effect of large volume injection of hydrophobic solvents on the retention of less hydrophobic...

J. Sep. Sci. 2008, 31, 2939 –2945 S. Udrescu et al. 2939

Stefan Udrescu1

Andrei Medvedovici2

Victor David2

1LaborMed Pharma S.A., SplaiulIndependentei, Bucharest,Romania

2Faculty of Chemistry,Department of AnalyticalChemistry, University ofBucharest, Bucharest, Romania

Original Paper

Effect of large volume injection of hydrophobicsolvents on the retention of less hydrophobicpharmaceutical solutes in RP-LC

Injection of large volumes of samples in solvents other than mobile phase composi-tion has been proved for some less hydrophobic compounds. Thus, the retentionbehavior of several compounds of pharmaceutical interest (isosorbide-2-nitrate, iso-sorbide-5-nitrate, tropicamide, pentoxifylline, and methyl p-hydroxybenzoate) wasstudied by using different hydrophobic solvents (n-hexane, n-heptane, or i-octane) assample solvents. Two types of stationary phases were used: octyl and octadecyl modi-fied silica (both of Zorbax Eclipse type). The experiments showed a linear depend-ence between capacity factor of each solute and sample injection volume, up to max-imum volume values of about 680 lL for C8 stationary phase and 580 lL for C18 sta-tionary phase, when the solutes are no longer retained in stationary phase. Injectionof large volumes of these hydrophobic solvents is thus possible in RP-LC with a grad-ual reduction of retention and peak efficiency. Two major conditions are howevernecessary in order to apply such an injection approach: the solutes must have aproper solubility in hydrophobic solvents and meanwhile they have to be less hydro-phobic than the sample solvent in order to avoid competition with solvent mole-cules in partitioning between mobile and stationary phases.

Keywords: Hydrophobic solvents / Large volume injection / Pharmaceutical compounds / Re-versed-phase LC /

Received: May 17, 2008; revised: June 24, 2008; accepted: June 24, 2008

DOI 10.1002/jssc.200800299

1 Introduction

Enhancement of sensitivity for spectrometric detectionin LC is obtained when increased sample volumes areloaded into the chromatographic column [1]. For thispurpose, it is usually recommended that the sample com-position is injected into analytical column to have a sim-ilar composition with mobile phase.

Large volume injection in a solvent similar to themobile phase composition in RP-LC has been applied tothe determination of chiral pesticides [2], pyrethroids insoil samples [3], simazine, terbuthylazine, atrazine, andhydroxyatrazine in soil [4, 5], or naphthalene derivativepesticides [6]. Several utilizations of this approach havebeen reported to the determination of bisphenol A and 4-octylphenol [7], acetylcholine esterase (ACE) inhibitingpeptides [8], perfluorooctane sulfonate and perfluorooc-tanoic acid [9], and fenofibric acid [10] in human plasma

by LC with different detection techniques. Large volumeinjection has systematically been studied to improve LC-MS/MS sensitivity and LODs. This method can be com-bined with online SPE for the analysis of compoundsdirectly in plasma samples [11]. Large volume injectionup to 500 lL was used with no negative effect on the sep-aration profile of trace carcinogenic polycyclic aromatichydrocarbons in microcolumn LC [12]. Enhancement ofthe detection sensitivity in LC by large volume injectionup to 50 lL by using packed capillary columns was alsoreported [13]. Separation of ceramides by temperature-programmed packed capillary LC has been performedusing subambient temperature-assisted large volumeinjection [14].

Injection of large volume of solvents nonmiscible withmobile phase and having no affinity to the stationaryphase induces a strong perturbation of the retentionprocess, resulting in focusing effects of analytes in sam-ple solvents [1]. Such solvents are chlorinated hydrocar-bons, ethers, or esters. Recently, it has been shown thatsamples of solvents nonmiscible with mobile phase, buthaving affinity to the stationary phase, can be injected inlarge volumes (hundreds of microliters) in RP-LC if some

Correspondence: Professor Victor David, Faculty of Chemistry,Department of Analytical Chemistry, University of Bucharest,Sos. Panduri no. 90, Bucharest 050663, RomaniaE-mail: [email protected]: +40-21-4102279

i 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com

2940 S. Udrescu et al. J. Sep. Sci. 2008, 31, 2939 – 2945

conditions are fulfilled [15]. The main condition is thestrong interaction of the solvent with the stationaryphase (pentane, hexane, heptane, or upper hydrocar-bons). The other condition is related to the solubility ofanalyte in these hydrophobic solvents. That means thesolute has a certain hydrophobic character, but it hasbeen proved that their hydrophobicity must not behigher than solvent hydrophobicity. From this point ofview the application of this approach can be limited tothose compounds having a moderate hydrophobicity,and meanwhile, to the solutes having an acceptable solu-bility in sample solvent. It is the purpose of this work toshow some new potential utilizations of large volumeinjection of hydrophobic solvent samples to the RP-LCseparation and determination of pharmaceutical com-pounds as well as the retention effects induced by thisapproach during the RP chromatographic elution proc-ess.

2 Experimental

2.1 Reagents and column

All solvents (ACN, methanol, n-hexane, n-heptane, i-octane) were of HPLC grade from Merck (Darmstadt, Ger-many).

Two types of stationary phases were studied in thiswork. The experiments performed on a C8 stationaryphase were carried out using Zorbax Eclipse XDB-C8,150 mm length, 4.6 mm id, and 3.5 lm particle size (Agi-lent Technologies) as the chromatographic column. Theexperiments performed on a C18 stationary phase werecarried out using Zorbax Eclipse XDB-C18, 150 mmlength, 4.6 mm id, and 5.0 lm particle size (Agilent Tech-nologies) as the chromatographic column.

Several target solutes have been used (their structuresand data related to octanol/water partition constant,log Kow are given in Table 1): isosorbide-2-nitrate, isosor-bide-5-nitrate, pentoxifylline, methyl p-hydroxybenzoate(MPHB), and tropicamide. For reducing the number ofexperiments, the target solutes were coupled accordingto their hydrophobicity. In order to promote the dissolu-tion of target solutes in the hydrophobic solvents, con-centrated stock solutions were made in methanol, fol-lowed by their subsequent dilutions in the adequate sol-vent. Methanol percentage in the working solutions wasless than 1%. Working stock solutions of individual tar-get compound in hydrophobic solvents were at 100 lg/mL concentration level. Further dilutions in the hydro-phobic solvents were made when necessary. Whenincreasing the injected volume, concentrations of thetarget compounds were progressively decreased, in order


Table 1. Structures of target analytes and experimental values for their log Kow

Target compound Structure Experimental log Kow

Isosorbide-2-nitrate –0.40

Isosorbide-5-nitrate –0.15

Pentoxifylline 0.56

MPHB 1.96

Tropicamide 1.19

J. Sep. Sci. 2008, 31, 2939 –2945 Liquid Chromatography 2941

to keep almost constant their absolute amount loadedonto the column. The injection volumes applied in thisstudy were the following: 5, 10, 25, 50, 100, 200, 300,400, and 500 lL.

2.2 Instrumentation and chromatographicconditions

Experiments were performed on Agilent 1100 seriesliquid chromatograph containing degasser, binarypump, autosampler (with large volume injection option),column thermostat, and diode-array detector. Detectionwas made at 210 l 2 nm for isosorbide-2-nitrate, isosor-bide-5-nitrate, and tropicamide, and at 270 l 2 nm forpentoxifylline and MPHB. For all target compounds, thereference wavelength was 480 l 10 nm. Chromato-graphic data were acquired by means of the Chemstationsoftware (Agilent Technologies, Waldbronn, Germany).

The study was carried out under isocratic conditions.For the pair isosorbide-2-nitrate/isosorbide-5-nitrate, themobile phase consists of 5% ACN and 95% water. For thepair pentoxifylline/MPHB, the mobile phase consists of20% ACN and 80% water. For tropicamide, the mobilephase consists of 22% ACN and 78% aqueous component(aqueous 0.1% H3PO4 brought to pH = 7 with triethyl-amine).

Column preparation for a consecutive injection in asequence consists of the following operations: a fast stepgradient to 100% ACN (in 0.05 min); 10 min needed forthe elimination of the hydrophobic sample solventloaded to column during the previous run; a step gra-dient back to initial elution conditions in 0.05 min;10 min for adequate column re-equilibration. Note thatthese operations are made only for column conditioningand for removal of all interferences transferred from pre-vious separation stages.

Flow rates of 1.0 mL/min were used. The mobile phaseand the column were thermostated at 308C using an Agi-lent Peltier based heater (G1316A).

2.3 Preliminary data

Dead time (t0) was determined by means of aqueous KNO3

injections (concentration was 0.1 mg/mL, injected vol-ume 5 lL, monitoring wavelength 210 nm). Calculation

of the capacity factors (k9) was made according to the rela-tion k9 = (tR – t0)/t0, where tR (min) is the absolute reten-tion time of the target solute.

Functional fitting and representation were made withOriginm version 7.0 (OriginLab, Northampton, MA, USA).Some of the characteristics related to solvents and targetcompound used for calculations were taken from TheMerck Index, version 12.3 (Merck Whitehouse Station,NJ, USA), or computed via KowwinTM version 1.67 (copy-right� 2000, US Environmental Protection Agency) andgiven in Tables 1 and 2.

3 Results and discussion

Injections of large volumes with hydrophobic solventsrely on stronger affinity of solvent molecules towardsilica-modified stationary phase than the solute mole-cules from the injected sample [15, 16]. Adsorptionmodel as the only possibility to explain retention phe-nomena occurring during injecting large volume ofhydrophobic solvents nonmiscible with mobile phase isbased on the interaction between solute molecules (A)and hydrocarbonaceous chains from stationary phase(denoted by L): L + nA$L N An that is in competition withthe equilibrium of interaction between sample solventmolecules (S), i. e. L + mS$ L N Sm. Such events take placeeven in case of organic modifiers usually used in RP-LC,as resulting from studying their adsorption isothermsfrom water on chemically bonded stationary phases withdifferent lengths [17]. The value of m should be independ-ent of the nature of sample solutes, but it is likely todepend on the nature of sample solvent and hydrocarbo-naceous ligand bound on silica (i. e., m for C18 could belarger than for C8). However, the molecular length ofhexane, heptane, and i-octane does not vary significantlysuch that the value of m is expected not to vary for thesesolvents. As a conclusion, the stationary phase surfacethat is allowed to the solute molecules will decrease withthe increase in the sample solvent volume. Thus, thecapacity factor (k9) for the solute A depends linearly onthe volume of sample solvent injected into the chromato-graphic column:

k9 = a – b VS (1)

where a and b are regression parameters, which can becorrelated to molecular (octanol/water partition coeffi-cient of solute A, sample solvent density, number of sol-vent molecules m in interaction with one chain L in sta-tionary phase) and experimental parameters (mobilephase volume, the chain load in stationary phase, par-ticle size of stationary phase influencing the contact sur-face). This relationship allows the estimation of the sam-ple (solvent) volume for which k9 = 0, denoted by Vk9¼0

S .Beyond this value the solute(s) will spend no time in sta-


Table 2. Some data on hydrophobic solvents used in thisstudy

Solvent Density(mg/mL)

Mw

(g/mol)Calculatedlog Kow

Experimen-tal log Kow

n-Hexane 0.660 86.18 3.28 3.9n-Heptane 0.684 100.2 3.78 4.66i-Octane 0.692 114.23 4.09 –


tionary phase. For this solvent volume injected into chro-matographic column, the entire number of hydrocarbo-naceous chains from stationary phase is “blocked” bymeans of interactions with solvent molecules. The newlyadded hydrophobic layer does not exhibit any affinity tothe solute molecules, and hence the partition model isno longer valid to explain the retention process in RP-LCcarried out by these experiments [18].

Three hydrophobic solvents were studied: n-hexane, n-heptane, and i-octane. High volumes of samples contain-ing target solutes (described in Section 2) were injectedand two chromatographic parameters were processed:capacity factor and efficiency. The chromatographicparameters (k9 and N) were calculated as an average valueof three consecutive injections for a given sample vol-ume injected onto the column. Linear representations


Figure 1. Linear representations for the dependence ofcapacity factor (k9) of target analytes on injected sample vol-ume (VS) for the three mentioned solvents and C8 as station-ary phase.

Figure 2. Linear representations for the dependence ofcapacity factor (k9) of target analytes on injected sample vol-ume (VS) for the three mentioned solvents and C18 as sta-tionary phase.


for the dependence between capacity factor (k9) of targetsolutes and injected sample volume (VS) for the above-mentioned solvents loaded on both stationary phasesused are given in Figs. 1 and 2.

The linear dependences between capacity factor andinjected sample volume were obtained for all studied sol-utes and for the three solvents, in both cases of stationaryphase used for chromatographic separations: C8 and C18modified silica. The results obtained from the lineardependences described by Eq. (1) for all investigatedexperimental conditions are given in Tables 3 and 4.

As an important remark we may note that for bothtypes of stationary phases the extrapolated parameterVk9¼0

S did not vary significantly from one solute toanother. Besides that, this value does not depend on thenature of sample solvent, a result that can be consideredin accordance with initial supposition based on solventadsorption onto the stationary phase. The mean valuefor Vk9¼0

S calculated for all solutes from Table 3 in case ofC8 stationary phase led to 682 lL (with an acceptableRSD of 5.88%). In case of C18 stationary phase the meanfor Vk9¼0

S calculated for all solutes given in Table 4 led toan unexpected lower value, of approximately 586 lL, butwith a better RSD (2.87%). Obviously, a higher Vk9¼0

S

should be expected for a C18 stationary phase, owing tothe higher length (surface) of L, potentially interacting

with a higher number of solvent molecules. This contra-diction may be explained, if considering the granulome-try of the packing material (3.5 lm for C8, and 5 lm forC18). Smaller particle size favors interactions by meansof a higher contact surface.

The chromatographic efficiency depends on the injec-tion volume by means of a similar relationship as in Eq.(1), characterized by relatively high correlation coeffi-cients (given in Tables 3 and 4); the theoretical platenumber (N) decreased linearly with the increased valueof injection volume. Such dependence is somehow unex-pected, as efficiency depends on the square of the abso-lute retention. However, a decrease in retention reduceslongitudinal diffusion, and consequently reduces thepeak width.

On the other hand, the peak shape may be affected athigher injection volumes of the used hydrophobic sol-vents. For instance, some fronting effects were observedfor pentoxifylline and MPHB when dissolved in n-hexane,for injected volumes higher than 200 lL. However, suchbehavior was not observed for samples dissolved in hep-tane or i-octane. For injection volumes close to calculatedVk9¼0

S values, adverse effects affecting peak symmetry aremore evident (fronting and peak splitting, in some cases,such as in the case of tropicamide dissolved in n-hexane).These effects can be explained by the possibility of


Figure 3. Overlaid chromato-grams for different volumes of asample containing pentoxifylline,and MPHB in n-hexane on a C18stationary phase.

Figure 4. Overlaid chromato-grams for different volumes of asample containing pentoxifyllineand MPHB in n-heptane on a C18stationary phase.


adsorption of both solute and solvent molecules on thesame site in the stationary phase [19], according to theequilibrium L + (m – k)S + kA$ L N Sm–kAk (k a m), result-ing in nonhomogenous coverage of the adsorption sites[15]. Some of the peak symmetry distortion effects arisingat high injected volumes of samples into the chromato-graphic column are illustrated in Figs. 3–5, for the C18stationary phase.

From 500 lL onwards, the baseline of chromatogramsfor experiments carried out on C18 stationary phasebegins to be drastically disturbed (as can be seen fromthe above figures) as a consequence of the emulsificationphenomena, which become more evident in case ofmobile phases with higher content in ACN (above 20%

ACN). The emulsification phenomenon is due to the mix-ing process between desorbed molecules of hydrophobicorganic solvent and mobile phase. However, during theentire chromatographic run this process has not beenobserved when C8 stationary phase was used.

4 Concluding remarks

Injection of large volumes of samples (up to 500 lL) usinghydrophobic solvents, such as hexane, heptane, or i-octane, could be a method of choice in RP-LC. The experi-ments carried out for some compounds having low ormoderate hydrophobicity showed that their retention isdependent on the injected sample volume: the higher is


Figure 5. Overlaid chromatogramsfor different volumes of a samplecontaining pentoxifylline andMPHB in i-octane on a C18 sta-tionary phase.

Table 4. Regression parameters for the dependences ofcapacity factor and efficiency on the solvent volume injectedonto chromatographic column with C18 silica

Target compound r2 for ca-pacityfactor

Vk9¼0S

(lL)r2 forefficiency

n-HexaneIsosorbide-2-nitrate 0.9994 566 0.9612Isosorbide-5-nitrate 0.9994 565 0.9844Pentoxifylline 0.9990 597 0.9067a)

MPHB 0.9994 581 0.9288a)

Tropicamide 0.9998 580 0.9368a)

n-HeptaneIsosorbide-2-nitrate 0.9997 567 0.9540Isosorbide-5-nitrate 0.9996 568 0.9837Pentoxifylline 0.9985 611 0.9229a)

MPHB 0.9994 596 0.9627a)

Tropicamide 0.9997 571 0.9621a)

i-OctaneIsosorbide-2-nitrate 0.9997 594 0.9877Isosorbide-5-nitrate 0.9998 592 0.9900Pentoxifylline 0.9981 615 0.9080a)

MPHB 0.9992 604 0.9696a)

Tropicamide 0.9997 581 0.9848

a) Indicates appearance of fronting/splitting effects onpeak shapes.

Table 3. Regression parameters for the dependences ofcapacity factor and efficiency on the solvent volume injectedonto chromatographic column with C8 silica

Target compound r2 for ca-pacityfactor

Vk9¼0S

(lL)r2 forefficiency

n-HexaneIsosorbide-2-nitrate 0.9973 658 0.9731Isosorbide-5-nitrate 0.9955 662 0.9922Pentoxifylline 0.9951 726 0.9318a)

MPHB 0.9997 673 0.9685a)

Tropicamide 0.9994 678 0.9032a)

n-HeptaneIsosorbide-2-nitrate 0.9988 700 0.9965Isosorbide-5-nitrate 0.9990 692 0.9963Pentoxifylline 0.9980 715 0.9813MPHB 0.9995 687 0.9960Tropicamide 0.9900 584 0.9765a)

i-OctaneIsosorbide-2-nitrate 0.9969 711 0.9973Isosorbide-5-nitrate 0.9988 684 0.9988Pentoxifylline 0.9991 742 0.9982MPHB 0.9995 697 0.9907Tropicamide 0.9993 621 0.9364a)

a) Indicates appearance of fronting/splitting effects onpeak shapes.


the volume of the injected sample, less retention andchromatographic efficiency is obtained for target sol-utes. One analytical advantage of this approach could befound in potential applications in sample preparation byavoiding tedious procedures of solvent evaporation andresidue solubilization in mobile phase solvent. Thus,samples resulting from liquid –liquid extraction (LLE)using these solvents can be directly injected into analyti-cal column, operated under RP conditions [16]. From thispoint of view injection volumes of 100–200 lL can be uti-lized without a significant loss of a retention time andefficiency for the solutes of interest.

The authors acknowledge and they highly appreciate the financialsupport of this study given by the Romanian Agency CNCSIS forthe grant PN2-ID no.55/2007.

The authors declared no conflict of interest.

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