Help on: HPLC Dwell Volume and Dead Volume

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:

1. Remove the column from the system and use a short length of 0.010″ tubing to connect the injector directly to the detector.
2. 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).
3. Set the detector wavelength to 265nm.
4. 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.
5. 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

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

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

Preparation of Mobile Phase for HPLC

PEAK SOLUTIONS
A resource for chromatographers

The following is an excerpt from 'An Introduction to HPLC for Pharmaceutical Analysis' by Oona McPolin, 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’.

  Tweet

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

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

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

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

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

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

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.”

Free e-Learning Module on HPLC Instruments

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

'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.

MTS Recommends... From Molecule to Medicine

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

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

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:

1. Understand how HPLC works and the role of each component in an HPLC system
2. Understand how problems can arise in the individual components of an HPLC system
3. Implement measures which prevent problems occurring
4. Use a systematic problem-solving approach to HPLC troubleshooting
5. 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

Help on: Peak Purity Assessment

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.”

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

‘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

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: Retention Time Shifts in NP-HPLC

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

Lab Tech Guy on a Delicate Problem

View more presentations from Oona McPolin.

Help on: Investigating Out of Specification (OOS) Results

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

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

‘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

'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

Help on: Number of Injections for HPLC Columns

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

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:

1. A series of training videos (totalling over 3 hours where each video is approximately 25 minutes),
2. Exercises on which you will receive feedback from your personal tutor and,
3. 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

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

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’

‘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'

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

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

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

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

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

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

‘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

‘%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

Lab Tech Guy on the Effect of Heating Volumetric Glassware

View more presentations from Oona McPolin.

HPLC Calculator for Mobile Phase Buffer Strength

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

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

‘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

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."

MTS Recommends... A History of LC-MS

‘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?

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

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

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

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.