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.”