparison of commercially available reversed-phase liquid chromatographic columns using principal component analysis’.This research group has a collaboration with Advanced Chemistry Development (ACD/Labs) to provide a column selection tool (http://www.acdlabs.com/).

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

Help on: Gradient programs for HPLC

2011-05-20T10:27:00.006+01:00

MTS HELPDESK

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:
“How do you calculate the gradient program in HPLC method development?”

Answer:
“The aim of an HPLC method is to enable the separation of a mixture of components. This may be achieved by selecting a suitable mobile phase composition for a particular column which results in a peak for each component that is separated from other peaks, and is retained at a suitable retention time. The composition of the mobile phase may be isocratic, where it is held constant throughout each injection, or gradient, where the amount of the stronger solvent is increased throughout each injection. Whether you use isocratic or gradient conditions depends on the nature of your mixture of analytes. For example in reversed phase HPLC a mixture of analytes which have widely differing hydrophobicity is likely to require an unfeasibly long run time under isocratic conditions and will need to be analysed under gradient conditions.

For RP-HPLC, I suggest that you run your test sample using a full range gradient, e.g. 5 to 95 %B. An inspection of the resulting chromatogram will indicate whether the method is suitable for isocratic analysis (if the difference between the first and last peak is less than 25% of the gradient time) and if not, provides a starting point for gradient method development. Changing the gradient time, tG will make the gradient more or less steep, corresponding to stronger and weaker mobile phase composition in isocratic analysis.

For a detailed discussion on gradient method development you may wish to attend one of my training courses. ‘How to Develop HPLC Methods’ will be held next week in London and Milton Keynes. Future events will be publicised on this blog.”

How to Develop HPLC Methods

2011-05-19T12:01:00.021+01:00

This HPLC method development training course, sponsored by Phenomenex, was held on Tuesday and Wednesday of this week, in Crewe and Edinburgh respectively. The aim of the training was to describe a strategy which can be put in place to develop a new method quickly and easily. The feedback from the delegates was very positive. Comments included:

“Excellent course, found it very informative and useful."

"Very enjoyable & informative, Oona was extremely helpful.”

“I enjoyed the exercises as they made you think and apply what we just learnt.”

There are two more dates for this course next week, in London on Tuesday and in Milton Keynes on Wednesday. If you are interested in attending, contact Phenomenex (course code SS0-5947) to check if there are any places left. Tel: UK: 01625 501367 or email: ukinfo@phenomenex.com

Course Details:
Summary
Learn how to select appropriate method conditions and perform suitable investigative experiments to obtain a set of method parameters which enables the desired separation for mixtures of analytes.
This course is ideal for those who have experience of running HPLC methods and now want to learn how to develop new methods.

Course Outline
Developing an HPLC method using a 5-step strategy:
 Step 1: Setting suitable objectives for method development
 Step 2: Assessing all available information
 Step 3: Selecting suitable samples
 Step 4: Performing scouting experiments to select suitable initial conditions
 Step 5: Optimising the method to define method parameters which achieve the desired separation
This course focuses on reversed phase mode separations.

Practical Skills Acquired
This course will enable you to take a strategic approach to developing HPLC methods with an understanding of the factors which can be adjusted to manipulate the retention time of analytes. In addition you will be able to:
1. Define the objectives for the development of a HPLC analytical method.
2. Effectively assess all the available relevant information relating to the desired method, e.g. pKa of the analyte.
3. Select and prepare a suitable sample or samples to be used for the method development.
4. Select suitable scouting conditions to find a suitable column and mobile phase system.
5. Optimise the chromatographic conditions to result in the best possible separation.

Course price is £195 + VAT per delegate. The price includes: Full day training (including post training assessment), course literature, technical brochures, lunch and refreshments.

A follow up course: ‘How to Develop HPLC Methods for Challenging Separations’ (course code SS0-5946) is planned for September, at Crewe on the 13th and Milton Keynes on the 20th. The aim of this course is to enable development of methods for problem samples and analytes, examples being very polar molecules and samples containing numerous analytes.

Help on: Forced Degradation Studies

2011-05-03T10:29:00.011+01:00

MTS HELPDESK

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:

What is meant by forced degradation studies?

Answer:

In a forced degradation study a molecule is subjected to extremes of heat, humidity, pH, light, etc. so that the molecule degrades. This study is commonly performed for pharmaceutical active pharmaceutical ingredients and formulations and is typically done for one of two reasons:

1. It provides information on the possible degradation pathways of the molecule and thus an assessment of its stability under different types of conditions. The information obtained is usually included in the marketing dossier for the drug.

2. It provides samples which can be used to develop a stability indicating method, i.e. a method which can detect and quantify degradation products. This method is used to assess shelf life during stability studies.

A problem with forced degradation studies is that the extreme conditions used may not be representative of the degradation under ‘normal’ conditions. For this reason the aim is to degrade the molecule of interest by no more than 10%.

System Suitability Failures

2011-04-25T11:39:00.046+01:00

The purpose of system suitability testing is summarised by the USP as follows: "System suitability tests are an integral part of gas and liquid chromatographic methods. They are used to verify that the chromatographic system is adequate for the intended analysis. The tests are based on the concept that the equipment, electronics, analytical operations and samples to be analyzed constitute an integral system that can be evaluated as such."[1] In practice the testing consists of measurements performed on the chromatograms obtained for particular injections during the analysis which provide an indication of whether the HPLC method and system is performing as would be expected. Typical system suitability parameters, as defined by the FDA[2], are summarised in Table 1, with definition of terms for the parameters provided in Figure 1. Acceptance criteria based on recommendations provided by the FDA[2] are provided in Table 2, these are often implemented as a ‘generic’ set of conditions when new methods are developed. Additional requirements may be added as required, e.g. a measure of detector sensitivity may be assessed for a low level impurity method.

Table 1

Figure 1

Table 2

When a system suitability test fails, an investigation into the cause is required. Data generated from the analysis cannot be used. Ideally analytical results should not be generated until the system suitability test results have been shown to pass. In practice however, the processing for an HPLC analysis is usually automated and thus the results are often generated before the results of the system suitability test have been evaluated. In this situation it is very important that the documentation accompanying the analysis is very clear about why the results have been rejected. The available regulatory guidance[3,4] for the investigation of out of specification (OOS) and out of trend (OOT) results involves inspection of the system suitability conditions for the analysis, even though it should already have been assessed prior to accepting the analysis as valid. This may seem redundant but it is important to review all available data during this type of laboratory investigation. It is also possible that the system suitability testing for the method is inadequate and may need to be reviewed and updated.

It is desirable to minimise the occurrence of system suitability failures since each failure represents wasted time spent on analysis and additional time spent conducting an investigation into the failure. The consequences of the failure are not severe: the cause is documented and the analysis repeated. However, regular failures are of concern and may indicate a more serious root cause. The test measures the operation of an HPLC method in a holistic fashion and the reasons for failure of the test are varied. The most common reasons are related to:
1. Degradation of the HPLC column,
2. Competence of the analyst, and
3. Maintenance of the HPLC system.

Degradation of the HPLC column
The column has been singled out since it is a consumable item and it is expected that over time it will degrade and in the process have an effect on the chromatographic performance of the method. Resolution and efficiency are likely to decrease and tailing to increase over time. Under ideal conditions the primary purpose of the system suitability test would be to indicate when the column was no longer performing adequately for the method and should be replaced. In many laboratories the expected lifetime of a HPLC column for a particular method is not investigated and thus there is a risk that using a generic set of acceptance criteria for system suitability testing may result in a failure of the test when in fact the column is still performing adequately, or potentially (though less likely) a pass when the column has actually degraded past the point where acceptable results can be obtained.

Typically the number of injections that can be obtained for a column depends on the combination of the mobile phase, the system conditions, and the sample being analysed. Columns which are subjected to method conditions which include inorganic buffers, high pH or high temperature may result in a reduced number of injections, as will columns which are used for ‘dirty’ samples. An evaluation of the aging of the column for a method is advisable to set appropriate system suitability criteria. This may be carried out as a dedicated study during method development, or a column may be monitored in normal use of the method. Either way the data gained will provide a sound scientific basis for a set of relevant system suitability criteria. In addition, warning limits may be set which alert the operator that the column needs to be replaced soon, thus eliminating any system suitability failure due to the aging column.

Competence of the analyst
The analyst performing HPLC analysis must not only be competent in the preparation of mobile phase and test solutions but also in setting up the HPLC system. Errors in mobile phase preparation can affect the capacity factor and thus the retention time of analytes. If the analyst introduces contamination in the mobile phase this may introduce extra (unwanted) peaks, decrease resolution and degrade the overall chromatography. Errors in preparation of test solutions can affect the accuracy and precision of the analytical results. Examples of problems due to incorrect set up of the HPLC system include unsteady and noisy baselines if the pump is not adequately primed, peak broadening effects (decreased efficiency) due to unsatisfactory connections and drifting baselines and retention times if the temperature is not controlled.

If the operator has a good understanding of the method, this will help to ensure that important method parameters are controlled, e.g. in some HPLC methods the retention times of the analytes are very sensitive to the pH of the buffer in the mobile phase and thus strict control of the pH is critical. If the analyst is aware of this requirement they will be more careful during the pH adjustment task. Suitable training is essential to ensure that system suitability failures are not due to the competence of the analyst.

Maintenance of the HPLC System
If not correctly maintained the HPLC instrumentation can contribute to system suitability testing failures, e.g. worn injector parts may result in repeatability failure, an aged lamp in a UV detector can cause baseline noise and have a detrimental effect on the quantification of low level analytes. The system suitability test is not a substitute for the performance qualification of the HPLC instrument since it is method based rather than instrument based. Although it provides assurance that the method may be used satisfactorily at that time it does not test that the instrument is functioning correctly, e.g. accuracy of flow rate, wavelength, injection volume, temperature, etc. Regular maintenance and performance verification should be scheduled to ensure that the HPLC system does not contribute to system suitability testing failures.

In summary, system suitability testing failures can be reduced by a combination of three measures:

1. Set system suitability criteria which relate specifically to the method in use. A column degradation study will identify the parameters of resolution, tailing and efficiency which indicate that a new column should be used.

2. Ensure that HPLC operators have received suitable training and are familiar with the method in use, and in particular, the robustness issues relating to the method.

3. Implement a regular maintenance and performance verification procedure for HPLC systems.

References:
1. United States Pharmacopeia (USP), Chromatography <621>
2. Reviewer Guidance: Validation of Chromatographic Methods, US Food and Drug Administration, 1994
3. Guidance for Industry: Investigating Out-of-Specification (OOS) Test Results fror Pharmaceutical Production US Food and Drug Administration, 2006
4. Out of Specification Investigations, MHRA

MTS Recommends... Measuring Volume - Beakers, Cylinders, Erlenmeyer Flasks, & Volumetric Flasks

2011-04-11T11:57:00.002+01:00

This free video is a useful learning resource which describes the different types of volumetric glassware and how to use them.

MTS Recommends... Chiral Method Development Screening Techniques

2011-03-31T10:18:00.002+01:00

Chiral Method Development Screening Techniques: A practical guide and new approaches in LC-MS by David S Bell & Denise Wallworth, Chromatography Today, September 2009

When faced with developing a chiral HPLC method it is difficult to predict which type of chiral column will give the best results for your analyte. Screening a range of columns and conditions allows you to pick the most suitable. This article describes a screening approach for chiral HPLC.

Phenomenex Press Release for HPLC Training Courses in UK

2011-03-21T09:05:00.001Z

The MTS training consultant, Oona McPolin, will be delivering a series of HPLC training courses in partnership with Phenomenex over 2011. The following press release about the courses has been released by Phenomenex:

These new training courses are designed to cater for novice chromatographers through to experienced method developers. This new series of courses will provide you with practical skills and knowledge to utilise immediately in your own working environment.
At locations across the UK: London, Milton Keynes, Crewe, Glasgow & Edinburgh

Courses being run in 2011 are:

How to….
Course 1: Run HPLC methods.
Course 2: Develop HPLC methods
Course 3: Validate chromatographic methods
Course 4: Develop HPLC methods for challenging separations
Course 5: Troubleshoot HPLC

All courses cost just £195+ VAT (this includes full course materials, refreshments, lunch and post course assessment with certificate of completion).

Download Course Brochure: Click Here

Although sponsored by Phenomenex, these courses are written and presented by Mrs Oona McPolin (BSC, MSc, CSci, CChem, MRSC) who has considerable experience and is fully qualified in the areas of both pharmaceutical analysis and training practice. She worked as an analytical chemist for over 10 years at a major global pharmaceutical company, on a range of drug development projects also with responsibility for many pharmaceutical analysis training programmes. Mrs McPolin has obtained the industry standard qualification for training, the Certificate in Training Practice, from the Chartered Institute of Personnel and Development (CIPD).

For more information or to book a place, please contact Phenomenex Today.
Tel: 01625 501367(UK) 01 247 5405(Eire) Email: ukinfo@phenomenex.com

Troubleshooting Tip: Baseline noise

2011-03-18T13:22:00.009Z

PEAK SOLUTIONS A resource for chromatographers If there is regular noise in your HPLC baseline then you need to figure out what is causing it before you can find a solution. A good first step is to turn off the pump and monitor the output from your detector. If the noise is still present then the problem is not linked to the flow of mobile phase. For a UV detector the most likely source of the noise is the lamp, either it has aged to a point where it needs replaced, or it is defective. Replacing the lamp should resolve the problem. If the noise stops when the pump is stopped then it is linked to the flow of mobile phase. Likely causes are:

Trapped air in the flow cell - try flushing with degassed mobile phase, or a strong solvent

A leak - carefully check your system for leaks and refit or replace the affected part

Temperature fluctuations - control the temperature

Incomplete mobile phase mixing - if you are using the HPLC system to mix the mobile phase, try mixing the mobile phase by hand and see if the noise goes away.

MTS Recommends... LC Modelling using DryLab

2011-03-15T12:57:00.004Z

3-Dimensional Retention Modelling of Gradient Time, Ternary Solvent-Strength and Temperature of the Reversed-phase Gradient Liquid Chromatography of a Complex Mixture of 22 Basic and Neutral Analytes using DryLab® 2010 by Melvin R Euerby, Gesa Schad, Hans-Jürgen Rieger & Imre Molnár, Chromatography Today, December 2010

In this article the HPLC modelling software, DryLab, is used to model a separation of pharmaceutical analytes using gradient time, temperature and ternary composition. Probably the most common approach for computer modelling is using a combination of gradient time and temperature, so this approach adds the potential use of ternary mixtures of mobile phases and brings the increased selectivity options that this will provide. This article will be of interest to anyone using computer simulation as part of their method development strategy.

HPLC Training in Belfast

2011-03-11T15:09:00.004Z

Last Tuesday, the MTS training consultant, Oona McPolin, delivered the training course ‘How to Troubleshoot HPLC’ at the Culloden Hotel in Belfast. The training was well received generating very positive feedback. This one day course is designed with two aims, to provide: 1 - a full understanding of how HPLC systems work so that users can prevent problems from occurring, and 2 - knowledge of the symptoms of common problems to allow successful troubleshooting. Thank you to everyone who took part and don’t forget to get in touch if you have any HPLC troubleshooting questions.

We plan to organise further training sessions later this year on the topics of HPLC method development and method validation at venues in Ireland. Get in touch if you are interested in attending these courses.

Help on: ‘Dry’ solvents for HPLC?

2011-02-28T13:12:00.003Z

MTS HELPDESK
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:
"I have observed that in one of your answers for a previous helpdesk blog, ‘Retention time shifts in NP-HPLC’, you used the term ‘dry’ solvents. What is the difference between dry solvents and ordinary HPLC solvents, and what is the use of anhydrous sodium sulfite in mobile phase?"

Answer:
"Anhydrous or dry solvents have been treated to remove trace amounts of water that may be present. It is possible to buy anhydrous solvents from your normal solvent supplier or you can use a variety of methods to dry solvents in your laboratory. The most suitable method depends on the actual solvent in question but typical methods include addition of anhydrous salts, distillation and use of a molecular sieve (usually 0.4nm). The suggested use of solvents which are half saturated with water (in the previous blog post) is a method by which retention times can be kept reasonably constant by controlling the amount of water present in the mobile phase, since the amount of residual water present in mobile phase can affect retention times considerably.

Another source of trace amounts of water is the sample which is injected onto your HPLC column, thus the suggestion to consider drying this sample by the addition of anhydrous sodium sulphate. This salt is widely used as an inert drying agent for removing traces of water from organic solutions in the laboratory. I would not recommend adding it to the mobile phase. The aim in NP-HPLC is to achieve a fairly consistent amount of water in the mobile phase rather than using completely dry solvents. Once opened dry solvents will absorb water over time and this change will affect retention times in NP-HPLC."

MTS Recommends... How to Calibrate Pipettors

2011-02-23T11:48:00.003Z

HPLC Troubleshooting Training Course in Belfast

2011-02-01T10:03:00.014Z

Does HPLC ever leave you feeling like this?
You need our training course How To Troubleshoot HPLC.

Learn how to find solutions for problems encountered when running HPLC analysis by diagnosing symptoms and implementing appropriate preventative measures. This course is ideal for those who have experience of using HPLC and now want to develop their skills further.

New date now available:
8th March 2011
The Culloden Hotel, Belfast, Northern Ireland.
Book online or contact us for more information

Just £195 + VAT per person if booked before 22nd February 2011. Discounts for groups are available. This includes: Comprehensive handouts; access to online training resources; post training assessment and certificate of training; lunch and refreshments.
This course is eligible for Refer Your Friends.

Let us know if you would like this training course at a location near you.

MTS Website Updates

2011-01-28T15:55:00.005Z

We have made some updates to the MTS website. There is now a Course List so that you can easily view the range of courses that are available. In addition to a brief course summary and information on costs, the delivery options for each course are listed so that you can see at a glance whether the course is available by eLearning, on-site, at an external location, etc. The Resources section has also been updated and all past issues of Analyse This are now available on a dedicated webpage. We also have a webpage for our new Refer Your Friends scheme.

MTS Recommends... Using Temperature in LC Method Development

2011-01-20T15:32:00.003Z

The Use of Temperature for Method Development in LC by Gerd Vanhoenacker , Frank David & Pat Sandra, Chromatography Today, September 2010

Since the ionisation equilibria of analytes and mobile phase for polar and ionisable analytes is temperature dependent, temperature can be used as a useful selectivity parameter when developing methods for these types of analytes. This technical note discusses the range of temperatures which can be used for method development and the different types of available LC columns which can be used at high temperatures.

Help on: Equilibration of HPLC columns between gradient method injections

2011-01-18T14:28:00.005Z

MTS HELPDESK
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:
“I have a question about column re-equilibration after each injection ready for the next one. Firstly, is there a way how to calculate how long the re-equilibration step should be (or is it more of experience and trial and error)? I am using a 30 x 4.6mm column (2.7 particle size), HP1100 pump, 1.7ml/min flow rate, and 2 min gradient method (as you can see I’m after a fast turn-around!). I’m just a bit worried if I’m giving the column enough time to re-equilibrate ready for the following injection. My gradient is as follows:

Time - %A - %B
0.00 - 90.0 - 10.0
0.10 - 90.0 - 10.0
0.50 - 10.0 - 90.0
1.50 - 10.0 - 90.0
1.60 - 90.0 - 10.0
2.00 - 90.0 - 10.0

I am also taking advantage of the next injection’s injection time (25 seconds) which I’m assuming can also be used as part of the re-equilibration step? I have done a series of injections (~10) and the retention times are pretty constant using this 2 min method. Though I was also wondering over how many injections could you justify the method reliable, would 10 be enough?”

Answer:
“A general rule for equilibration is to allow 10 column volumes of mobile phase to pass through the column. Column volume refers to the amount of space in a column which can be taken up by the mobile phase, or the space not taken up by stationary phase packing. A previous blog on column equilibration describes the calculations for determination of this value and the MTS HPLC calculator can convert this to a time value, you may find these helpful.

For your method 10 column volumes is equal to 2.1 minutes. Given the length of your analysis I think that this would be too much time to spend on re-equilibration. Your current equilibration time is half a minute in the gradient table and another 25 sec for the injection which equates to about 5 column volumes. I feel that this should be sufficient if the method can be demonstrated to be working reproducibly. The signs of insufficient equilibration are baseline problems and variation in retention time so if you can show that these are constant then you can justify re-equilibration using 5 column volumes.

The method conditions will determine whether 5 column volumes is enough for re-equilibration, i.e. how different the starting and end conditions of the gradient are. Although your gradient is quite steep which means it has to go back from 90% B to 10% B in the re-equilibration, the 10 injections that you have performed give a good indication that the method is working fine. It would probably be good to back this up with some results over a longer time frame. Rather than setting up an experiment to test this I would suggest monitoring the use of the method when you analyse samples.”

HPLC Training Video Wins Silver Award in Tutorial Competition

2011-01-04T13:34:00.003Z

The training video ‘A Brief Guide to HPLC Instruments’ has won a Silver Award in the Year of Education in Separation Science Tutorial Competition. Click here to read more and view the three winning entries.

Help on: HPLC Dwell Volume and Dead Volume

2010-12-10T10:23:00.003Z

MTS HELPDESK
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:
“In HPLC, how can I calculate dwell volume for my system, and what is the main difference between dead volume and dwell volume?”

Answer:
“The dwell volume for an HPLC system refers to the volume in the system from the point where the mobile phase is mixed to the inlet of the HPLC column. This volume is important when using gradient elution since there will be a delay between the time when a change in mobile phase is applied and when it actually reaches the column. The size of the dwell volume varies between different systems; it can be calculated as follows:

Remove the column from the system and use a short length of 0.010″ tubing to connect the injector directly to the detector.
For solvent A, use HPLC grade water; for solvent B, add about 0.1% acetone to water (methanol or acetonitrile can be used instead of water).
Set the detector wavelength to 265nm.
Run a typical gradient from 0 to 100% B (e.g. 0-100% in 20 minutes at 3 mL/min flow). Record the detector signal during this gradient.
Print out or display the "chromatogram" from the gradient run. It should look like Figure 1. Draw the best straight line fit to the flat portion at the beginning of the plot. Draw the best straight line fit to the linear ramp of the gradient. The time at which these two lines intersect is the dwell time (tD). The dwell volume is the product of the dwell time and the flow rate used for the test.

Figure 1

The term ‘system dead volume’ is used to refer to the volume in a HPLC system from the point of injection to that of detection. Think of it as the volume of mobile phase between these points. The ‘column dead volume’ is the volume of space in the column not taken up by stationary phase (sometimes also referred to as void volume), or you can also think of it as the volume of mobile phase that fits in the column. Typically the difference between the system dead volume and column dead volume is very small and so you may find the terms being used interchangeably. Unfortunately chromatographers can sometimes be a bit sloppy about using consistent terminology but the context should make it clear what is being referred to.”

Help on: HPLC Baseline Problem

2010-12-08T11:26:00.005Z

MTS HELPDESK

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:
“The problem is the baseline looks lumpy (not steady) instead of being normal or steady. I have tried all the procedures and therefore eliminated the column, the mobile phase and detector. The system has been thoroughly flushed and purged but still my baseline is not as expected. I have tried the column on a different system under the same conditions (ambient temp, flow rate 1.0ml/mins, inj vol 2ul) and the baseline is normal as expected. I have attached a copy for you to see if you can help. Any ideas what could be happening on this system?”

Answer:
“The baseline in your chromatogram is very far from what you normally expect. The regular cycling pattern in the chromatogram makes me think that the problem is most likely related to the pumping of the mobile phase. Since you have ruled out the detector, and have transferred the mobile phase and column successfully to another system this means that the pump is the most likely source of the problem. Even though it is thoroughly flushed and purged there is still a small chance that it may be due to trapped air, but if not then a faulty check valve is likely. Try flushing and purging the pump with methanol (remove the column) and then with your mobile phase. If this doesn’t work try replacing the inlet check valve (this is the one which usually causes problems), or clean it by placing in a beaker and covering with methanol or isopropanol, then sonicate for about 5 minutes and replace.”

HPLC Training Video Shortlisted in Tutorial Competition

2010-12-03T15:15:00.004Z

We are delighted to announce that the training video ‘A Brief Guide to HPLC Instruments’ has reached the shortlist of the Year of Education in Separation Science Tutorial Competition. The Year of Education in Separation Science is an initiative run in conjunction by separationsNOW.com (Wiley-Blackwell's separation science website) and Chromedia (an online community and database of tutorials and other educational content). The tutorial initiative is partnered by PITTCON 2011. The purpose of the competition was to facilitate and encourage the creation and sharing of educational material. The tutorials were judged on their content and how effective they are at communicating their message. The winner will be announced in the next few weeks.

MTS Recommends... How to Weigh Out Small Amounts

2010-12-01T16:40:00.004Z

Preparation of Mobile Phase for HPLC

2010-11-29T10:14:00.006Z

PEAK SOLUTIONS
A resource for chromatographers

The following is an excerpt from 'An Introduction to HPLC for Pharmaceutical Analysis' by Oona McPolin, now at a reduced price of only £20 (that’s a discount of over 30%),available to purchase through the MTS website.

Solvent grades
There are HPLC grade solvents available for the common solvents used and it is recommended that these are used for the best results. They are typically ≥ 99.9% pure. Within the HPLC grade there are different types available from some suppliers. These include ‘gradient grade’, and ‘LC-MS’. When running gradient elution and also when using a mass spectrometer as a detector the solvent used needs to be very pure to eliminate interferences which might affect the results. Acetonitrile is also available in a ‘far UV’ grade, this relates to the use of a UV detector in the far UV range, i.e. below 200nm. The difference in these grades relates to purity, very high purity solvents are usually obtained by multiple distillations. If a specialised grade is required for an analysis then this information should be included in the details of the HPLC analytical method.

Water for HPLC may be sourced from solvent suppliers in HPLC grade bottles or some type of water purification system can be used[1]. A water purification system for HPLC should generate ultra pure water (18 MΩ resistivity), it is extremely important that the system is well maintained.
Different types of methods have different tolerances to the grade of the solvents used in the mobile phase. This should be assessed thoroughly during the method development process and any requirements included in the analytical method.

Measuring solvents
When measuring out the solvents to be used in a mobile phase each solvent is measured separately using a measuring cylinder of an appropriate size rather than measuring one and making up to volume with the other. The reason for this is that the volume of the mixture is smaller than the individual volumes due to a contraction effect. This is particularly applicable to methanol but is true for other water miscible solvents to some extent.

Mixing the mobile phase
When the mobile phase contains added buffer, ion-pair reagent or other additive, it is customary to weigh out these substances using HPLC grade reagents and add them to the aqueous portion of the mobile phase mixture. The pH is then adjusted on the aqueous portion prior to mixing with the organic portion. There are a number of options about how to perform the mixing of the mobile phase:

Isocratic methods
The aqueous and the organic portions of the mobile phase may be measured separately and mixed in a container by the analyst (premixing) or each portion may be placed on the HPLC system and mixed in the correct proportions by the HPLC pump (online mixing).

Gradient methods
The aqueous and the organic portions of the mobile phase for both the starting conditions and for the final conditions of the gradient may be measured separately and mixed in a container by the analyst (premixing). This will result in two mobile phases, e.g. for the gradient 20 to 80 %B over 20 minutes, a mobile phase of 20 %B and a mobile phase of 80 %B are prepared by the analyst, these are then placed on the HPLC system and mixed during the analysis by the HPLC pump. The other option, as in the isocratic method, is to place the aqueous and organic portions on the HPLC system and mix in the correct proportions by the HPLC pump (online mixing).

There are advantages and disadvantages to each approach. Premixing means that the pump does not have to perform mixing in the isocratic case and performs the least amount of mixing in the gradient case. If the pump being used is not capable of this type of mixing then the premixing option is preferred. Premixed mobile phases are subject to analyst error and different batches will be slightly different to each other. Online mixing removes the analyst error and is often more convenient to operate. Bottles received from the supplier containing solvent and premade reagent can be placed directly on the system preventing contamination associated with measurement. Plastic coated bottles are available for this purpose. However, if the pump is unable to deliver a well mixed mobile phase then online mixing is not suitable. The final decision depends on the pump capabilities, the analyst preference and the application.

Reference:
1. S. Mabic, C.Regnault, J. Krol, LC•GC Eur., 18(7), 2005, ‘The Misunderstood Laboratory Solvent: Reagent Water for HPLC’.

Huge Savings on MTS Training Books - Now Only £20 or Both Books for Just £35

2010-11-15T10:40:00.011Z

In a special end of year offer you can purchase each of the MTS training books, 'An Introduction to HPLC for Pharmaceutical Analysis' by Oona McPolin, and 'Validation of Analytical Methods for Pharmaceutical Analysis' by Oona McPolin, for only £20. Or you can buy both books for just £35. The offer runs until 31st December 2010. Usual shipping rates apply.

Visit the MTS website now, click here, to take advantage of this huge saving.

How to Troubleshoot HPLC – External Training Courses

2010-11-12T16:28:00.010Z

The best way to avoid and resolve problems when using HPLC is to have a sound understanding of how the technique works and the role of each component in the HPLC system. On the course How to Troubleshoot HPLC, sponsored by Phenomenex, we look at each component of a HPLC system in turn, explaining the role that it plays and how it works, and then identifying the causes of potential problems. This allows you to implement appropriate preventative measures and apply what you have learned to any brand of HPLC instrumentation. You will receive lots of helpful advice and tips on how to consistently achieve trouble free HPLC analyses.

We will focus on the practical application of your new skills and knowledge so that you are fully equipped to effectively troubleshoot HPLC when you return to your lab. This is achieved by putting your newfound problem solving skills into practice using numerous real-life case studies. You are encouraged to bring along your HPLC problems for discussion during the training.

The available dates are as follows:

Thursday 18th November
Louis Fitzgerald Hotel, Naas Road, Dublin

Tuesday 23rd November
De Vere Wychwood Park, Weston, Crewe, Cheshire

Tuesday 30th November
The Lensbury, Broom Road, Teddington, West London

The course costs £195 which includes the full day of training (including post training assessment), course literature, technical brochures, lunch & refreshments. To register click here.

Help on: Large Peak at the End of a Gradient Analysis

2010-11-10T10:13:00.008Z

MTS HELPDESK
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:
“I am running a gradient method of 20 to 45% B over 55 minutes, where B is acetonitrile and A is acetate buffer at pH 4.8. There is a large peak at the end of the gradient as shown below. This peak is observed for a blank injection of the sample diluent so I think it must be due to a contaminant in the mobile phase. I have tried preparing fresh mobile phase from different reagent batches but it is still observed. Can you suggest what could be causing it?”

Answer:
“Contamination of the mobile phase is usually one of the primary suspects when unexpected peaks occur in gradient analysis. However, it is commonly observed that a peak such as that shown in the chromatogram occurs when the strong solvent conditions at the end of the gradient are rapidly changed back to the original conditions. This only occurs with acetonitrile and is not yet fully explained to everyone’s satisfaction. It occurs after the separation is complete and thus is not normally a problem. Simply ignore the peak or set your CDS to stop collecting data after the last peak of interest.”

MTS Recommends... Scaling LC Methods

2010-11-09T12:47:00.002Z

Separation Scaling by Uwe D. Neue, Separation Science, Volume 2, Issue 13, October 2010, page 3

This article from Uwe Neue provides helpful advice for scaling methods between columns of different particle size and column dimensions, such as HPLC and UHPLC.

MTS Recommends... A Review of High Temperature LC

2010-11-08T12:26:00.001Z

High temperature liquid chromatography - a brief review about an emerging technique by Thorsten Teutenberg, Chromatography Today, September 2010

This review presents a general overview of high-temperature liquid chromatography. It starts with a brief definition and then explains the necessary requirements for this emerging technique. Also, the advantages of high-temperature liquid chromatography such as the reduction in the mobile phase’s viscosity and the possibility to replace toxic organic solvents with water are outlined.

Help on: Developing a HPLC Method for Veterinary Drugs

2010-10-28T17:24:00.003+01:00

MTS HELPDESK

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:
“I am trying to develop a HPLC method for some veterinary drugs. I am using a C18 column 250mm x 4.6 mm, 5u particle size. The mobile phase I’m using has a composition MeOH: 5% GAA in water, 98:2 (v/v). During the analysis I'm observing that between analyses of the same sample, the RT for the drug shifts in each run. The RTs for 5 successive injections are: 8.1 min, 8.2 min, 8.3 min, 8.4 min and 8.2 min.”

Answer:
“The retention time shifts that you are experiencing could be due to a number of different factors, the most common three being variation in temperature (are you controlling the column temperature?), insufficient column equilibration (prior to any injections and between injections), and changes in mobile phase composition (how are you mixing the mobile phase? If it is premixed this is unlikely to be the source of the retention time variation).

Regarding your choice of mobile phase, the proportion of organic is very high at 98% methanol. The analytes must interact very strongly with the column. Have you considered using a less retentive phase, such as C8? This may reduce your consumption of organic solvent and give you more room to manoeuvre when selecting optimum conditions for your method.”

HPLC e-Learning: CD-ROM option for UTrain now available

2010-10-26T13:36:00.011+01:00

The MTS in-house e-Learning training solution, UTrain, is now available on CD-ROM. Training videos, exercises and an assessment are provided on one CD and the worked solutions for the exercises and answers to the assessment questions on another. The currently available course, ‘Basic HPLC for Pharmaceutical Analysis’ is comprised of 4 modules (over 3 hours of video) and costs just GBP £500 (+ VAT where applicable).

The UTrain page on the MTS website will be updated soon with information on the CD-ROM option. Contact us to order your CDs or send us your queries about the product.

Free e-Learning Module on HPLC Instruments

2010-10-13T15:08:00.005+01:00

We are pleased to introduce a new format to our UTrain e-Learning portfolio: interactive training modules. Consisting of video combined with interactive content, these modules will be available to purchase as web-based training, either hosted by us on e-MTS or on your own learning management system, and also on CD-ROM.

View the demo now and contact us to discuss your requirements.

MTS Recommends... Quantitative Open-Access HPLC

2010-10-12T13:55:00.001+01:00

'Quantitative open-access HPLC analysis: a new calibration approach' by Phil Borman, John Roberts, Barbara O’Reilly, Robin Attrill, Ian Barylski & Keith Freebairn, Pharmaceutical Technology Europe, October 2010

This article describes a calibration technique used by GSK which enables the estimation of yields in solutions and the assay of isolated solids, so that synthetic chemists can use open access instruments for these analyses. The response factors for a specially selected external standard and that of the analyte are used in the calculation. This will be a useful read for labs using open access instruments.

HPLC e-Learning Offer Ends 31st October

2010-10-11T11:25:00.005+01:00

The special ‘Back-to-school’ offer for our iLearn course, ‘Basic HPLC for Pharmaceuticals’ will end on the 31st October. Book now and receive a 20% discount: only £100 (+ VAT where applicable). iLearn is an open enrolment e-learning course which is delivered online. Visit the iLearn webpage for more information.

MTS Recommends... From Molecule to Medicine

2010-10-08T15:55:00.001+01:00

This 6 minute film explains the various stages for a pharmaceutical company to develop new medical cures. It clearly shows what processes occur in conditions of sickness and how scientists use their knowledge to discover new, innovative drugs to help patients worldwide. Made by NORVELL JEFFERSON, a Belgian audiovisual production company.

Help on: Retention Time Variability

2010-09-24T11:01:00.009+01:00

MTS HELPDESK

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:
“I have a question regarding retention time variability between different types of HPLC systems. An experiment was performed on an Agilent 1200 HPLC and on a Waters 717 instrument using a gradient profile. On the Agilent, the retention time was 14.7 minutes, and on the Waters, the retention time was 19.6 minutes. The same mobile phase was used for each system along with the same sample preparations, which seems to point to a problem with the Waters instrument (the Agilent gives typical results). What are some issues that could cause such variability with the retention times?”

Answer:
“The most probable reason for the difference in retention time that you are observing is that the dwell volume for the two HPLC systems being used is different. The dwell volume relates to the time taken for a change made in the mobile phase proportions to reach the column. The dwell volume for modern instruments like the Agilent 1200 is quite small (typically less than 1 mL) but for older instruments it can be in the region of 2 to 3 mL or larger. The Waters 717 is an autosampler but I assume that it is connected to other Waters components of a similar age. It is likely that the dwell volume for this system is greater than that for the Agilent and thus is the cause of the larger retention time.

You can easily check if the dwell volumes are different by determining this value for each system. I have provided directions for this in a previous blog post. You may also be interested in the reply to another MTS Helpdesk question which was similar to yours where I discuss further the reasons for differing retention times for different systems.“

'How to Troubleshoot HPLC' - New External Training Course

2010-09-13T12:01:00.017+01:00

Sponsored by

MTS is delighted to announce a series of training dates for a new course ‘How to Troubleshoot HPLC’. These one day external training courses are sponsored by Phenomenex. The available dates are as follows:

Thursday 18th November
Louis Fitzgerald Hotel, Naas Road, Dublin

Tuesday 23rd November
De Vere Wychwood Park, Weston, Crewe, Cheshire

Tuesday 30th November
The Lensbury, Broom Road, Teddington, West London

COURSE SUMMARY

Learn how to find solutions for problems encountered when running HPLC analysis by diagnosing symptoms and implementing appropriate preventative measures. This course is ideal for those who have experience of using HPLC and now want to develop their skills further.

COURSE OUTLINE

Overview of the HPLC and how it works:
Mobile phase, pumps, injectors, columns, detectors and connections
Common problems and preventative measures
Problem solving strategy:
Assessing the symptoms
Making diagnosis
Finding the appropriate solution

PRACTICAL SKILLS ACQUIRED

This course will enable you to go back to your lab with a full understanding of why problems may arise with your HPLC system and give you the skills and knowledge to both prevent and resolve those problems. In addition you will be able to:

Understand how HPLC works and the role of each component in an HPLC system
Understand how problems can arise in the individual components of an HPLC system
Implement measures which prevent problems occurring
Use a systematic problem-solving approach to HPLC troubleshooting
Diagnose and resolve problems associated with HPLC

The course costs £195 which includes the full day of training (including post training assessment), course literature, technical brochures, lunch & refreshments. To register click here or contact Phenomenex and quote course number SS0-7449.
Telephone: 01625 501 367 (UK) or 01 247 5405 (Eire)
Or email: ukinfo@phenomenex.com

New MTS Telephone Number

2010-09-07T12:10:00.011+01:00

Please note that the MTS contact telephone number has now reverted back to our previous number:

028 41773724

No other contact details have been affected.

Help on: Peak Purity Assessment

2010-09-06T11:51:00.002+01:00

MTS HELPDESK

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:
“While performing peak purity assessment (or doing force degradation studies), if any unknown impurity with similar spectra co-elutes with the peak of interest, then software will show that the peak is pure. But is there any trick or any method to get correct results...?”

Answer:
“Peak purity assessment software can only interpret the information that is collected from the UV diode array. Unfortunately organic impurities often produce very similar spectra to analytes of interest because the difference in the molecules does not relate to the functional groups which are producing the UV response. Although software may report that a peak is pure you can never reach this conclusion when investigating peak purity by this method. All you can conclude is that you have not found any impurities co-eluting with the peak of interest. To differentiate between analytes of interest and their impurities where the structure is very similar is not within the capability of this UV technique so there are no tricks or methods to make it possible. Mass spectrometry may also be used for peak purity assessment but it also suffers from some limitations.”

iLearn 'Back-to-School' Offer: 20% Discount for Bookings made in September & October

2010-09-01T09:39:00.003+01:00

We are offering a special 'Back-to-School' discount on all iLearn bookings which are made in September and October this year. The already low price of £125 GBP is now reduced to only £100 (+VAT where applicable). For this you get:

Access to HPLC training videos which describe how the technique works;
Exercises to support your learning;
Support from the MTS tutor who will give you feedback and answer your questions;
A copy of the book 'An Introduction to HPLC for Pharmaceutical Analysis' (as reviewed by Chemistry World) and,
A training certificate recognised by the Royal Society of Chemistry for the purposes of continuing professional development.

Visit the iLearn page on the MTS website to find out more and book now.

MTS Recommends... ‘Holistic LC strategies, from UHPLC to HPLC and back’

2010-08-25T16:02:00.001+01:00

‘Holistic LC strategies, from UHPLC to HPLC and back’ by Adrian Clarke, John Nightingale, Partha Mukherjee and Patrik Petersson in Chromatography Today, May/June 2010

One of the problems when introducing new technologies, such as UHPLC, is that although they may result in dramatic performance improvements, the methods utilised may not be easily transferred to other laboratories. In large pharma companies analytical methods may be transferred for stability studies, to contract research organisations, and to quality assurance departments, among others, and so this limits the use that can be made of a new technology. In this article, scientists at AstraZeneca describe the methods that they have used to introduce UHPLC alongside traditional HPLC techniques in a global strategy.

MTS Recommends...Video on Clinical Research

2010-08-17T15:50:00.001+01:00

This short animation introduces the stages of clinical research in drug development, useful if you work in pharmaceutical analysis and want to know more about the industry you work in, or may be useful as a resource in your pharmaceuticals training programme.

Help on: Expiry Dates for HPLC Mobile Phase

2010-07-23T10:52:00.006+01:00

MTS HELPDESK

Question:
“What is the typical (industrial “norm”) for the expiration date of mobile phases for HPLC, i.e. with buffer and without buffer. Also, what should you do (e.g. measure the pH or run and verify the retention time) if you need to extend the expiration date in extenuating circumstance e.g. shortage of acetonitrile or delay of the orders from the vendor, etc. ”

Answer:
“There is no industrial norm for the expiration date assigned to mobile phases because the composition of mobile phase varies so greatly. However, a typical expiry date is one month after preparation. In general, preparing fresh mobile phase is best and if possible it is a good idea to prepare as much as you are likely to need. Since it is wasteful (in terms of both cost and environment) to dispose of solvents if not necessary I would make the following recommendations:

Buffers are most likely to cause problems on storage due to bacterial and fungal growth but all buffers do not behave in the same way. An example of a buffer in which growth occurs very soon after preparation is a phosphate buffer at pH 7. You should assess how long a buffer should be kept for your particular method. If the buffer is not mixed with organic solvent (for example when you use the HPLC system to mix the mobile phase for you) then it will probably not last as long. Assessing the buffer for a suitable expiry date consists of checking for visual growth (dispose of the buffer if you can see any), and checking the chromatographic performance.

The easiest way to check chromatographic performance is to use the system suitability test for the method, this includes any standard checks. If the system suitability is well designed it should be able to confirm that a particular mobile phase is giving the expected results and is suitable for use.

Typically for blends of aqueous buffers and organic solvents, where the aqueous content is less than 20%, an expiry date of 1 month after preparation is suitable and it is likely that you could extend this to 6 months. For blends where the buffer content is greater than 20% assess visually and for chromatographic performance. Where your mobile phase consists of blends of organic solvents you can easily set an expiry date of 6 months, but watch out for changes in the relative amounts of solvents due to evaporation. My personal preference is to use the HPLC system to blend solvents and thus a single solvent is in each mobile phase reservoir and changes in the composition cannot occur.”

Help on: Retention Time Shifts in NP-HPLC

2010-07-19T15:35:00.010+01:00

MTS HELPDESK

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:
“I am using NP-HPLC, my analyte needs 100% hexane. I am using pure material with un-known RT. The RT shifts to the right with every injection, something like 2-2.5 min for each injection. I started at 9 min and get to 16 min in 6 injections. Then I decided to clean the column overnight with 100% hexane and now I started from 27 min and it's shifting ... Any advice will be helpful. The temperature is constant, there is only one peak for my analyte.”

Answer:
“The most common problem when using NP-HPLC is retention time variability. This is normally due to the presence of very polar molecules such as water in the mobile phase. A small amount of water can significantly alter retention times. Once your column has equilibrated with the water content in your mobile phase any alteration in this amount will cause shifts in retention time. Reaching full equilibration is very time consuming in normal phase systems and can take anything from a few hours to a few days.

It sounds like your original injections had drifting retention times because the column had not reached equilibration. A strategy of running the mobile phase through the column overnight should help to achieve equilibration but since the RTs have not settled down either the column has not yet equilibrated or perhaps the mobile phase was altered. Is the mobile phase 100% hexane?

Controlling the water content of the mobile phase is desirable for reproducible results. Working with dry solvents is usually difficult and results in very long equilibration times. Using solvents which are fully saturated with water causes water to build up in the pores of the column. A common approach is to use ‘half-saturated’ solvents. Add 1 or 2 mL of water to 500mL of dry solvent and stir for 30 minutes, then remove excess water and combine with 500mL of dry solvent. Use this as your mobile phase and your equilibration should be quicker (although it may still take a few hours). If you can dedicate the column to your method then equilibration should be quicker when you use the column again in the future.

A possible solution for shifting retention times and equilibration problems that I have heard works well (but have not personally tried) is to use a magnetic stirrer to stir the mobile phase gently throughout the run, making sure not to place it close to the detector since it may cause baseline ripples.

Another potential source of water is your sample, if you suspect it may contain water then it is a good idea to dry it with anhydrous sodium sulfate of a high purity grade.”

Lab Tech Guy on a Delicate Problem

2010-07-13T12:40:00.003+01:00

Lab Tech Guy on a Delicate Problem

View more presentations from Oona McPolin.

Help on: Investigating Out of Specification (OOS) Results

2010-07-12T15:37:00.004+01:00

MTS HELPDESK

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:
“If any sample fails in testing, i.e. OOS or OOT, then what sort of thing does an analyst have to do, and what is basic difference between OOS &OOT?”

Answer:
“The difference between out of specification (OOS) and out of trend (OOT) results was discussed in a previous MTS helpdesk enquiry. I recommend that you refer to the FDA Guideline ‘Investigating Out-of-Specification (OOS) Test Results for Pharmaceutical Production’ for a detailed description of the procedure for investigation of an OOS or OOT result.”

Tips to Enhance your Learning Experience

2010-07-08T12:34:00.006+01:00

LEARNING AT WORK

When you attend a training course you hope to improve your knowledge and skills on the subject matter in question so that you can use your newfound competence to do your job better, and potentially enhance your career prospects. No matter how good training is, there are ways in which you can make the most out of the learning experience. The following tips are based on my experience of delivering and evaluating training. They should help you to retain what you learn on your training course and thus enable implementation of the learning in your day to day tasks.

1. Pre-training preparation
It is worth taking some time before you go on the course to think about exactly why you are going on the training and what you hope to get out of it. This means that you arrive at the training with clear expectations and can make sure that these are met. It also gets you thinking about the subject matter in terms of what you already know, providing a good starting point for improving your knowledge. Even if you have been ‘volunteered’ for the course you will still want to get the most out of the time you invest in attending so some preparation will pay off.

2. Take notes in your own words
How many times have you attended a presentation where the presenter says ‘Don’t worry, you don’t need to write anything down, there are handouts’? Research has shown that making some notes in your own words will help you to retain what you have learned. This doesn’t mean that you have to write down everything that you hear. Simply take down some quick notes about the main points of the presentation. The important thing is that it is in your words. This will help you to retain what has been said. Also, reading through these notes later will help you to remember the content.

3. Ask questions
Throughout your training it is important that you have a clear understanding of the content, so don’t be afraid to ask questions. You are in a learning environment so you are not expected to have the answers. Most trainers will encourage questions since it gives an opportunity for interaction and allows them to gauge how well their training is working.

4. Participate fully in the training
Most training events will include some type of session where you get a chance to do something other than just listen to the trainer. This may be in form of exercises, discussions, case studies, workshops, group exercises etc. These sessions allow you to apply what you have learned so try to throw yourself into them to get the most out of the experience. If you find the session is a bit uncomfortable (role-play springs to mind) then try to focus on what you want to get from the training.

5. Review what you have learned
At the end of the training you will probably summarise what you have learned with the trainer and may have an assessment to measure the learning. This review of the main points of the training is very useful for helping you to retain your new knowledge and skills. You should also use this review to visualise how you will apply your learning when back in work. If you can plan how you are going to use the training then it is more likely to happen.

6. Apply learning as soon as possible
When you get back to work look through all the notes associated with the course and remember the material that was covered. Then try to apply the learning. This will reinforce the learning so that you are less likely to forget.

MTS Recommends... QbD for Chromatography

2010-07-07T13:32:00.001+01:00

‘Practical Implications of Quality by Design to Chromatographic Method Development’ by Mike McBrien in Chromatography Today, Volume 3, Issue 2, June 2010

A good discussion of the practical implications of applying Quality by Design to chromatographic methods.

MTS Recommends... Headspace GC for Pharmaceutical Analysis

2010-07-07T13:12:00.002+01:00

'Experimental Considerations in Headspace Gas Chromatography' by Laila Kott, Hong Ming Chen in Pharmaceutical Technology, Volume 34, Issue 5, pp. 76-79 May 2, 2010

In this article a case study of amines is used to consider the important experimental parameters when developing a GC headspace method.

iLearn: HPLC Training by e-Learning

2010-06-26T18:26:00.002+01:00

Help on: Number of Injections for HPLC Columns

2010-06-14T11:18:00.000+01:00

MTS HELPDESK

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:
"How many injections should I expect to get out of my reversed phase C18 HPLC columns?"

Answer:
“Put simply, it depends. This is probably not the definitive answer you were hoping for but there are a number of different factors which influence the total number of injections that you can expect for a HPLC column. These relate to the actual column in question, the way you are using the column (the method operating conditions), and also the nature of the sample which is being injected.

Factors relating to the column include the type of packing, e.g. silica, zirconia, etc., and how the stationary phase, in this case C18, is bonded to the packing. Factors relating to the operating conditions include the pH, temperature, and mobile phase components such as solvents and buffers. Factors relating to the sample include the cleanliness of the sample, the volume, pH, impurities present and the nature of the actual sample molecules. ‘Clean’ samples are usually straightforward preparations such as standards, simple drug formulations and mixtures, whereas ‘dirty’ samples include biological fluids and environmental samples.

You can maximise the number of injections for a column by using it within its recommended operating conditions, e.g. pH and flow rate, and by flushing the column routinely to remove strongly retained impurities. Use of a guard column should also help to prolong column life.

As a rough guide you can expect to achieve at least 1000 injections if the column is used within its recommended range of operating conditions and the sample being injected is ‘clean’. You may even be able to perform 5000 injections for some methods. If using ‘dirty’ samples then you should expect to achieve less than 1000 injections but the actual number can vary considerably and may even be as low as 50 injections.

Of course if you do not count the number of injections performed on your columns then you will not know how many injections are possible. It is helpful to keep a record, if only for budgeting purposes in these lean times. Modern chromatography data systems and autosamplers make it simple to count injections for a particular column as long as you keep a note of a column identifier such as serial number.”

iLearn: e-Learning HPLC Training Course

2010-06-11T13:45:00.025+01:00

Mourne Training Services is pleased to announce that our e-Learning HPLC training course is now available in a distance learning option: iLearn. This course introduces the important concepts of HPLC and will enable you to fully understand: the different types of HPLC and how they differ; the different types of available HPLC columns and how they differ; why there are so many different mobile phase components and the ways in which they are combined to effect a chromatographic separation; and how components such as the pump, injector, detector and processor are combined to make a HPLC instrument.

The course is delivered using our virtual environment for learning, e-MTS, and consists of:

A series of training videos (totalling over 3 hours where each video is approximately 25 minutes),
Exercises on which you will receive feedback from your personal tutor and,
An assessment which enables accreditation of the training - It is recognised by the Royal Society of Chemistry for the purposes of continuing professional development.

The cost of this course is only GBP £125 + VAT and includes a copy of our training book, An Introduction to HPLC for Pharmaceutical Analysis (which normally costs GBP £29.27). This is great value for a course which equates to a full day of training.

So, how does it work?

All you need is access to the internet. The training is undertaken over a period of 1 week. You sign up and select a week which suits you. Each day training videos and exercises are uploaded to your virtual classroom which you can watch and submit answers for the exercises. This allows you to access the training materials at a time which suits you within the 24 hour period (you should allow about 90 minutes since there are 2 videos, each of length approximately 25 minutes and the exercises should take about 40 minutes). At the end of each session feedback from your personal tutor and full solutions for the exercises is sent to you by email. The assessment is completed on the last day and a certificate is sent out for your training records.

Contact us if you would like to sign up for this training course or if you would like more information. Full details will be posted on the MTS website soon.

HPLC Calculator for working out how much mobile phase to prepare - Gradient Methods

2010-06-01T09:03:00.004+01:00

PEAK SOLUTIONS
A resource for chromatographers

As promised, the MTS HPLC calculator can now work out how much mobile phase you need to prepare for gradient methods. Simply fill in the gradient table making sure to match up A, B, C & D to the solvent lines on your HPLC instrument and include any solvent holds and re-equilibration steps at the end/beginning of each injection.

Let us know your suggestions for HPLC calculations that you would like to be added to the calculator.

Help on: Selecting Columns for HPLC

2010-05-26T13:33:00.007+01:00

MTS HELPDESK

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:
“Can you advise me on how to select a column for a particular molecule?
e.g. Molecule is acidic in nature; Molecule is basic in nature; Molecule is neutral in nature; In which cases would we prefer cyano and amino columns; When we should use an amino column for the analysis of sugars on a Refractive Index detector; Why a lot of noise is produced which is not produced while using a union; What is the best solvent for preservation of amino columns, excepting hexane?"

Answer:
“The process of selecting a column for a particular analysis is quite complex and requires a good knowledge of HPLC method development. I recommend the book ‘Practical HPLC Method Development’ by Lloyd Snyder, Joseph Kirkland and Joseph Glajch or, if you can, attend a good training course on HPLC method development such as those offered by MTS. As a general rule the type of column that you select will depend on the type of HPLC that you are using and the selection of the type of HPLC is based on the polarity and molecular weight of the molecule you wish to analyse. Types of HPLC include: partition, adsorption, size exclusion chromatography (SEC) and ion exchange chromatography (IC). Of these partition in the reversed phase (polar mobile phase) is the most commonly used. Acidic, basic and neutral molecules may be analysed using all these types of HPLC and so many columns can be used. Sorry that my answer cannot be more precise but the fact is that it is not just the acidic or basic nature of the molecule but also its other features that dictates the choice of column. My first choice with a new molecule is reversed phase partition HPLC if possible, using a C18 column and only move to other phases if necessary.

Both amino and cyano columns may be used in reversed phase and normal phase modes. Amino columns are extensively used for the analysis of sugars and saccharides. Cyano columns can be used for a range of molecules. Cyano offers an alternative selectivity to alkyl columns such as C18 and C8 but in past it has been difficult to obtain reproducible chromatography using cyano columns. Recent developments in column technology have improved this phase. You might try a cyano column at the outset of a method development to investigate which phase gives the best separation for a particular set of analytes. There is no specific best use for cyano columns in the way that amino columns are used for sugars.

I would agree that your problem with detector noise appears to be related to the column since it is absent when the column is absent, however the union does not provide back pressure comparable to a column and it may be that the problem is in the HPLC system. Can you try other columns? If it is due to your column then it may be contaminated and need cleaned, since RI detection is universal it is very sensitive to any compounds in the mobile phase. Unfortunately RI detection is very prone to baseline instability, controlling the temperature of the column may help.

I suggest isopropanol for the storage of your amino columns.”

MTS Recommends... ‘Kinetic Plots Made Easy’

2010-05-21T13:37:00.004+01:00

‘Kinetic Plots Made Easy’ by Uwe D. Neue in LCGC North America, November 2009

Kinetic plots, sometimes called ‘Poppe plots’ have become a popular way to compare HPLC columns. In particular they have been used extensively in discussions of sub 2 micron particle size materials. This article by Uwe Neue presents a simple description of how kinetic plots are generated and why they are useful.

Book Reviews in 'Chemistry World'

2010-05-07T09:16:00.013+01:00

The training books from Mourne Training Services have received very positive reviews in this month's issue of the Royal Society of Chemistry magazine, Chemistry World.

An Introduction to HPLC for Pharmaceutical Analysis

Chemistry World review:
"A brief well-written guide and resource to assist the analyst in the use of HPLC in a pharmaceutical analysis environment."

Validation of Analytical Methods for Pharmaceutical Analysis

Chemistry World review:
"A nicely written handbook on how to perform validation of methods used in pharmaceutical analysis."

Read the reviews on the RSC website

Ask Lab Tech Guy is now on YouTube!

2010-05-06T10:55:00.002+01:00

Ask Lab Tech Guy has made his first appearance on YouTube. The popular reply to the question on HPLC wash solvents has been uploaded, accompanied by a soundtrack of his choosing. Let us know if you have any comments. New Ask Lab Tech Guy questions coming soon!

HPLC Calculator for working out how much mobile phase to prepare - Isocratic Methods

2010-05-04T13:59:00.007+01:00

PEAK SOLUTIONS
A resource for chromatographers

The HPLC calculator from MTS has been updated to include working out how much mobile phase you need to prepare for your analysis. Currently you can use it to calculate mobile phase requirements for isocratic methods and next month it will be revised to include gradient methods.

Let us know your suggestions for HPLC calculations that you would like to be added to the calculator.

Help on: Peak Purity by PDA

2010-05-04T11:18:00.010+01:00

MTS HELPDESK

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:
“I want to know why we are not able to determine Peak purity by use of a UV detector instead of Photodiode Array detecion (PDA)?”

Answer:
“The HPLC detectors, variable wavelength UV detectors and photodiode array detectors, both work on the principle that molecules of interest are detected by the absorption of UV light. However the design of the instruments differs.

In a variable wavelength detector the instrument can be set up to monitor a particular wavelength and thus a chromatogram is obtained which corresponds to the absorption of UV light at that wavelength at each time point during the injection. Some UV detectors are capable of acquiring multiple wavelengths and so chromatograms can be obtained for several wavelengths.

A photodiode array detector collects information on all the wavelengths within a range specified by the operator, thus obtaining a UV spectrum for each time point during the injection. An individual wavelength may be extracted so that a chromatogram can be displayed for a particular wavelength of interest if required.

To measure peak purity it is necessary to be able to inspect the UV spectrum at a number of time points across the peak of interest. In the example shown in Figure 1, spectra are extracted at retention times of 8.11, 8.21 and 8.31. Since the UV spectra are not comparable it can be concluded that the peak is not pure. Only the photodiode array detector is able to provide this information.

Figure 1

A word of caution, although peak purity by UV can be very useful it cannot conclusively prove that a peak is pure. Molecules which have very similar structures may produce identical UV spectra and thus cannot be differentiated using this technique.”

Help on: Out of Specification (OOS) vs Out of Trend (OOT) Results

2010-05-03T16:44:00.004+01:00

MTS HELPDESK

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:
"I want to know how to differentiate between Out of Specification (OOS) results and Out of Trend (OOT) results?”

Answer:
“An Out of Specification (OOS) result refers to any test result that falls outside the specifications or acceptance criteria established in drug applications, drug master files (DMFs), official compendia, or by the manufacturer. The term also applies to all in-process laboratory tests that are outside of established specifications. An Out of Trend (OOT) result is a result that does not follow the expected trend, typically in a stability study. This can be either in comparison with other batches or with respect to previous results collected during a stability study. The result is not necessarily OOS but does not look like a typical data point. Both OOS and OOT results can be investigated using the same procedure.”

Help on: Use of Caffeine for Wavelength Accuracy

2010-04-30T10:29:00.003+01:00

MTS HELPDESK

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:
“Can you tell me why caffeine is used for the calibration of HPLC systems? I know it shows two maxima (205 nm & 272nm) & minima at245, apart from this property why is it used?”

Answer:
“The main reason that caffeine is used for the wavelength accuracy test is as you have already identified, it contains chromophores which absorb at the wavelengths you have quoted. Other reasons are that it is easily available and relatively safe to handle, this makes it an ideal choice for this test. In addition organisations which certify calibration standards, such as NIST in America, specify the use of caffeine for the test and thus the calibration can be performed in a NIST-traceable manner.”

MTS Recommends... Reduced Robustness Testing for Analytical Methods

2010-04-23T10:26:00.007+01:00

‘Reduced method robustness testing of analytical methods driven by a risk-based approach’ by Phil Borman, Marion Chatfield, Patrick Jackson, Alice Laures, and George Okafo in Pharmaceutical Technology Europe, Volume 4, Issue 22

This article details an interesting approach to reducing the number of factors investigated during a robustness study. Basically the factors which are chosen to be studied in an experimental design are further reduced by performing a risk analysis of each factor and combining factors where possible. As we hear more and more about addressing method robustness earlier in the method development process as part of Quality by Design, more efficient approaches to performing these studies become desirable.

The thing that stands out for me about this article, and indeed all discussions of robustness, is that although the statistics and design of experiments is very important (and I think this article describes these quite well) the most important contribution to robustness testing comes from the experience of the analyst who understands the actual effects of the factors involved and can identify those which are most important. The risk assessment performed in the case study described in this article was performed by ‘GC experts’. Without this contribution the studies cannot produce meaningful results.

MTS Recommends... Using %RSD Correctly

2010-04-23T10:08:00.002+01:00

‘%RSD: Friend or Foe? When to use percent relative standard deviation—and how to do so correctly.’ By Lynn D. Torbeck in Pharmaceutical Technology, January 2010, Volume 34, Issue 1, pp. 37-38

In most analytical laboratories percent relative standard deviation
(%RSD) is used extensively as a measure of variability. It is important to understand how to use, and how not to use, this useful statistic. This article from Lynn Torbeck provides a good guide to proper use.

Lab Tech Guy on the Effect of Heating Volumetric Glassware

2010-04-09T16:55:00.004+01:00

Lab Tech Guy on the Effect of Heating Volumetric Glassware

View more presentations from Oona McPolin.

And the winner is...

2010-04-09T11:57:00.002+01:00

Congratulations to Martin from Stockport in England who has received a limited edition Ask Lab Tech Guy T-shirt for submitting a question on the effect of heating on volumetric glassware.

You can view Lab Tech Guy's response here.

HPLC Calculator for Mobile Phase Buffer Strength

2010-04-06T11:43:00.008+01:00

PEAK SOLUTIONS
A resource for chromatographers

The HPLC calculator from MTS has been updated to include working out how much buffer salt to weigh out when preparing mobile phase.

Let us know your suggestions for HPLC calculations that you would like to be added to the calculator.

Help on: TOC vs HPLC for Cleaning Validation

2010-03-31T23:18:00.013+01:00

MTS HELPDESK

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:
"I want to know whether TOC analysis is better than HPLC for cleaning validation?"

Answer:
"The use of Total Organic Carbon (TOC) as an analytical test for cleaning validation has increased rapidly in recent years. This is due to a number of factors including:

It is very easy to use;
The actual testing can be carried out very quickly;
TOC testing requires very little method development and is easy to validate;
One method can be used for all cleaning samples; and
It is a cost effective analytical technique.

TOC measures all the carbon in the sample and is thus a non-specific technique; this can be viewed as both an advantage and disadvantage. On one hand it means that all carbon which can be oxidised under TOC conditions can be detected and thus the result provides a measure of all contaminants. On the other hand it does not determine the amount due to the active substance. The result may be considered as ‘worst case’, assuming that the entire residue is due to the active substance.

To use TOC for cleaning analysis, you need to establish that the contaminating material is organic and contains carbon that can be oxidised under the TOC test conditions; some organic compounds cannot be reliably detected using TOC. Use of TOC requires that the contaminant is at least slightly soluble in water.

As to which is better, TOC or HPLC, it will depend on the contaminating substances you are testing for. The advantages of TOC make it preferable but there are times when it will not be suitable, in which case HPLC may provide a better option. The effort involved in validation of cleaning means that it is not usually beneficial to switch established processes from HPLC to TOC. However for new processes TOC may provide a good solution.”

MTS Recommends... LC Trends at Pittcon

2010-03-25T12:13:00.004Z

‘New Chromatography Columns and Accessories at Pittcon 2010: Part I’ by Ronald E. Majors in the March 2010 issue of LCGC North America.

Every year after Pittcon, Ron Majors writes a great summary of the trends and new introductions at the conference relating to chromatography. This provides an easy way to keep up to date with the latest developments in LC so that you can evaluate their usefulness in your laboratory.

This year Ron observes ‘that there was a further increase in columns and accessories designed to work with ultrahigh-pressure liquid chromatography (UHPLC) products’. New column introductions included more sub 2 micron columns for UHPLC but also a number of manufacturers have introduced superficially porous particles (SPP) which have a solid core of non-porous silica coated with a thin shell of silica and can achieve improved efficiency comparable to UHPLC using standard HPLC systems. There were over 57 new reversed phase bonded phases added in Pittcon 2010, dominated, as in previous years, by silica based columns with C18 bonded phases.

All these identified trends in the very latest LC developments are already incorporated into training courses available from MTS so you can be sure of up to date and relevant training when you register for one of our courses.

Help on: Developing a HPLC Method for Ertapenem

2010-03-18T11:53:00.012Z

MTS HELPDESK

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:
"Can you suggest a HPLC method for Ertapenem Injection?”

Answer:
“A quick search on an internet search engine reveals a multitude of HPLC methods in the literature developed for the antibiotic Ertapenem in a variety of samples, but mostly biological matrices. This type of information can be very useful when developing a new HPLC method: it’s reassuring to know that HPLC can be used for analysis of your molecule and; the conditions used by other operators can point you in the direction of the most suitable conditions for your requirements. I recommend a 5 step strategy for HPLC method development, as taught on the MTS training course ‘HPLC Analytical Method Development for Pharmaceutical Analysis’. The 5 steps involved are:

Step 1: What are your goals?
Step 2: What do you already know?
Step 3: What sample(s) will you use to develop the method?
Step 4: What conditions will you use for the method?
Step 5: What method parameters will you use?

Step 1
In the case of Ertapenem injection the first step involves a consideration of the goals of the required method. This simply involves establishing the following for the required method: the analyte(s); the type of method (e.g. assay, impurities, etc.); the nature of the samples; and the purpose of the method (e.g. stability analysis, clinical study, etc.). You will also want to evaluate the level of effort you want to invest in the method and the resources that you have available, together with any specific requirements, such as a short analysis time.

Step 2
The second step involves assessing all the information you currently have on the analyte(s) of interest. For example, the structure of Ertapenem is shown below.

The size of the molecule and the nature of its functional groups means that it will probably be amenable to reversed phase HPLC and this will usually be our first preference. From the structure we can deduce that it is an ionisable compound since it contains amine groups and carboxylic acids groups and thus requires a pH controlled mobile phase for reproducible chromatography. The actual pKas of the molecule may be helpful to select a suitable buffer pH but this information is not always known. You can use predictive software to calculate pKa if it is available. The aromatic nature of the molecule together with the presence of double bonds indicates that the molecule will be suitable for detection by UV.

Other information that you may have access to relating to previous work, sample preparation, and impurities (to give just a few examples) should be evaluated fully. From a brief literature search I found a method for Ertapenem using a C18 column with a mobile phase of methanol and phosphate buffer at pH 6.5 using UV detection at 300nm. This could provide a convenient starting point for method development.

Step 3
The third step involves considering the nature of your samples which will be analysed by the new method and considering what samples you will use for the actual method development. This is likely to be more complex when you have multiple analytes, e.g. an impurities method. You may need to use specific samples containing analytes of interest or you may need to degrade your main analyte to obtain samples of degradation products.

Step 4
The fourth step involves a simple gradient run to test combinations of stationary phase and mobile phase for the analyte(s) of interest. In complex mixtures a number of different combinations may be tried to investigate which gives the best separation. Each set of conditions will be chosen to provide very different environments for the sample and thus provide a range of different selectivities. For a simple analysis involving a single analyte a good first choice is to run a gradient scouting run on a familiar C18 column, in the case of Ertapenem the conditions found in the literature would be a suitable choice using a gradient of 5 to 90% methanol. The information from this run indicates whether a gradient or isocratic run will be possible. If you have a single analyte and no interferences then it is likely that you will be able to run an isocratic method, the most suitable mobile phase proportions can be determined from the gradient scouting run.

Step 5
In the final step the most promising conditions from the scouting experiments are investigated fully and the best mobile phase conditions selected. Other system parameters such as temperature, injection volume, flow rate and column dimensions can be optimised.

This 5 step strategy allows the analyst to develop a suitable method for their purpose, maximising the potential for success by taking a flexible but structured approach to the task."

UTrain: Online HPLC e-Learning for in-house use

2010-03-02T16:02:00.010Z

Mourne Training Services, the specialist in training relating to pharmaceutical analysis, has launched a new training solution: UTrain - an online e-Learning HPLC training course designed for in-house use. UTrain combines theoretical knowledge with practical know-how to provide workplace learning which translates straight to the laboratory. It is very reasonably priced and, with a standard licence, a full day training course can cost as little as £25 +VAT per person. You can try it out; just visit the MTS website to set up your free trial.

This short video summarises the UTrain package:

The UTrain package consists of three key elements:

1. Training videos are used to provide all the necessary subject information. They can be used either by an individual as self-paced learning or in a group training setting. The videos can be viewed as often as required by individual learners. Click here to view a sample of a training video.

2. Exercises and instructions for practical experiments (where applicable) are provided so that learners can apply their new skills. Detailed solutions for the exercises are also provided. The exercises and solutions are designed to be printed off as required.

3. The assessment questions used to measure the learning for the training are administered in an e-learning module and are combined with a review of the learning. A training certificate is awarded by Mourne Training Services on correct completion of all the questions. The certificate contains a space for an in-house signature to certify that all the training was completed satisfactorily.

All three UTrain elements are accessed online via e-MTS, our virtual environment for learning. The UTrain package is comprised of a series of modules, where four modules is equivalent to a full day's training. Individual modules may be selected to suit your training needs.

The first available UTrain modules together make up an introductory HPLC training course. This e-learning HPLC course is the best possible starting point for any HPLC operator to develop their knowledge and skills. The course is based on the MTS course ‘An Introduction to HPLC for Pharmaceutical Analysis’ and is recognised by the Royal Society of Chemistry for the purposes of Continuing Professional Development. Further modules on HPLC, including troubleshooting HPLC and HPLC analytical method development, as well as modules on the validation of analytical methods, will be available in the future.

Visits the MTS website or contact us for more information.

MTS Recommends... A History of LC-MS

2010-03-01T09:46:00.002Z

‘The fascinating history of the development of LC-MS; a personal perspective’, by Frank Pullen, in the March 2010 issue of Chromatography Today.

HPLC Calculator: How can we help?

2010-02-16T15:40:00.004Z

PEAK SOLUTIONS
A resource for chromatographers

So far, our free HPLC calculator allows you to work out column equilibration times and convert between different pressure units. We would like to add to the calculator to provide a genuinely useful tool. Have you any suggestions which would make your life just a little bit easier? Suggestions so far include calculation of mobile phase, both the amount required for an analysis, particularly gradients, and weighing out the mobile phase additives. Would this help you? Please let us know using the comments on this post.

Help on: Installing an Analytical Balance

2010-01-21T10:30:00.013Z

MTS HELPDESK

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:
"Recently I have ordered a new analytical balance for my lab. I want to know what precautions I will have to take for proper installation. And as for calibration, up to what range of weights should I verify?”

Answer:
“The instructions supplied with a new analytical balance are usually quite comprehensive and provide information on how to set up the balance correctly taking into account suitable location and internal calibration capabilities. Some other considerations (as provided by Scientech Balances) are as follows:

Set up the balance on a firm weighing table and even surface. Turn the adjustable feet until the balance is level using the spirit level/bubble indicator as guidance.
Avoid exposing the balance to vibrations during weighing. Corners of rooms are usually less prone to vibrations.
Best operating temperature is about 20°C/68°F at about 50% relative humidity. If you transfer the balance to a warmer area, make sure to condition the balance for about 2 hours at room temperature, leaving the unit unplugged from AC power. This is because the moisture in the air can condense on the surfaces of a cold balance whenever it is brought into a substantially warmer place. Never expose the balance to extreme moisture over long periods.
Protect the balance from drafts that come from open windows or doors. Heat and air-conditioning ducts will also product draft resulting in unstable readings.
Use caution when using weigh containers made of plastic since plastic is more prone to holding a electrostatic charge. If the samples being weighed hold static electricity an aftermarket static ionizer maybe needed for ionization for static removal. Static charges tend to develop when different materials rub against one another. Some materials can pick up excess electrons, resulting in negative static charges while other materials give up electrons, resulting in a positive charge. If the charged material is non-conducting (as are films, glass lenses and plastics), then the static charge remains.
Allow sufficient space around the balance for ease of operation and keep away from radiating heat sources.

Modern analytical balances typically include internal calibration features but regularly checking the accuracy against external calibrated masses is good practice. The calibration weights which you should verify will depend on the operating range of the balance so choose weights which bracket the range of weights which you will use the balance to measure and perhaps one or two spaced out throughout the range."

Help on: Troubleshooting in pharmaceutical analysis

2010-01-20T16:38:00.008Z

MTS HELPDESK

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:
"What are the general problems occurring in HPLC, GC and spectroscopy during operation in the pharmaceutical industry?”

Answer:
“A full discussion of the general problems which can occur when operating HPLC, GC and spectroscopy for applications in the pharmaceutical industry could run to several books! In my experience the greatest problems are usually due to a lack of experience, knowledge and skills on the part of the operator (that’s probably the answer you would expect from a trainer!).

Modern analytical instruments have developed to such an extent that they can be fairly simple to use, with sophisticated software that lulls you into a false sense of security about your own abilities. It pays to remember what is actually happening during analysis and a good background in the theory of the technique will enable effective troubleshooting.

If you consider HPLC, setting up a run and then processing it can be quite a straightforward task. But, if the results are not as expected then a solid understanding of the method will help to assess the available information and diagnose the problem. This knowledge extends to an understanding of how the instrumentation works, an example being the selection and use of a suitable wash solvent to prevent carryover of your analyte from one injection to another. Most often, problems will occur when developing methods for new compounds, since that is when knowledge and skills are tested most.

The answer to building suitable skills and knowledge is primarily through effective training (which Mourne Training Services is always happy to provide) but developing experience after the training is equally important. Making sure that learning is fully implemented and kept up to date is a significant challenge for both individuals and managers in the pharma industry. Troubleshooting problems actually helps to develop expertise.

There is a wealth of information to be found on the web relating to troubleshooting of all these techniques. For example, type ‘HPLC troubleshooting’ into a search engine and you will be presented with numerous helpful resources. I recommend chromatography magazines such as LCGC which has regular troubleshooting columns for both LC and GC. There are also many books available which provide more in-depth knowledge regarding troubleshooting.”

MTS Website Updates

2010-01-19T09:04:00.005Z

The MTS website has been updated!

You can now access more information about our new online training package for in-house use: UTrain. This includes a request form for a free trial of the package to allow you to try it out.

We have also updated the free training resources page including a Useful Links section. Please send us your suggestions for links.

Lab Tech Guy on resolutions for the New Year

2009-12-18T15:31:00.003Z

Lab Tech Guy on resolutions for the New Year

View more presentations from Oona McPolin.

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

2009-12-17T12:29:00.009Z

LEARNING AT WORK
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:

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.
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.

Feedback from free HPLC training video

2009-12-16T16:26:00.012Z

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.

HPLC calculator for pressure conversion

2009-12-01T09:00:00.001Z

PEAK SOLUTIONS
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.

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

2009-11-30T12:25:00.011Z

MTS HELPDESK

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:
"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?”

Answer:
“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.

Thoughts on applying QbD to analytical methods

2009-11-27T13:42:00.006Z

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.

Quality by Design for analytical methods

2009-11-11T16:30:00.012Z

ANALYTICAL TOPICS

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.

References:
1. 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.

Tips for trainers - Icebreakers

2009-11-10T11:55:00.009Z

LEARNING AT WORK
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.