Peak purity analysis in HPLC and CEusing diode-array technology

Application

Abstract

In terms of quality control and for all quantitative analysis peak purity

is a major task, which can be addressed in different ways. Often only

peak shapes and chromatograms are taken into consideration but a

very elegant possibility without the need to use a mass spectrometric

detector is the comparison of spectra recorded with a diode-array

detector during the registration of a chromatographic peak. Due to the

differences of these spectra within a single chromatographic peak, its

purity can be decided easily. The Agilent ChemStation software offers

an easy to use tool to perform these tasks routinely. In this note, the

theoretical background of peak purity determination is presented as

well as the practical use of the ChemStation software for these purposes.

Mark Stahl

In the elution dimension SignalsA valuable tool in peak purityanalysis is the overlay of separa-tion signals at different wave-lengths to discover dissimilaritiesof peak profiles. The availability ofspectral data in the three-dimen-sional matrix generated by thediode-array detector enables sig-nals at any desired wavelength tobe selected and reconstructed forpeak purity determination afterthe analysis. A set of signals ca beinterpreted by the observer bestwhen, before being displayed, it isnormalized to maximumabsorbance or to equal areas. Agood overlap, where peak shapeand retention or migration timematch, indicate a pure peak whilea poor overlap indicates animpure peak, as demonstrated infigure 1.

In addition to overlaying signals,their ratios ca be calculated andplotted. The resulting ratiogramsare sensitive indicators of peakpurity (figure 2). Any significantdistortion of the ideal rectangularform of the ratiogram indicates animpurity. However, there are somelimitations to be considered whenusing signals for peak purity deter-mination: The UV-Visible spectra of both

the main compound and theimpurity must be well known inorder to select the most suitablewavelengths for the peak profilecomparison. This fact reducesthe applicability of this type ofimpurity detection to a routinelike search of known impuritiesin known main peaks.

ty detector, peak purity can only bejudged from the peak profile of thissignal. Peak profile however isinfluenced by a variety of parame-ters and depends heavily on chro-matographic or electrophoretic res-olution. Therefore peak puritydetermination based on the peakprofile of a single signal is a veryunreliable and insensitive method.This is especially true in CE, wheremismatched sample and bufferzones always result in peak skew-ing2. As a consequence, a secondapproach involving multiple wave-length detectors acquiring morethan one signal in parallel has beenadopted. Such detectors enableimpurities to be uncovered bymethods that involve overlaying sig-nals to compare peak profiles andcalculate signal ratios. These detec-tors have some disadvantageswhich will be discussed in the fol-lowing section. They can be elimi-nated easily by a third approachbased on diode-array technology1: on-line acquisition of UV-Visible

spectra during the peaks elution, several signals at different wave-

lengths in parallel, signal extracts from a 3-D data

matrix, containing spectral dataand separation signals, for peakpurity analysis..

Peak purity determination can beperformed in different levels, tai-lored to the complexity of the sepa-ration and the users needs.Detailed descriptions of the variouspeak purity routines for signals andspectra, such as normalization andoverlay, the mathematical opera-tions and the different displaymodes will be given in the follow-ing sections.

Introduction

An essential requisite of a separa-tion analysis is the ability to verifythe purity of the separated species,that is, to ensure that no coelutingor comigrating impurity contri-butes to the peaks response. Theconfirmation of peak purity shouldbe performed before quantitativeinformation from a chromato-graphic or electrophoretic peak isused for further calculations.Neglecting peak purity confirma-tion means, in quality control, animpurity hidden under a peakcould falsify the results or, inresearch analysis, important infor-mation might be lost or scientificobservations rendered void shouldan impurity remain undiscovered.Validated analytical methods usu-ally include the peak purity checkas a major item in the list of theirmethod validation criteria (table 1).

Techniques for peak puritydetermination

Several techniques are currentlyused for peak purity determinationin high performance liquid chro-matography (HPLC) and in capil-lary electrophoresis (CE)1. With aconventional single wavelengthdetector (or a monitor providingjust one single output signal) suchas a refractive index- or conductivi-

2

Method validation criteria

Selectivity (peak purity determination) Linearity Limits of detection and quantification Quality of data (accuracy and precision) Ruggedness

Table 1Peak purity determination a major criterion in method validation

The technique is not directlyapplicable to research work ormethod development. The riskof overlooking an unknownimpurity remains even when several wavelengths are selectedin parallel.

Peak purity determination in the thirddimension SpectraComparing peak spectra is probablythe most popular method to discover an impurity. If a peak is pure all UV-visible spectraacquired during the peaks elutionor migration should be identical,allowing for amplitude differencesdue to concentration. The resultsobtained by comparison of thesespectra against each other shouldbe very close to a perfect 100%match. Significant deviations canbe considered as an indication of impurity. Unfortunately theinverse is not necessarily true. Ifthe spectra are not significantlydifferent, the peak can still beimpure for one or more of threepossible reasons:1. The impurity is present in much

lower concentrations than thatof the main compound.

2. The spectrum of the impurityand the spectrum of the maincompound are identical or verysimilar.

3. The impurity completely coe-lutes or comigrates with the maincompound, with both havingexactly the same peak profile.

Any peak purity algorithm canonly confirm the presence ofimpurities and never proveabsolutely that the peak is pure.The likelihood of discovering animpurity rises with increasing dis-

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tinction between spectra and peakprofile, higher resolution betweenthe main compound and the impu-rity, and with increasing absoluteand relative concentration of theimpurity.

There are no hard and fast rulesas to which concentrations ofimpurities can be detected andwhich not. In general, less than 0.5- 1 % can be determined if thespectra are distinct enough. If thespectra resemble each other very

signal A signal B

impure pure

200

150

100

50

1.61.51.41.31.21.1

7.6 7.8 8.0 8.2 8.4 8.6Time [min]

7.6 7.8 8.0 8.2 8.4 8.6

Ratio A-to-B

mAU

Time [min]

Figure 2 Signal ratiograms for impure and pure peaks

signal Bsignal A

pure impure

Time [min] Time [min]7.5 8.1 7.5 8.1

signal A(offset)

signal B

Figure 1 Normalized signals for pure and impure peaks

closely, and the column or capil-lary system does not resolve theimpurity and main componentwell, then only 5 % impurity maybe feasible.

Before proceeding, the differencebetween the terms spectral impu-rity and peak impurity should beclearly defined. Spectral impurityindicates a distortion of the ana-lyte spectrum by the near - con-stant presence of backgroundabsorbance from solvents, and/ormatrix compounds and/or animpurity. Peak impurity, in con-trast, refers to a distortion of theanalyte spectrum by an additionalcomponent which partially orcompletely coelutes or comigrateswith the major compound3.

Background correction of the peakspectraBefore the spectra are used forthe peak purity analysis, theyshould be corrected for back-ground absorption caused by themobile phase or matrix compounds,by subtracting the appropriate reference spectra. Whether such abackground correction needs tobe applied depends on the separa-tion system employed. For isocraticseparations with a properly bal-anced diode-array detector, thesolvents constant spectral contri-bution will be eliminated by theautomatic subtraction of the sol-vent spectrum at the beginning ofthe run. For gradient separationswhere the mobile phases contri-bution to absorbance may changewith time, background correctionsshould be made for each peakindividually. The Agilent ChemSta-tion software offers three differentmodes for setting reference spectra(figure 3):

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Figure 3 Spectral Options, Reference Spectrum window of the ChemStation

without reference spectrum

using nearest baseline

using apex as reference

apex spectrum

baselinespectrum

peak start

peakend

Figure 4 Apex and baseline spectra of a peak

1. No reference:Spectral operations are per-formed without any reference(recommended for exceptionalsituations only, even isocraticseparations should use a refer-ence spectrum).

2. Manual reference:One or two reference spectra can be specified by the user.This mode is used for interac-tive spectral evaluations of non-

baseline separated peaks. Onlythe spectra in between the twoselected reference points areused for the purity evaluation.

3. Automatic background correction:Depending on the acquisitionparameters chosen either A, Bor C is performed automatically.A) All spectra: For peak purity, the spectra at the actual peak start and end

are linearly interpolated and subtracted fr