Factors Affecting Sensitivity Evaporative Light Scattering...

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Factors Affecting Sensitivity of Evaporative Light Scattering Deteotion Sylvie H^ron,^ Michel Dreux^ and Alain Tchapla,^ 'Groupe de Chimie Analytigue de Paris-Sud., Orsay, France, 2Sedere, Aifortville, France. An evaporative light scattering (ELS) detector is a powerful detection tool if the solutes are less volatile than the eluent. Three main processes occur successively: nebulization, evaporation of the liquid chromatographic (LC) effluent and measurement of the light scattering by the residual particles. This leads to a non-linear calibration curve such as, A = a.m^ where A is the peak area, m the sample mass and b the response coefficient measured as the slope of Log A - bLogm i Log a. The goal of this paper is to demonstrate that ELS response is similar to ultraviolet (UV) or fluorescence (F) detection and can be nfiodified by adjusting certain parameters. For triacylglycerol (TG) analysis, we show that we can alter the b value by modifying the nature of the mobile phase by post-column addition. It was found that fa greatly varies (from 1.89-1.02) depending on the nature of the added compounds. Solvents that favour interactions that modify the particle size distribution of the three diverse aerosols characterizing the three processes were chosen as added compounds. This allows the value of b to be monitored in direct relation with the scattering particle size as predicted by theory that fa should be between 0.66 for big particles to 2 for small particles of the Rayleigh domain. 414 In the field of liquid chromatography (LC) detectors, the evaporative light scattering (ELS) detector is becoming more popular and can be considered as a quasi-universal detector as long as the solutes are less volatile than the LC eiuent.'-2 The response is determined by A = a.m" [1] where A is the peak area, m the sample mass, a and b are numerical coefficients that depend on droplet size, concentration and the nature of solute, gas and liquid flow-rates, molar volatility, and soon. The response coefficient b values listed in the literature are between 0.9-1.9 which results in non-linear calibration cun/es (b ^ 1) and corresponds to the slope of Log A = bLogm + Log a 121 The goal of this paper is to demonstrate that ELS response is similar to UV or fluorescence (F) detection and can be modified by adjusting certain parameters. Ultraviolet (UV) detection depends on the selected wavelength of absorption (which is related to the chromophore of the solute to LC'GC Europe July 2007

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Factors Affecting Sensitivity ofEvaporative Light Scattering DeteotionSylvie H^ron,̂ Michel Dreux^ and Alain Tchapla,^'Groupe de Chimie Analytigue de Paris-Sud., Orsay, France,2Sedere, Aifortville, France.

An evaporative light scattering (ELS) detector is a powerful detection tool if the solutes areless volatile than the eluent. Three main processes occur successively: nebulization,evaporation of the liquid chromatographic (LC) effluent and measurement of the lightscattering by the residual particles. This leads to a non-linear calibration curve such as,A = a.m^ where A is the peak area, m the sample mass and b the response coefficientmeasured as the slope of Log A - bLogm i Log a.

The goal of this paper is to demonstrate that ELS response is similar to ultraviolet (UV) orfluorescence (F) detection and can be nfiodified by adjusting certain parameters.

For triacylglycerol (TG) analysis, we show that we can alter the b value by modifying thenature of the mobile phase by post-column addition. It was found that fa greatly varies (from1.89-1.02) depending on the nature of the added compounds. Solvents that favourinteractions that modify the particle size distribution of the three diverse aerosolscharacterizing the three processes were chosen as added compounds. This allows the valueof b to be monitored in direct relation with the scattering particle size as predicted by theorythat fa should be between 0.66 for big particles to 2 for small particles of the Rayleigh domain.

414

In the field of liquid chromatography (LC) detectors, theevaporative light scattering (ELS) detector is becoming morepopular and can be considered as a quasi-universal detector aslong as the solutes are less volatile than the LC eiuent.'-2 Theresponse is determined by

A = a.m" [1]

where A is the peak area, m the sample mass, a and b arenumerical coefficients that depend on droplet size, concentrationand the nature of solute, gas and liquid flow-rates, molar volatility,and soon.

The response coefficient b values listed in the literature arebetween 0.9-1.9 which results in non-linear calibration cun/es(b ̂ 1) and corresponds to the slope of

Log A = bLogm + Log a 121

The goal of this paper is to demonstrate that ELS response issimilar to UV or fluorescence (F) detection and can be modified byadjusting certain parameters.

Ultraviolet (UV) detection depends on the selected wavelengthof absorption (which is related to the chromophore of the solute to

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be analysed) and may be compromised by the mobile phaseabsorption, which must be low at this wavelength. Evaporative lightscattering detection (ELSD) is easy when there is a large differencebetween the mobile phase volatility and the solute voiatiiityConsequently, the possibility of evaporating the mobile phase at lowtemperature allows the analysis of semi-volatile compounds v>/ithnon-volatile compounds. The wavelength choice for UV detectioncorresponds to the drift tube temperature chosen to evaporate themobile phase in ELSD, The perfomnance of ELS detectors, like UVdetectors, depends on the model and manufacturer.

UV or ELSD can analyse a wide range of compounds and maybe more sensitive when the appropriate parameters are optimized.These factors can be divided into three different categories;instrumental parameters, such as the characteristics of the UV cellor ELSD nebulizer and eiuent flow-rate; solute properties, such aschemical derivatization or transfomiation by complexation; and LC •separations conditions, such as the nature of the mobile phase.

In the first set of parameters, the UV cell design influencesbackground noise, signal intensity via the cell length, geometryand volume, so the UV response depends on these parameters.3''For a given wavelength, the response factor (RF) is lowered with acell length and volume decrease. Such a decrease iscompensated by an increase in the solute concentration at the topof the peak when capillary chromatography replaces conventionalchromatography. So, for an amino acid analysis, Barrett et al.observed an increase in signal to noise ratio (S/N) and a decreasein the limit of detection (LOD) with a decrease in internai columndiameter with a UV detector,^ but such an increase is moreimportant for the ELSD than for the UV detector. Cur interpretationof this result, which highlights ELSD, is mainly based on thefollowing considerations: It is easier to evaporate low quantities ofsolvent and additive. In these analysis conditions the semi-volatileion-pairing agent represents the major component of thebackground noise and the quantity to evaporate decreases withthe mobile phase flow-rate.

Explanations for the ELS signal and S/N increase in capillarychromatography may result from the increase in the soluteconcentration that directly induces an increase in the droplet sizeof the scatterer as published by Chaminade et al.^ Thisexplanation agrees with a decrease in the b value that goestowards the unit.^ Consequently, in capillary chromatography adirect linearity may characterize the ELS response asi^-'O

A = a.m [3]

Chemical derivatization is the second category that stronglymodifies the detector response and signal intensity.' ^ With UVdetection, off-line and on-line derivatization may transform anon-UV-absort)ing solute into a UV-absorbing solute and also assistsolute detection.^2 Currently, in ELSD the solute transformation is notcommonly practised, even though it was clearly mentioned in the firstpaper in 1966.'^ However, the modification of the mobile phase pH isthe classical parameter used to make ELSD of basic compoundseasier because ionization reduces the compound's volatility.

Also, in a paper published in 2004, two different solutecomplexations using a post-column on-line complexation oftriacylglycerols were reported.^'* One corresponds to the formationof a cholesterol-TG complex, which increases the particle size ofscatterer compared with the non-complexed TG or assooiated TC.The other corresponds to a 1/1 TG-Ag ^ complex, which avoidssolute association between solute as well as solute-mobile phase

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association. As expected, an increase in the scatterer sizedecreases the b value (b decreases from 1.3-0.6 with diversecholesterol concentrations) while a decrease in the scatterer sizedoes increase the b value {b increases from 1.3-1.8). This showsthe importance of the particle size of the scatterer and theconsequent variations in thei) values.

When smalt quantities of impurities in asolvent alters solute-solvent interactionsthe b slope and the log a value maychange as well as the reproducibility.

As mentioned in a paper by Engelhardt and Schultz,'^ we donot know what the detected particles are (i.e., what is the nature ofthe scatterer in the tertiary aerosol?) Dry particles, wetted particlesof bare solute or of associated solutes? Factors such as mobilephase nature and evaporation temperature of the drift tube mayhave a strong influence on the scattering particles.^-'^'^'' Recently,Chaminade shows a response enhancement in ELSD of diversecompounds with a triethylamine-formic acid additive to the mobilephase.'8''9 The proposed mechanism involves a "clusterformation" which changes the b value slightly and the a value inequation 1 strongly. Such a result was also obsen/ed with atriethylamine-trifluoroacetic acid mixture added to the mobilephase to determine anions using LC-ELSD.20

In this paper, we report changes in the aerosol distribution(primary aerosol) or in the tertiary aerosol (the scatterer) using anon-line post-column addition after a chromatographic separation.Some modification of the mobile phase properties, such asviscosity and surface tension, when using PEG 4000 atmicromoiar concentrations may change the aerosol size anddroplet distribution. A specific property of other solvents used inthe mobile phase may influence the size of the scatterer anddroplet size distribution of the primary aerosol. Another wayconsists in using moiar concentration of a solvent of specificproperties that may influence the size of the scatterer.Triacylgycerols (TG) were chosen as molecular model to involvespecific interactions with molar concentration of solvent, whichdiffer from the mobile phase constituents.

Experimental DataChemicals: TGs [tricaprin (TG 10) and thiinolein (TG 18:2)] wereobtained from Sigma (Saint-Cuentin Fallavier, France). Acetonitrile(MeCN) [Acros, Noisy le Grand, France], methylene chloride(CHgCi^), methanol (MeOH) [Cario Erba, Milan, Italy] and water(H2O) [SDS, Peypin, France] were of high perfomiance liquidchromatography (HPLC) grade. Pentanol and nitropropane werepurchased from Merck (Darmstadt, Germany): ethanol (EtOH) andisopropanol (iPrCH) from Prolabo (VWR internationai,Fontenay-sous-bois, France); tri methyl phosphate (IMP) andpolyethylene glycol (PEG) 4000 from Fluka (Buchs, Switzeriand).Equipment: The chromatographic system consisted of a Model1050 pump (Hewlett-Packard, Palo-Aito, California, USA), a Model7125 injection vaive with a 20 pL loop (Rheodyne, Cotati,California, USA) and a Model Sedex 75 light-scattering detector(Sedere, Alfortville, France). The nebulizing gas was air at 3.6 bars(corresponding to a flow-rate of 1.92 L/min), the nebulizationtemperature was 37 or 55 °C, according to the solventpost-column used, and the gain (Pfvl) was maintained at 11.

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Chromatograms were recorded with Azur (v 3.0) acquisitionsoftware (Datalys, Saint-Martin d'Heres, France),

The column temperature was controlled using an Igloo-cil oven(Cluzeau, Sainte-Foy-la-Grande, France) and set at 20 °C.

A Kromasil C,8 (5 Mm) 150 mm x 4.6 mm column was used(Touzart and Matignon, Les Ulis, France), The mobile phaseflow-rate was 1 mlVmin,

For the post-column addition, a model 114 M pump in micromode (Beckman, Gagny. France) and a pulsation damper (Touzartand Matignon) were used and connected between the columnoutput and the ELSD nebulizer via a polyether ether ketone (PEEK)tee in which the main flow comes from the orthogonal branch oftee and the added flow from the top branch of tee. Thepost-column addition flow-rate was 50 tjLVmin and such a deviceallows good mixture of the two flows.Methods: The mobiie phase composition MeCN/CH2Cl2 (65/35)was chosen to give moderate retention times for the TGs.^^ For TG10 and TG 18:2 the retention factors were 1.9 and 6,4 respectively.

The TGs were dissolved in MeCN/GHjClg (50/50). The calibrationcurves were established using between five (range 5-40 ppm) andeight (range 5-100 ppm) different TGs concentration levels. Thedifference in the calibration ranges was a result of the saturation ofthe signai with some of the post-column solvent.

We found that irrespective of the number of concentration levels[i.e., (5-100) or (5-40) ppm]. the b value was the same. Each areavalue was the average of three reproducible injections. To avoidmodification of the solutes retention, the tested solvents wereadded using a post-column addition, using a polyether etherketcne (PEEK) tee.

The post-column addition flow-rate was maintained at50 pLVmin — corresponding to a slight increase in the mobilephase flow-rate (5%).

Results and DiscussionStudies without post-column addition: First, the study ofpeak area versus the amount of injected TGs was made withoutpost-column addition. The mean b vaiue was 1.5 (1.48-1,55 forTG 10 and 1.45-1.54 for TG 18:2), showing no significantdifference between the two compounds. As previously reported,the b values are closed for compounds belonging to only onechemical family (library), ̂ Normal b values for NARP are generallyhigher than 1.5 while in aqueous liquid chromatography (LC) bvalues lower than 1.5 are commonly reported.'' According totheory, a low b value is consistent with a big particle size scafterer.So, the low b value (1,5) means that the tertiary aerosol is notcomposed of "dry" solutes but composed by "wet" solutes (i,e., byparticles bigger than particles of dry solutes).'^'^^Studies with post-column addition of molarconcentration of different compounds: The study of peakarea versus the amount of injected TGs was pertormed withpost-column addition of four different alcohols. The sameobservations can be made about the two TGs:

When the post-column addition is made of alcohols ofincreasing carbon atom number (MeOH, EtOH, iPrOH, Pentanol),the b value decreases [Figure 1 (a)].

With post-column addition of methanol, a common b value(1,8-1,9) is observed. When the b value decreases, log aincreases (results not shown) as noted before.""'"•22-23 These resultscan be linked to the variations of the interactions between thesolute (TG) and alcohol: interactions between aikyi chains of thesolute (TG) and the alcohol increase with the alkyl chain length ofthe alcohol and an increase in the resulting particle size contributes

to the decrease of the b value. Moreover, the different volatilities cfadded alcohols may modify the size of the alcoho!-TG aggregate.

With MeGH, the b vaiue agrees with the dry solute particlesproduced in the tertiary aerosol, The same study and the sameobservations have been made with other additives: b values arelower than 1,55 (the b value obtained without post-columnaddition) [Figure 1(b)],

This corresponds to differences in solute-solvent aggregation. Itdemonstrates a solute "wetting" dependent of the additive nature.The differences in b values correspond to solute-solventinteraction variations that directly Influence the tertiary aerosolnature, as well as size distribution.Studies with post-column addition of micromolarconcentrations of PEG: The study of peak area versus theamount of injected TCs was pertormed with post-column addition

• of PEG 4000 dissolved in MeCN/HjO (50/50). The b valuesobtained are shown in Figure 2. PEG 4000 is known to changephysical properties of water, particularly viscosity and wettability.2''

A miscible ternary phase composed of MeCN/CH2Cl2/H2O wasobtained by dissolving the PEG 4000 in MeCN/H2C (50/50),Without PEG 4000 (0 pM), the b value decreases from 1,5[Figure 1(a) and 1(b), without] to 1.3 [Figure 1(b). MeCN/H20(50/50)], Such a decrease corresponds to some interactionscaused by the water content.

With the addition of PEG 4000 at 0,3 xiO-6M(corresponding4 ppm w/w), the b value decreases more strongly, tending towards1, showing the specific effect of the polymer. The presence of PEG4000 and water leads to a mobile phase, which spreads over thesolutes better, making bigger and more stable (solute-solvent)aggregates. For PEG 4000 with smaller concentration

Figure 1: Values of b obtained for the post-column addition ofmolar concentrations of different additives for the twoTGs, A: TG 10 +:TG18:2

(a)

Without MeOH EtOH iPrOHMobile phase addition

Pentanol

(b)

C0

0

1owo

esp

CC

2,0

1.8

1.6

1.4

1.2

1.0

.p/

Mobile phase addition

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(0,16 X 10-6 M), the b value goes up, leading to conclude thatthere is not enough polymer in the solution to modify the viscosityand/or the wettability as well as the maximum amount ofinteractions with the solute.

An explanation for this behaviour may be that the nebulizationprocess is affected by the presence of polymers in the solution,because of change in the viscosity. At different analyteconcentrations the mean droplet size may be different, giving riseto a different number of particles. Moreover, the efficiency of thetransport of droplets through the evaporation chamber maydepend on their mean size.

When the b value decreases, the peak width increases

(o>,/2 = 0.15-0,17 with TG10;a),^2 = 0,27-0,34 with TG18:2). Asdemonstrated by Gulochon 21 years ago,^^ this result is inagreement with the standard deviation a corresponding to aGaussian curve in ELSD,

We may conclude to a 6 variation given by a micromolarcompound addition that modifies the primary and the tertiaryaerosol produced in an ELS detector. Such a micromolarconcentration of solvent added to the output of the column cannotmodify the b value while, as we have seen previously, some molarsolvent addition will produce an important b variation.

ConclusionsThese results agree with previous research showing that thesensitivity of the ELS detector depends on the nature andconcentration of the additive,"'•18.20,23,26 as well as the instrumentsettings. ̂ .̂26.27 Some variations may be a result of the presence ofvery low concentrations (micromolar level) of additive and mayexplain the lack of reproducibility of some of the results obtainedfrom an ELS detector. When small quantities of impurities in asolvent alters solute-solvent interactions the b slope or the log avalue may change, as well as the reproducibility.

Currently, there is no specific grade of solvent purity for ELSDbut further investigation into this is required to obtain betterreproducibility and/or a better limit of detection. The dependencyof tbe parameters a and b was mentioned earlier by Guiochon'"'and confirmed recently by Chaminade,22-23 |_ane et al.,28 and ourown unpublished results.

A decrease in b is "compensated" by an increase in a or log a,so we may conclude that these parameters are notindependent.29 These results offer a new approach for ELSDquantitative ^o

Figure 2: The effect of the post-column addition of polyethyleneglygol (PEG 4000) dissolved in MeCN/HjO (50/50) at differentconcentrations on the response of the two triacylglycerols,A:TG 10 +:TG18:2,

effic

ient

Res

pons

e c

o

2.0 n

1,8-

1,4-

1,2-

1.0-

A t

0.30 0.16 0

Concentration of PEG 4000 (uM)

References1. M. Dreux and M. Lalosse. In Carbohydrate Analysis. Z. El Rassi. Ed

{Journal of Chromatography Library. Vol. 58, Elseviar, Amsterdam, 1995)pp. 515.

2. J.M, Chariesworth.Aia/. Chem.. 50. 1414-1420(1978),3. A.M. Krstulovic, RR, Brown, In Reversed-Phase High Performance Liquid

Chromatography: theory, practice, and biomedical applications. (J,V\'ileyand sons. New York. 1982, chap. III.) pp. 32.

4. J.N. Done, In High Performance Liquid Chromatography. C.F. Simpson,Ed. (Heyden and Sons Ltd, London, 1978) pp, 79.

5. Z. Cobb et al., J, Microcolumn Separations. 13(4), 169-175 (2001)6. K. Gaudin et al., 17th IntemationalSymposium an Microscale Separations

and Capillary Electrophoresis, Feb S-12, 2004 Salzburg, Austria, posler P176.

7. S, Heron and A. Tchapla, J. Chromatogr. A, 648, 95-104 (1999),8. M.B.O. Andersson and L.G. Blomberg, J. Microcol. Sep.. 10, 249-254

(1998),9. R, Tranes, et al., J. Chromatogr. A, 814, 55-61 (1998)10. J.N, Alexander IV, J. Microcoi. Sep.. 10. 491-502 (199B),11. T Toyo'oka, Ed, Modem Derivalization Methods for Separation Sciences.

(J.Wiley and Sons. Chichester, 1999) pp. 233.12. K. Zattsu, M Sai and K. Hamase. In Derivatization Methods for Separation

Sciences. I Toyo'oka, Ed. (Wiley and Sons, Chtchester. 1999) pp 64,13 D. L, Ford and W. Kennard, J. a/Cotour Chem,/^ss,, 49, 299-304 (1966).14. S, Heron, M. Dreux and A, Tchapla, J. Chromatogr. A.. 1035. 221-225

(2004),15. R, Schult2 and H. Engelhardt, Chromatographia, 29. 517-522 (1990),16. G. Guiochon, A. Moysan and C. Holiey, J. Liq. Chrom, 11(12), 2547-2570

(1988),17. W. Righezza and G. Guiochon. J, LyqC/iram. 11(9-10), 1967-2004

(1988).18. K. Gaudin et al., J. Liq. Chromatogr., 23, 387-397 (2000).19. RS, Deschamps et al., Chromatographia, 54, 607-611 (2001),20. C. Elfakir. R Chaimbault and M, Dreux, J, Chromatogr. A. 829, 193-199,

(1998).21. S, H6ron, J, Bleton and A. Tchapla, In New Trends in Upid and Upoprotein

Analysis. E,G, Perkins and J.L. StoSdio, Eds, (/American Oil Chem. Soc.Press Publ. Champaign, illinais. USA, 1995) pp.205

22. F.S. Deschamps, R Chaminade, A, Baillei, The Analyst. 127, 35-41(2002).

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24. D, Bonn, V. Bergeron and L. Vovelte, La recherche, Mars. 3, 40-43 (2002).25. A. Stolyhwo et aL, J. Chromatogr.. 288, 253-275 (1984).26. H. Gangloff et al., US Rending Ratent 10/167, 820 (2002).27. H, Gangloftetal., French Ratent n" 01 09 202(07-11-2001)26, S. Lane et al.. 25th International Symposium on Chromatogtaphy,

4-8 October 2004, Paris, France, Poster.29, S. Heron et al., LC*GC Eur. 19, 864-672 (2006).30. S, Heron et al., J. Chrom. A, accepted 2007,

Sylvie Heron is assistant professor of analytical chemistry in theGroup of Analytical Chemistry of Paris Sud (LETIAM) at theUniversity of Paris Sud (lUT Orsay). Her current research interestsinclude development and optimization of LC methods for lipidsanalysis with Evaporative Light Scattering Detector and MassSpectrometry.

Michel Dreux was a professor in separation sciences at theInstitute of Organic and Analytical Chemistry of the University ofOrleans, France. He was at the head of the Analytical ResearchGroup, His main research interests concern chromatography,capillary electrophoresis and capillary electrochromatography. HeIs now the Scientific Director at SEDERE.

Alain Tchapla is professor of analytical chemistry in the Group ofAnalytical Chemistry of Paris Sud (LETIAM) at the University ofParis Sud (lUT Orsay). He is head of the LETIAM research group.His main research interests concern chromatography, massspectrometry and liquid stationary phases caracterization.

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