Calibration of migration times of variable salinity samples with internal standards in capillary...

Asif RiazDoo Soo Chung

School of Chemistry,Seoul National University,Seoul, Korea

Received August 15, 2005Revised November 5, 2005Accepted November 5, 2005

Research Article

Calibration of migration times of variablesalinity samples with internal standards incapillary electrophoresis

A practical approach is presented for identifying the analyte peaks stacked by transientITP (TITP) in samples of uncontrolled salinity. For TITP with chloride ions acting as theleading electrolyte, the effect of matrix chloride of an unknown concentration wascalibrated using multiple internal standards to predict the migration times of weaklyacidic anionic analytes behaving as strong electrolytes to an accuracy of over 99.9%.The calibration equations for the migration time of an analyte are given as a function ofthe migration times of internal standards using the mobilities of the relevant ions asparameters. The effects of matrix chloride and various separation conditions such asthe temperature, plug length, ionic strength, and pH of the BGE were completelyeliminated from the calibration equations. In addition, the actual mobilities, determinedfor a standard saline sample under the working conditions, were used, and thus, therewas no need to conduct supplementary experiments to determine the absolute mo-bilities at infinite dilution. The internal standards were dyes, which were easily identifiedin an auxiliary channel monitoring the absorbance at a longer wavelength. For fivestandard saline matrices containing 100–300 mM NaCl at intervals of 50 mM, the meanabsolute error (MAE) in migration times calibrated with two internal standards was0.4 s (n = 5613). For an electropherogram of a real standard reference urine sample,peaks of spiked analytes were identified with an MAE of 0.9 s (n = 13) without con-ductivity normalizing or desalting of the sample.

Keywords: Internal standards / Migration time standardization / Transient iso-tachophoresis of anions/ Variable matrix DOI 10.1002/elps.200500595

1 Introduction

The many advantages of CE over other chromatographictechniques, such as its high efficiency, high speed, sim-plicity, small sample volume requirement, and low con-sumption of chemicals have enabled it to be an indis-

pensable tool in modern separation science [1–7]. How-ever, CE has poor concentration sensitivity for the widelyused absorbance detection and is incompatible with highconductivity samples such as highly saline samples ofbiological origin. Although highly conductive saline sam-ples induce band broadening in conventional CE, a largevolume of such a saline sample can be stacked underproper conditions to improve the concentration sensitivityby means of transient ITP (TITP) [8–12]. For example,chloride ions in a saline sample can act as a transientleading electrolyte to induce stacking of minor anions ofsuitable mobility in a proper BGE [13–15]. The majoradvantages, such as online desalting and the pre-concentration of real samples after minimal treatmentslike filtration or mixing with other reagents (e.g. derivatiz-ing agents) make TITP a powerful method in CE.Enhancements in the sensitivity with TITP by several 100-fold have been reported for saline samples using com-mercial or microchip-based CE instruments [14, 16–21].

Correspondence: Professor Doo Soo Chung, School of Chemistry,Seoul National University, Seoul 151-747, KoreaE-mail: [email protected]: 182-2-877-3025

Abbreviations: AB, 3-aminobenzoic acid; AMB, 2-aminomethylbenzoic acid; AMP, adenosine 5’-monophosphate; BB, 3-bromoben-zoic acid; CFL, carboxyfluorescein; DCF, 2,7-dichlorofluorescein;DMB, 3,4-dimethylbenzoic acid; FL, fluorescein; GMP, guanosine5’-monophosphate; IB, 4-iodobenzoic acid; MAE, mean absoluteerror; NAN, 5-nitroanthranilic acid; 1-NAP, 1-naphthoic acid; 2-NAP,2-naphthoic acid; NB, 4-nitrobenzoic acid; TAPS, N-tris(hydroxy-methyl)methyl-3-aminopropanesulfonic acid; TEA, triethylamine;TITP, transient ITP; TMB, 2,4,5-trimethylbenzoic acid

Electrophoresis 2006, 27, 553–562 553

© 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com

554 A. Riaz and D. S. Chung Electrophoresis 2006, 27, 553–562

In CE, the comparison of the migration time of an analytein a sample with that in a standard solution analyzedunder the same conditions is a general practice for peakidentification. However, the parameters affecting migra-tion times directly or indirectly, such as the temperature,ionic strength, pH of the sample and the BGE, samplematrix, and condition and history of the capillary innersurface, are difficult to apprehend or control. Thus, it iscommon to observe low precision in migration times fromrun-to-run, day-to-day, and from interlaboratory datacomparisons. This critical aspect of CE has beenaddressed by a number of authors and various methodsto improve migration time precision have been reported[22–30]. However, the reported methods are not suitablewhen a saline sample is subjected to TITP, especially be-cause the migration time of an analyte shows a strongdependence on the sample matrix conditions such as thestacker concentration and the sample plug length. Toovercome this problem the chloride concentration in realsaline samples can be normalized to a certain value byadding appropriate amounts of NaCl, often requiringconductivity measurement of each sample [15].

Here, we report a simple but excellent method of predict-ing the migration times of anions of weak acidic analytesstacked by TITP using multiple internal standards. Inhighly saline samples of uncontrolled pH the analytesbehaved as strong electrolytes due to the elevation of pHin the sample compartment during TITP [16] and using aBGE of high pH (9.0). Equations for predicting migrationtimes of analytes under various TITP conditions werederived for one, two, and three internal standards. Select-ed internal standards were colored compounds coveringthe mobility range of a number of metabolically importantbenzoic acid derivatives [31] and two nucleotides. Theinternal standards were easily identified by monitoring theabsorbance at an auxiliary channel in the visible regionwhere none of our analytes has a significant absorbance.Inserting the migration times of the internal standards intothe calibration equations, the migration times of the ana-lytes became predictable irrespective of the matrix salin-ity. Using a single internal standard was found to be aninefficient means of predicting the migration times, as theresults were accompanied by significant deviations andfailed to assign peak identity. However, by using two orthree internal standards the accuracy of migration timeprediction was dramatically increased and peaks instandard saline samples were identified within migrationtime windows of less than a second. The method wassuccessfully used to identify peaks in an electro-pherogram of a standard reference urine sample. In addi-tion to the variations in sample salinity, the tested varia-tions in parameters such as the temperature, plug length,ionic strength, and pH of the BGE could be compensated

for to an acceptable level with two internal standards, andto a better level with three internal standards. With ourcalibration methods, knowledge of the absolute mobilitiesof the internal standards and the analytes under the pre-vailing matrix salinity is unnecessary, since merely theactual mobilities [32] obtained by analyzing a standardsample under the working conditions are sufficient topredict migration times.

2 Materials and methods

2.1 Chemicals

Highly pure sodium chloride (99.999%) was obtainedfrom Aldrich (Milwaukee, WI, USA). N-Tris(hydroxy-methyl)methyl-3-aminopropanesulfonic acid (TAPS),guanosine 5’-monophosphate (GMP), adenosine 5’-monophosphate (AMP), 4-nitrobenzoic acid (NB), 5-nitroanthranilic acid (NAN), 3-aminobenzoic acid (AB), 3-bromobenzoic acid (BB), 2-aminomethyl benzoic acid(AMB), 1-naphthoic acid (1-NAP), 3,4-dimethylbenzoicacid (DMB), 4-iodobenzoic acid (IB), 2-naphthoic acid (2-NAP), and 2,4,5-trimethylbenzoic acid (TMB) were fromSigma (St. Louis, MO, USA). Triethylamine (TEA) was fromYakuri (Osaka, Japan). Colored compounds 2,7-dichloro-fluorescein (DCF) from Merck (Darmstadt, Germany) and6-carboxyfluorescein (CFL) and fluorescein (FL) fromSigma were used as internal standards. An aqueous so-lution (50 g/L) of fluorocarbon polymer neutral (FC-PN) ofFC-480 was from 3 M (St. Paul, MN, USA). All reagentswere of analytical grade or better and used as received.Reference urine (ME 28351) was obtained from Pro-mochem (Wesel, Germany). Since some of the analyteswere not readily soluble in water, all the stocks (0.01 M)were prepared in 0.1 M NaOH solutions. Standard solu-tions were made by adding appropriate volumes of theanalyte stocks and the matrix solution composed of 1 MNaCl, and then diluting to 200 mL with water. Similarly, aspiked urine sample was made by mixing appropriatevolumes of analyte stocks, ME28351, and 40 mL of thematrix solution, and then diluting to 200 mL with water.BGE was prepared by dissolving an appropriate amountof TAPS in 20 mL of water, and then 100 mL of FC-PN so-lution was added. Then the pH was adjusted to 9.0 bytitrating with TEA and the volume was adjusted to 25 mLwith water. The resulting BGE was composed of100 mM TAPS, 80 mM TEA, and 0.02 vol% FC-PN atpH 9.0. The FC-PN in BGE provided dynamic coating ofthe inner wall of a m-Sil-FC coated capillary (Agilent,Waldbronn, Germany) to protect the permanent coating.All the solutions were filtered through a 0.45-mm syringefilter (Whatman, Clifton, NJ, USA) and degassed by soni-cation prior to CE runs.


Electrophoresis 2006, 27, 553–562 CE and CEC 555

2.2 Instrumentation

CE analyses were carried out with a P/ACE 5500 system(Beckman, Fullerton, CA) having a diode array detector. A57-cm m-Sil-FC-coated capillary (50 mm ID, 365 mm OD,and 50 cm to the detector) was used as described pre-viously [14] under a suppressed EOF in a reverse polarityelectric field (anode at detector end). A new capillary wasfilled with BGE containing 0.05 vol% FC-PN and leftovernight for preconditioning. The preconditioned capil-lary was rinsed with BGE for 10 min at 1.386105 Pa.Samples were introduced hydrodynamically at 36103 Pafor 240 s, unless otherwise mentioned. Between runs thecapillary was rinsed with BGE for 3 min at 1.386105 Pa.Separation was carried out using the constant currentmode at 207C. Absorbance at 254 and 500 nm was mon-itored for the detection of analytes and internal standards,respectively. After use, the capillary was stored accordingto the protocol described by the manufacturer.

2.3 Theory

In order to predict the migration times for samples ofuncontrolled salinity we derived three calibration equa-tions including one, two, and three internal standards. Theconsiderations described and all further experimentsrefer to the case of chloride acting as the leading electro-lyte in TITP, stacking all anionic analytes having lowermobilities. Due to the application of coated capillaries andaddition of FC-PN to the BGE, the EOF is negligible andBGE anions (TAPS) serve as the terminating electrolyte[14, 16]. The pH of the injected sample was not controlled.The specific conductivity of the sample solution ks isgiven by [33]

ks ¼ FCMðjzMjmM þ jzRjmRÞ (1)

where F is the Faraday constant, CM is the concentrationof the matrix ions, and mM and mR are the absolute elec-trophoretic mobilities of the matrix anion of charge zM andcation of charge zR, respectively. When an electric field isapplied across a capillary containing a relatively long plugof the sample, the rear boundary of the sample zonemigrates as an isotachophoretic boundary with a self-sharpening effect [9] as shown in Fig. 1. By contrast, thefront will spread into a diffused transitional zone due toelectromigrative dispersion [34].

The rear of the diffused matrix extends up to the rearboundary (isotachophoretic) of the matrix zone with aconcentration plateau. At the time tx the concentrationplateau of the matrix zone is lost

tx ¼lsks

iðmM � mBÞ(2)

Figure 1. Schematic diagram of the TITP process. M,matrix chloride; A, B, analytes. (a) Initial injection; (b) TITPprocess under an applied electric field showing a plateauMp and diffused front Md of matrix ion M; (c) transitionfrom TITP to zone electrophoresis; and (d) zone electro-phoresis predominates and separation of the analytesoccurs.

where, ls is the sample plug length, i is the current density,and mB is the electrophoretic mobility of the BGE co-ion.For an analyte of mobility ma, the time tITP at which it leavesthe stack is given by

tITP ¼ txðmM � mBÞ2

ðmM � maÞ2(3)

Then the overall migration time ta of the analyte can beobtained by adding tITP and the zone electrophoresis timefor the remaining length of the capillary as [9, 33]

ta ¼lDva� lsksðmM � mBÞ

imMðmM � maÞ(4)

where va is the average migration velocity of the analyte inthe BGE and lD the effective capillary length from start tothe detector. Equation (4) shows that the ks value affectsthe migration times even if the other parameters are fixed.Using one internal standard 1, the explicit dependence onks can be eliminated as

ta ¼lDvaþ mM � m1

mM � mat1 �

lDv1

� �� (5)

where t1 is the migration time of the internal standard. Inorder to further eliminate the influences of the remainingparameters lD/va and lD/v1, we need to consider an addi-tional internal standard 2. Replacing the average migra-tion velocity vi (i = a, 1, and 2) in Eq. (4) with the product mi

and the average electric field ,E. in zone electrophore-



sis, ta can be expressed in terms of the migration times ofthe two internal standards, t1 and t2, and the matrix mo-bility mM as

ta ¼m2ðm1 � maÞðmM � m2Þt2 � m1ðm2 � maÞðmM � m1Þt1

maðm1 � m2ÞðmM � maÞ(6)

Using a third internal standard 3, the dependence on mM

can be eliminated and ta is given as

ta ¼m1m2ðm1�m2Þðma�m3Þt1t2 þ m2m3ðm2�m3Þðma�m1Þt2t3 þ m3m1ðm3�m1Þðma�m2Þt1t3

�ma ½m1ðm2�m3Þðma�m1Þt1 þ m2ðm3�m1Þðma�m2Þt2 þ m3ðm1�m2Þðma�m3Þt3�

(7)

By inserting appropriate parameters in a spreadsheet, thecalibrated migration times for an electropherogram canbe conveniently calculated using Eqs. (5), (6), or (7).

3 Results and discussion

3.1 Effects of the sample plug on TITP migrationtimes

Samples of biological origin usually contain a sufficientamount of chloride, which can be used for online pre-concentration by TITP stacking of minor anionic analytesof suitable mobility using chloride as the leading electro-lyte and the BGE as the terminating electrolyte. The chlo-ride concentrations in a set of such samples are expectedto vary depending on the history of biological conditionsof the sample donors. According to Eq. (4), samples con-taining variable concentration of chloride or variablesample plug length will show variations in migration timesof the analytes stacked by TITP as shown in Fig. 2. Sam-

ples of 15 analytes having different NaCl concentrationsor plug lengths were analyzed in a BGE composed of100 mM TAPS, 80 mM TEA, 0.02 vol% FC-PN at pH 9.0.As expected, quite large variations (20–40%) of averagedmigration times for respective concentrations of NaCl inmatrix were observed in a series of electropherograms forsamples in which the NaCl concentrations ranged from100 to 300 mM (Figs. 1a–e). Furthermore, samples of pluglengths ranging from 14 to 35% of the effective capillarylength also exhibited substantial variations in migrationtimes (Figs. 1f and g). Therefore, careful identification ofthe peaks is required in view of the fact that, althoughTITP of such samples offers a convenient online pre-concentration, the matrix of uncontrolled salinity inducessubstantial variations in migration times and peakheights.

3.2 Application of internal standards

The raw actual mobilities of the 15 analytes, as given inTable 1, were obtained by CZE using the same BGE asthat of TITP. For this experiment a sample prepared in theBGE was injected for 1 s at 36103 Pa. In Fig. 2, peaknos. 1, 9, and 13 refer to three colored compounds forinternal standards, CFL, FL, and DCF, respectively, whichcould be easily identified by monitoring the absorbancenear 500 nm as shown in Fig. 3. In order to incorporatethe nonideal behaviors of the saline matrix, another set offitted actual mobilities (Table 1) of the analytes wereobtained from an electropherogram of a standard samplein 200 mM NaCl by solving Eq. (7) for ma for each analyte

Figure 2. Electropherograms ofstandard saline samples con-taining NaCl (a) 100 mM,(b) 150 mM, (c, f, g) 200 mM,(d) 250 mM, and (e) 300 mM.Conditions: 50/57 cm m-Sil-FCcoated capillary with 50 mm ID;Injection, (a–e) 240 s, (f) 120 s,and (g) 180 s at 36103 Pa; BGE,100 mM TAPS, 80 mM TEA,0.02 vol% FC-PN, pH 9.0; driv-

ing current, 225 mA; absorbance, 254 nm. Peak numbering and concentration (mM): 1, CFL (0.5); 2, GMP (5); 3, AMP (5); 4,NB (5); 5, NAN (5); 6, AB (5); 7, BB (10); 8, AMB (5); 9, FL (1.25); 10, IB (1.25); 11, 1-NAP (5); 12, DMB (10); 13, DCF (1.25);14, TMB (10); and 15, 2-NAP (2.5). Migration order was maintained for all runs. Peak nos. 1, 9, and 13 were internalstandards.



Figure 3. Identification of inter-nal standards in a run withabsorbance monitored at(a) 254 nm and (b) 500 nm.Standard sample in 200 mMNaCl; injection 240 s at36103 Pa, other conditions asin Fig. 2.

Table 1. Actual mobilities of the anions

Analyte 6109 m2V21s21

Rawa) Fittedb)

Chloride – 241.96c)

Iodide – 239.92c)

Nitrate – 239.14c)

CFLd) 222.38 –GMP 221.14 221.38AMP 220.35 220.48NB 219.96 220.07NAN 219.07 219.07AB 218.49 218.47BB 217.99 217.95AMB 217.63 217.61FLd) 217.29 –IB 216.97 216.941-NAP 216.61 216.60DMB 216.14 216.12DCFd) 215.83 –TMB 215.70 215.672-NAP 215.37 215.38

a) Actual mobilities obtained experimentally by CZE(details given in the text).

b) Actual mobilities obtained experimentally by applyingEq. (7) to TITP data.

c) Actual mobilities obtained experimentally by usingEq. (6).

d) Analyte used as the internal standard.

in terms of the raw actual mobilities and migration timesof the three internal standards. Similarly, Eq. (6) was usedto calculate the fitted actual mobilities of chloride, iodide,and nitrate (Table 1), when each of them was used as theleading electrolyte (200 mM) in three separate experi-ments (n = 363).

The calibration of migration times using one internalstandard (Eq. 5) was done by selecting FL as an internalstandard, whose peak was more or less in the middle ofthe analyte peaks. The two migration time terms (lD/va andlD/v1) in Eq. (5) were determined separately from CZE. Thetwo sets of mobilities (raw and fitted) as given in Table 1were used to obtain the two sets of migration times pre-dicted by Eq. (5), which were then compared with experi-mentally observed migration times, as listed in Table 2aand b, respectively, for various saline matrices. As shownin Table 2, for all the saline matrices the slopes were sub-stantially higher than “one” and the intercepts were sig-nificantly below “zero”, showing considerable deviationsin the predicted migration times from the experimentalmigration times. Figure 4a shows the differences betweenthe predicted and the experimental migration times asresiduals. These residuals were used to calculate themean absolute error (MAE = S)yi 2 xi)/n), which was 10 s(n = 5614) for the 14 analytes in five saline matricesranging from 100 to 300 mM at intervals of 50 mM. Al-though the migration times predicted by a single internalstandard calibration were correlated with the observedmigration times showing fairly high linear correlationcoefficients (0.9962–0.9984) as listed in Table 2, themethod was ineffective considering the large residuals foranalyte peaks having migration times quite different fromthat of internal standard peak, which could be attributedto the fact that the two terms, lD/va and lD/v1, in Eq. (5) arestrongly dependent on the experimental conditions.

Next we applied Eq. (6) using two internal standards CFLand DCF, eliminating the two terms, “lD/vi”. The standard-ization method utilizing two internal standards was foundto be highly effective and experimental migration timesdeviated less than 1% from the calculated ones. Theequations of regression lines obtained by plotting the



Figure 4. Comparison of resi-duals obtained by calibration ofmigration times in various salinematrices with (a) one (n = 5614),(b) two (n = 5613), and (c) three(n = 5612) internal standardsusing the fitted mobilities fromTable 3 in Eqs. (5)–(7), respec-tively. Internal standards: Peakno. 9 (a); 1 and 13 (b); 1, 9, and13 (c). Sample NaCl: 100 mM( ), 150 mM (u), 200 mM ( ),250 mM (j), and 300 mM ( ).u u

u

predicted against the experimental migration times invarious saline matrices are given in Table 2. Slopes andintercepts of the regression lines approached 1 and 0,respectively. As shown in Tables 2a and b, the predictedand the experimental migration times correlate excellentlywith each other, with narrow ranges of their respectivecorrelation coefficients from 0.9997 to 0.9999 and 0.9999to 1.0000, for the raw and the fitted actual mobilities of theanalytes, respectively. However, the use of the fitted overthe raw actual mobilities of the analytes was found to be

more efficient in predicting the migration times, whichwas reflected in the respective MAEs of 0.4 s (n = 5613),and 0.8 s (n = 5613), for the 13 analytes in the five salinematrices as described above. The peak numbers 1 and13 were excluded while calculating the MAE since thesewere used as internal standards. The residuals of thepredicted migration times, using fitted mobilities, and theexperimental migration times are shown in Fig. 4b.Obviously, Eq. (6) eliminates all the parameters in Eq. (4)contributing to deviations in migration time of an analyte,



Table 2. Equations of linea) and linear correlation coefficients (r) comparing the calculated and experimental migrationtimes with a matrix NaCl using (a) the raw actual mobilities of the analytes

Matrix NaCl Single internal standardb) Two internal standardsc) Three internal standardsd)

Slope Intercept r Slope Intercept r Slope Intercept r

100 mM 1.1909 21.9077 0.9984 0.9896 0.1150 0.9999 0.9954 0.0507 0.9999150 mM 1.2974 23.1731 0.9977 0.9881 0.1323 0.9999 0.9897 0.1142 0.9999200 mM 1.3058 23.5155 0.9973 0.9901 0.1164 0.9998 0.9903 0.1133 0.9998250 mM 1.3263 24.0253 0.9967 0.9972 0.0356 0.9997 0.9908 0.1195 0.9997300 mM 1.2955 23.7531 0.9968 0.9995 0.0071 0.9997 0.9919 0.1084 0.9998

(b) The fitted actual mobilities of the analytes

100 mM 1.1899 21.8980 0.9984 1.0020 20.0131 0.9999 1.0076 20.0754 0.9999150 mM 1.2946 23.1423 0.9976 0.9993 0.0109 1.0000 1.0008 20.0067 1.0000200 mM 1.3004 23.4508 0.9971 1.0000 0.0002 1.0000 1.0003 20.0028 1.0000250 mM 1.3174 23.9123 0.9963 1.0057 20.0701 0.9999 0.9994 0.0113 1.0000300 mM 1.2851 23.6181 0.9962 1.0075 20.0951 0.9999 1.0001 0.0032 1.0000

a) y = mx 1 c, y = calculated migration time, x = experimental migration time.b) Calculated migration time obtained by using Eq. (5) with one internal standard 9.c) Calculated migration time obtained by using Eq. (6) with two internal standards 1 and 13.d) Calculated migration time obtained by using Eq. (7) with three internal standards 1, 9, and 13.

except for the mobility of the leading electrolyte. In orderto eliminate this dependence, Eq. (7) could be used withan additional third internal standard.

Equation (7) required the three migration times, t1, t2, andt3, of the three internal standards CFL, FL, and DCF,respectively. Two sets of predicted migration times wereobtained using either the raw or the fitted mobilities of theanalytes. The predicted and the experimental migrationtimes were compared in Table 2 in terms of their linearregression parameters. Although standardization withtwo internal standards predicted the migration times withhigh accuracy, a slight improvement in the migration timepredictions was expected by using standardization withthree internal standards since Eq. (7) acquires the max-imum information from the same electropherogram topredict the migration times of the analytes. Thus, asexpected, we found the least values of MAE with threeinternal standards, for the respective raw and fitted mo-bilities of the 12 analytes, as 0.72 s (n = 5612) and 0.34 s(n = 5612), in the five saline matrices. The residuals of thepredicted (using fitted mobilities) and the observedmigration times are shown in Fig. 4c.

Practically, the methods using standardization with two/three internal standards were found to be equally effec-tive, having the advantage of gaining independence fromthe mobility of the leading electrolyte in the latter. Con-versely, the actual mobility of any leading electrolyteunder working conditions could be obtained throughEq. (6), as we were able to obtain for I2 and NO3

2 (Table 1)using the fitting procedure described. To verify those

numbers, two standard samples containing 200 mM ofthe respective leading electrolytes I2 and NO3

2 were an-alyzed under the conditions given in Fig. 2, and the cali-bration was done using Eq. (6). The comparison of thepredicted and the experimental migration times providedhighly impressive linear regression parameters, such as aslope, intercept, and linear correlation coefficient of0.9980, 0.0288, and 0.9999, respectively, where the MAEwas found to be 0.26 s (n = 2613). Since it requires alower number of internal standards and is easy to handleeven with a hand-held calculator, the method using stan-dardization with two internal standards was furtherinvestigated for its tolerance to variations from the select-ed separation parameters.

3.3 Effects of other experimental conditions

Conditions such as the temperature, plug length, ionicstrength, and pH of the BGE are prone to a certain level ofuncertainty directly or indirectly depending on the meth-ods of measurement or techniques of manual handling.Since they are difficult to reproduce exactly, the variationsin such parameters contribute substantially to the uncer-tainty of migration times. Although the mobilities dependon the separation parameters such as the working tem-perature and pH of the BGE, we found that a substantialdeparture in such parameters from the working condi-tions was acceptable when used in conjunction with ourinternal standardization method. By varying the parame-ters one at a time, standard samples containing 200 mM



NaCl were analyzed. The variations from the optimumconditions were: Temperature 610%, plug length 625%,ionic strength 65%, and pH of the BGE 60.2 U, whichresulted in substantial fluctuations in raw migration timesof 10, 8, 10, and 15%, respectively (n = 3). For each set ofmeasurements the internal standards CFL and DCF wereidentified and the calibration was performed to predict themigration times. The MAE values after calibrating thevariations in temperature, plug length, ionic strength, andpH of the BGE were 2.5, 0.41, 1.5, and 1.7 s (n = 2613),respectively. The errors were negligible and presented nohindrance in picking up the right peaks in the electro-pherogram in spite of substantial variations in such pa-rameters. Note that the MAE due to variations in pluglengths was an order of magnitude less than the others.This could be attributed to the fact that the plug lengthvariations do not affect the mobilities. In fact, our stand-ardization method with two internal standards was quiterobust even for variations in experimental conditions andthe peaks could be identified with a high level of con-fidence.

3.4 Quantification and application to realsample

Using TITP conditions, a several 100-fold sensitivityenhancement for our analytes in a standard salinesample was realized compared to conventional CZE

analysis. For CZE a standard sample was prepared inBGE and injected for 1 s at 36103 Pa, while for TITP asample contained 200 mM NaCl and was injected for240 s at 36103 Pa. The sensitivity enhancement factorfor each analyte is listed in Table 3. Standard samples in200 mM NaCl containing analytes of concentrationsfrom 0.05 to 100 mM were analyzed to obtain variousanalytical parameters under the TITP conditions, asgiven in Fig. 2c. Then the peak heights were plottedagainst the respective analyte concentrations to obtainthe linear regression lines. The detector responseshowed good linearity over two orders of magnitude, asreflected in the calibration equations and linear correla-tion coefficients in Table 3. The migration times of theanalytes in a standard sample were excellently repeat-able with RSD from 0.01 to 0.02% (n = 3). The repeat-ability in peak heights was good with RSD from 2 to 4%(n = 3) and the plate numbers were high as listed inTable 3. Since the peak heights showed a linear de-pendence on the chloride concentration in the samplematrix, one internal standard was successfully used forquantification. For quantification FL was used as theinternal standard [35]. A standard sample containing200 mM NaCl was used to calculate the response fac-tors, and various standard samples containing NaCl inthe range of 6100 mM showed a range of recoveriesfrom 90 to 110%, which were 60–80% better than theusual external calibration methods.

Table 3. Sensitivity enhancement factor (SEF), linear regression equation, linear correlation coeffi-cients (r), linear dynamic range (LDR), reproducibility in peak height, and efficiency (N)

Analyte SEFa) Regression lineb) r LDR, mM %RSDc) Nd)

CFL 740 x = (y20.85)/13.1 0.9994 0.05–5 2 1 800 000GMP 410 x = (y20.90)/2.06 0.9982 0.5–50 4 850 000AMP 470 x = (y20.68)/1.76 0.9987 0.5–50 4 800 000NB 300 x = (y20.62)/1.35 0.9997 0.5–50 3 740 000NAN 300 x = (y20.01)/1.36 0.9971 0.5–50 2 300 000AB 300 x = (y20.53)/0.74 0.9980 0.5–50 3 610 000BB 280 x = (y20.44)/0.32 0.9962 1–100 2 500 000AMB 280 x = (y20.35)/0.80 0.9985 0.5–50 4 620 000FL 400 x = (y20.20)/5.37 0.9997 0.1–10 2 830 000IB 280 x = (y20.26)/5.31 0.9999 0.1–10 4 600 0001-NAP 280 x = (y20.31)/1.14 0.9996 0.5–50 3 560 000DMB 270 x = (y20.24)/0.92 0.9996 1–100 4 540 000DCF 300 x = (y20.25)/7.19 0.9977 0.1–10 4 630 000TMB 320 x = (y20.13)/0.46 0.9981 1–100 3 340 0002-NAP 330 x = (y20.33)/4.28 0.9996 0.3–30 3 950 000

a) TITP, matrix 200 mM NaCl, injection 240 s at 36103 Pa; CZE, matrix 10 mM BGE, injection 1 s at36103 Pa; SEF = (dilution factor6peak height in TITP)/peak height in CZE.

b) x = concentration (mM), y = peak height (mAU).c) n = 3.d) Number of theoretical plates.



Figure 5. Electropherogram of1:10 diluted standard referenceurine ME28351 spiked with theanalytes and 200 mM NaClmatrix. Injection for 240 sat 36103 Pa. Absorbance(a) 254 nm, and (b) 500 nm.Peak numbering and analysisconditions as in Fig. 2. *Un-known peaks.

In order to demonstrate the applicability of our method ina real situation, a standard urine reference (ME28351) wasused. Figure 5 shows an electropherogram obtained byanalyzing a standard reference urine sample ME28351(1:10 dilution) spiked with our analytes and the internalstandards under the conditions given in Fig. 2c. The twointernal standards CFL and DCF were clearly identified inthe electropherogram from the auxiliary channel. Equa-tion (6) was applied to calibrate the migration times. Thenumbered peaks were identified by the predicted migra-tion times as well as substantiated by the spectrumcomparison. The predicted migration times were highlyaccurate, where the MAE in our predictions was 0.90 s(n = 12). Peak no. 8 could not be confirmed since a highlyconcentrated peak originating from the urine sample wasoverlapping the migration time range of the peak. More-over, in Fig. 5 peak no. 9 (FL) was also identifiable in theauxiliary channel, cross-checking the accuracy of thecalibrated migration times. Samples in ME28351 (1:10dilution), 200 mM NaCl, and 0.1 mM of FL as an internalstandard for quantification showed 80–95% recovery ofthe spiked analytes based on peak heights.

4 Concluding remarks

Variations in migration times of anionic analytes as strongelectrolytes stacked by matrix chloride, acting as a lead-ing type of stacker for samples of uncontrolled salinity,could be successfully calibrated using two or three inter-nal standards, as given in Eqs. (6), (7), respectively. FromEq. (4), which describes the migration time of an analyteunder TITP conditions, the parameters including thesample conductivity, plug length, analyte velocity in BGE,terminating electrolyte mobility, current density, andeffective length were eliminated to obtain Eq. (6). In addi-

tion, the mobility of the leading electrolyte could also beeliminated, as in Eq. (7). Since the actual mobilities,obtained by fitting the migration times of the analytes in astandard saline sample under the working conditions,were used, the need for supplementary CZE experimentswas significantly reduced. Practically, the two methods,using Eqs. (6) and (7), were equally efficient for the pre-diction of the migration times, with respective MAE valuesof 0.4 s (n = 5613) and 0.34 s (n = 5612) in five standardsaline matrices containing 100–300 mM NaCl at intervalsof 50 mM. The calibration method based on two internalstandards was further applied to identify peaks with anMAE of 0.9 s (n = 12) in an electropherogram of a standardreference urine matrix without conductivity normalizing,desalting, or cleaning the sample. Moreover, our calibra-tion methods were quite immune to substantial variationsin such parameters as the temperature, plug length, andionic strength, and pH of the BGE. Thus, it can be utilizedto great advantage in capillary array electrophoresiswhere a number of capillaries are used for high-through-put analyses. The proposed methods of calibration canbe used as a helpful means of identifying peaks in realsamples of uncontrolled salinity where the calculationsinvolved are quite simple, executable on a spreadsheet oreven a hand-held calculator.

Authors thank Y. O. Jang for his assistance in CE experi-ments. This work was supported by the KOSEF of Korea.

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Calibration of migration times of variable salinity samples with internal standards in capillary...

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