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’.

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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.