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Critical Evaluation of HPLC Methods

A recent chromacademy webcast looked at the critical evaluation of HPLC methods. This involves examining every facet of the HPLC method, deciding what impact it has on chromatography, and altering these parameters to provide the desired chromatographic results.

Our viewers posed some interesting questions during the live Q&A segment of this webcast, so we wanted to share some of them here with you to hopefully encourage some critical evaluation of your own methods.

Watch the Critical Evaluation of HPLC Methods webcast here »

How do you determine t0?


t0 can be determined in a few ways:

  1. Using the time at which a baseline disturbance is seen due to differences in absorbance or refractive index properties as the injection solvent passes through the detector

  2. By determining the retention time of a non-retained compound which has no affinity for the stationary phase - in reversed phase HPLC uracil can be used, and hexane can be used for normal phase HPLC.  Uracil is the most popular t0-marker, because it has good UV response and is unretained for mobile phases which are typically used for most column test conditions i.e. ≥60% methanol/water. Thiourea is good marker if weaker mobile phases are required for column testing

  3. By using the column dimensions - For 4.6 mm i.d. columns the estimate is simple, the column volume, VM, can be estimated by multiplying the column length (in cm) by 0.1.  Therefore, a 150 x 4.6 mm i.d. column would be 15 cm long, so VM ≈ 0.1 x 15 ≈ 1.5 mL. This should be within about 10% of the true column volume.  To convert column volume to dead-time (t0), divide by the flow rate, F (for this example we will use F = 2 mL/min) giving,

t0 ≈ (1.5 mL) / (2 mL/min) ≈ 0.75 min. 

For other column dimensions use the equations:

(all units in cm)


Is elution strength greater for acetonitrile or methanol in reversed phase HPLC?  Will acetonitrile cause faster elution of peaks than methanol?

Yes, acetonitrile is a stronger eluent than methanol in reversed phase HPLC; therefore, analytes will be eluted from the column faster when using acetonitrile.  Changing the organic modifier in a mobile phase can have a dramatic impact on chromatographic results - overall analysis time and analyte retention and selectivity will be affected.  The amount of organic modifier can also be modified to change analyte retention.   

Changing the organic modifier


See the following CHROMacademy pages for further information.

Reversed Phase Mobile Phase Solvents »

Mobile Phase Strength and Retention »

Changing the Organic Modifier »

What about when working with the non-ionizable analytes, do we still need to control pH?

As the analytes are non-ionizable (do not have ionizable functional groups) pH will not affect their ionization state.  The only time that pH control would be used with non-ionizable analytes, is to lower the pH in order to render the surface silanol groups in their neutral form so that they cannot interact with polar (but non-ionizable analytes), which would result in poor peak shape (often tailing).

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How do you determine the pKa value for an unknown compound?  What if it has more than 1 pKa value?

Calculated pKa values of compounds can be found at or

pKa determination for an unknown may not be entirely straightforward, and there are many methods discussed in the literature including, titration, potentiometry, conductometry, voltammetry, calorimetry, NMR, electrophoresis, HPLC, partition and distribution, solubility, UV-visible spectrometry, fluorometry, polarimetry, kinetic method, and computational method.


Many analytes have more than one pKa, which can make controlling their ionization state more complex, and you may not be able to completely render your analyte in its neutral form to promote retention in reversed phase HPLC.  In this case other modes of chromatography such as HILIC may be more appropriate, or you may have to utilize an ion pair reagent. 

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How can I reduce peak fronting?

peak fronting

Peak fronting has many causes such as, channeling in the column packing, inadequate sample preparation, void at the column inlet, sample diluent/eluent mismatch, large extra-column dead volume, etc., but is most commonly seen when there is a mass or volume overload of the sample (column overload), therefore, if you see peak fronting try reducing the amount of sample you inject on to the system by either reducing the concentration or the volume.  Depending on your sample diluent and your mobile phase you will have to consider your injection volume. 

Analyte loading

Table: HPLC column loading.  µg on column.

Analyte loading

Table: Injection volume based on sample diluent.

The sample diluent (injection solvent) should also be taken into account.  If the diluent is more highly eluotropic (contains a higher percentage of organic modifier, different buffer strength, different solvents, additives etc.) then the analytes will behave differently as they enter the column.   

The figure below demonstrates what happens the when the sample diluent is more highly eluting (contains more organic for reversed phase separations) than the eluent.  One can easily see that some of the analyte molecules elute through the packed bed until the sample solvent is diluted out by the eluent, whereupon the analytes partition more like their counterparts who were associated with the eluent rather than the diluent when they entered the column.   One can imagine that this would cause fronting or splitting issues when the analytes elute from the end of the column.

Analyte band

Figure: Travel through the column after injection of a sample solvent containing more organic than the eluent.

See the following CHROMacademy pages for further information.

Sample Diluent Effects in HPLC »

What should I consider when choosing the ideal ion pair reagent to improve the separation of compounds in a mixture?  What molarity should the ion pair reagent be?

The ideal ion pair reagent is no ion pair reagent, if you can get away without using one please do.  If you are using a standard HPLC-UV system it is often possible to arrange that your stationary phase is of a more polar nature and able to retain analytes which are ionized.  

With an ion pair reagent, retention is governed by the length or size of the alkyl chain on the ion pair reagent but you have to titrate the concentration of the ion pair to determine the correct conditions for your analysis.  As the concentration of the ion pair reagent is increased, the retention of the analyte increases until you reach a certain value then the analyte retention value will begin to decrease again (this is due to the ion pair reagent excluding the analyte from the stationary phase surface because it is ion pairing with itself).  Typical working concentration ranges are between 0.5 and 100 mM. 

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Ion pair reagents are often used to reduce the effective polarity of the analyte.  For the analysis of very polar molecules other modes of chromatography such as HILIC may be preferable over the use of ion pair reagents.  If you are carrying out LC-MS you may get substantial ion suppression or see no signal at all if you use an ion pair reagent.


See the following CHROMacademy pages for further information.

Ion Pair Chromatography »

You may also enjoy
Webcasts & Tutorials

What HPLC Operators Need to Know: Part 1 »

What HPLC Operators Need to Know: Part 2 »

Quick Guides

Why Is pH Important for HPLC Buffers »

HPLC Extra Column Volumes »

Setting HPLC Chromatographic Parameters »

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Dr. Dawn Watson

This article was written by Dr. Dawn Watson.

Dawn received her PhD in synthetic inorganic chemistry from the University of Strathclyde, Glasgow. The focus of her PhD thesis was the synthesis and application of soft scorpionate ligands. As well as synthetic skills, this work relied on the use of a wide variety of analytical techniques, such as, NMR, mass spectrometry (MS), Raman spectroscopy, infrared spectroscopy (IR), UV-visible spectroscopy, electrochemistry, and thermogravimetric analysis.

Following her PhD she spent two years as a postdoctoral research fellow at Princeton University studying the reaction kinetics of small molecule oxidation by catalysts based on Cytochrome P450. In order to monitor these reactions stopped-flow kinetics, NMR, HPLC, GC-MS, and LC-MS techniques were utilized.

Prior to joining the Crawford Scientific and CHROMacademy technical team she worked for Gilson providing sales and support for the entire product range including, HPLC (both analytical and preparative), solid phase extraction, automated liquid handling, mass spec, pipettes, and laboratory consumables.

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