NMR

Anal. Chem. 1998, 70, 590-595

Quantitative Measurements in Continuous-Flow HPLC/NMR Markus Godejohann and Alfred Preiss*

Fraunhofer-Institut fu¨ r Toxikologie und Aerosolforschung, Nikolai-Fuchs-Strasse 1, D-30625 Hannover, Germany Clemens Mu 1 gge

Institut fu¨ r Chemie, Humboldt-Universita¨ t zu Berlin, Hessische Strasse 1-2, D-10115 Berlin, Germany

Two methods for the quantitative determination of compounds in continuous-flow HPLC/NMR are described. The first method uses an internal standard (caffeine) of known concentration directly mixed into the mobile phase, while with the second method, a known amount of internal standard is injected onto the column during the chromatographic run. The latter method was validated using several nitroaromatic compounds and explosives. Deviations between the injected and calculated amounts of analytes are usually below 10% while the relative standard deviation ranges from 2% in the upper microgram range to 40% at the limit of detection. HPLC/NMR is a relatively new technique in mixture analysis which combines the separation efficiency of HPLC with the specificity of NMR. This method is mainly used to separate a compound of interest from a mixture to obtain its pure NMR spectrum for structure elucidation. In the past, this method was applied to the analysis of drug metabolites, polymers, and natural products, where in all instances no exact quantification was attempted. Recently, we demonstrated that the NMR method may be applied successfully in environmental analysis.1,2 In particular, we reported the application of continuous-flow HPLC/NMR coupling for the determination of pollutants in groundwater samples of an ammunition hazardous waste site.3 By injecting high volumes (400 µL) onto a short RP column and reducing the flow at 17 µL min-1 (in order to obtain intense signals and sufficient time for spectra accumulation), it was possible to identify pollutants down to the microgram per liter range after 1200-fold enrichment of the sample by solid-phase extraction. Such measurements are time-consuming, but the long acquisition time is justified if both compound identification and quantitation can be achieved in one step as reported in this study. Although commercial HPLC/NMR couplings have been available for only ∼10 years, the fundamental principles for quantitative measurements in continuous-flow LC/NMR were reported by Haw (1) Preiss, A.; Levsen, K.; Humpfer, E.; Spraul, M. Fresenius J. Anal. Chem. 1996, 356, 445-451. (2) Preiss, A.; Lewin, U.; Wennrich, L.; Findeisen, M.; Efer, J. Fresenius J. Anal. Chem. 1997, 357, 676-680. (3) Godejohann, M.; Preiss, A.; Mu ¨ gge, C.; Wu ¨ nsch, G. Anal. Chem. 1997, 69, 3832-3837.

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et al. in 1982,4 who suggested the use of an internal standard and discussed some sources of systematic errors. For quantitative HPLC/NMR measurements, some special requirements have to be considered: (a) HPLC pumps have to provide a constant and pulsation free flow; (b) the internal standard and the analytes must be sufficiently soluble in the mobile phase; (c) the real flow rate of the pump has to be known exactly; (d) the solvent suppression technique should not influence the intensity of the signals of the analyte or internal standard near the solvent resonance; (e) the protons selected for quantification have to be fully relaxed for each repetitive rf scan. In this paper, two methods for the quantitative determination of analytes will be described: (1) Internal Standard Method I. For gradient runs, the internal standard is dissolved in the mobile phase to compensate for the influence of the varying composition on the intensity of resonance lines. (2) Internal Standard Method II. For isocratic HPLC runs, a defined amount of the internal standard is injected onto the column at any time, preferentially at the end of the chromatographic run to avoid superimposition of its resonance lines with those of the analytes. THEORY Quantification of analytes in a mixture is based on the following equation:

ca )

csns FaMa FsMs na

(1.1)

Here, c stands for the concentration [µg mL-1], the subscript s and a stand for the internal standard and the analyte, respectively, and n represents the number of protons generating the NMR signal selected for quantification, F the area of the NMR signal, and M the molecular weight of the compound. In continuous-flow HPLC/NMR measurements, 1-D spectra are stored as rows into a serial file which can be represented in chronological order to give the complete pseudo-two-dimensional NMR chromatogram. Each row of the NMR chromatogram corresponds to a time interval, R, which is the time resolution of (4) Haw, J. F.; Glass, T. E.; Dorn, H. C. J. Magn. Reson. 1982, 49, 22-31. S0003-2700(97)00630-6 CCC: $15.00

© 1998 American Chemical Society Published on Web 02/01/1998

this NMR chromatogram (s row-1). When the time interval R is longer than the time width of the chromatographic peak, the compound representing the peak may in principal be measured within one row. Within the time interval R, a defined volume V depending on the flow of the HPLC pump (V˙ ) passes through the NMR cell of the LC/NMR probehead:

V ) V˙ R

(2.1)

According to Haw et al.,5 there is no difference if this volume is measured on-line or off-line by fraction collection. If an internal standard is added to the mobile phase with cs, the amount of analyte ma injected onto the column is given by eq 1.1, where

ca ) ma/V

(3.1)

The chromatographic peak is usually located in several rows. All rows showing the spectrum of the analyte can subsequently be coadded by the spectrometer software to a single 1-D spectrum. In this case, the detection volume V is given by

V ) V˙ RN

(2.2)

Here, N is the number of the coadded rows. The equation for the calculation of the amount of analytes injected onto the column by the internal standard method I is given by

ma )

csns FaMa V˙ RN FsMs na

(1.2)

The areas Fa and Fs in this equation are extracted from the 1-D spectrum obtained as a result of the coaddition. If the internal standard is injected separately onto the column, its corresponding peak is located in Ns rows, the analyte peak in Na rows. The number of rows for the analyte and for the internal standard may differ, leading to different detection volumes. In this case, the eqs 2.2 and 3.1 have to be replaced by a more general expression, where i may be replaced by the indices a for the analyte and s for the internal standard:

Vi ) V˙ RNi ci )

(2)

mi mi ) Vi V˙ RNi

(4)

Taking eq 4 into account, eq 1.1 can be transformed into eq 1.3, which allows the calculation of the amount of injected analyte with internal standard method II, where a known amount of internal standard ms is injected onto the column:

ma )

msns NaFaMa NsFsMs na

(1.3)

(5) Haw, J. F.; Glass, T. E.; Dorn, H. C. Anal. Chem. 1983, 55, 22-29.

Usually the peaks of the analyte and the internal standard have different retention times. Coaddition of the corresponding rows leads to two 1-D spectra, one giving the area of the internal standard signal Fs, the other the area of the analyte resonance line Fa. Thus, only the injected amount of internal standard ms must be known to calculate the amount of the analyte of known structure injected onto the column. Hence, no reference compounds and no calibration data are necessary for quantitative measurements as compared to other chromatographic methods. EXPERIMENTAL SECTION Instruments. Continuous-Flow HPLC-NMR. The mobile phase consisted of a methanol-D2O mixture (45/55 v/v) buffered to pH 2.3 by phosphate buffer. Caffeine was added as internal standard to this mixture to give a concentration of 31 µg mL-1. Chromatographic separation was carried out on a Merck RP Select B column (75-mm length, 4-mm i.d., 5-µm particle size) (internal standard method I) and on a Merck RP Select B column (125mm length, 4-mm i.d., 5-µm particle size, Darmstadt, Germany) (internal standard method II) at a flow rate of 0.1 mL min-1. The injection volumes were between 100 and 500 µL using a Rheodyne 7725i injection valve with a 1-mL sample loop (Rheodyne, Cotati, CA). The chromatographic system consisted of a LC gradient mixer from Bischoff (Leonberg, Germany) Model 1155, a Bischoff HPLC pump Model 2250, equipped with micropump heads, and a Bischoff UV detector Lambda 1000. Pseudo-2-D spectra were recorded on a Bruker AMX600 spectrometer at 600.13 MHz with a 1H-13C inverse LC probe head (4-mm i.d. of measuring cell with a detection volume of 120 µL). Solvent suppression was done by a 1-D version of the noesyprtp (Bruker, Rheinstetten, Germany) pulse sequence with presaturation during relaxation delay and mixing time on two frequencies simultaneously. The pseudo-2-D spectra have been recorded under the following conditions: In general, 32K data points in f2 with a sweep width of 14 706 Hz. The flip angle was 90° (9.6 µs). Internal Standard Method I. A total of 64 data points in f1 with a time resolution of 50 s row-1 and 8 scans per increment of the NMR chromatogram. The relaxation delay was 5 s plus an aquisition time of 1.1 s, which was found to be sufficient for protons 4, 5, and 6 of 2-nitrobenzoic acid at δ ∼ 7.8 ppm and the methyl group of caffeine at δ ) 3.9 ppm. Internal Standard Method II. A total of 32 data points in f1 with a time resolution of 361 s row-1 and 32 scans per increment. The relaxation delay was 10 s plus an aquisition time of 1.1 s, which is sufficient for the aromatic protons at high field values and for the methyl groups. Data were multiplied with an exponential function in f2 (LB ) 1 Hz) and processed with the XWINNMR software. Reagents. The origin and purity of the compounds are listed in Table 1. Deuterium oxide (99.9%) was from Promochem (Wesel, Germany). Standard Solution. The standard solution for internal standard method I contained 500 ng/µL-1 2-nitrobenzoic acid dissolved in the mobile phase (ca in eq 3.7). The standard solution for the internal standard method II contained compounds mentioned in Table 3 and was also dissolved in the mobile phase. The internal standard (2,6-diaminotoluene) Analytical Chemistry, Vol. 70, No. 3, February 1, 1998

591

Table 1. Assignment, Origin, and Purity of Compounds assignment 1 a

*2

3 4 5 6 7 8 9 10 11

compound 2-nitrobenzoic acid caffeine 2,6-diaminotoluene 2,4-dinitrobenzenesulfonic acid 1,3,5-trinitrohexahydro-1,3,5-triazine 4-nitrophenol 3,5-dinitrobenzoic acid 3,4-dinitrobenzoic acid 2,4,6-trinitrotoluene 1,2-dinitrobenzene 4-amino-2,6-dinitrotoluene 2,3-dinitrotoluene

abbrev

origin

2-NBA

Aldrichb

2,6-DAT 2,4-DNBSA RDX 4-NP 3,5-DNBA 3,4-DNBA 2,4,6-TNT 1,2-DNB 4-A-2,6-DNT 2,3-DNT

Flukac Aldrich Aldrich Promochemd Fluka Promochem Promochem Promochem Riedel-de-Hae¨ne Promochem Aldrich

purity (%) 96 99 97 98 96 99 99 99 99 99 99 99

a Internal standard. b Aldrich, Steinheim, Germany. c Fluka, Buchs, Switzerland. d Promochem, Wesel, Germany. e Riedel-de-Hae ¨n, Seelze, Germany.

was dissolved with the other compounds in the mobile phase at a concentration of 317 µg mL-1. Safety Consideration. Explosives and related nitroaromatic compounds are toxic (1,3,5-trinitrohexahydro-1,3,5-triazine, (RDX), 1,3,5,7-tetranitro-1,3,5,7-tetrazacyclooctane (HMX), 2,4,6-trinitrotoluene), carcinogenic (dinitrotoluenes, 2,4,6-trinitrotoluene), and mutagenic (dinitrotoluenes, 2,4,6-trinitrotoluene) and should therefore be handled with special care. Procedure. Internal Standard Method I. Different amounts of 2-nitrobenzoic acid were injected onto the column by varying the injection volume. Integrated areas are those of the methyl resonance line of the internal standard (caffeine) located at δ ) 3.9 ppm and the resonance lines of the protons 4, 5, and 6 of the 2-nitrobenzoic acid at δ ∼ 7.8 ppm (cf. Figure 1c). Internal Standard Method II. The internal standard may be injected at any time of the chromatographic run. In this study, the internal standard, 2,6-diaminotoluene, was dissolved together with the other analytes in the mobile phase. The integral area Fs corresponds to the methyl group at δ ) 2.2 ppm, while the protons corresponding to the areas Fa of the compounds in Table 3 are listed in that table. RESULTS AND DISCUSSION Internal Standard Method I. Figure 1a shows the UV chromatogram of 2-nitrobenzoic acid (200 µg injected onto a short RP column under isocratic conditions); the column is overloaded, leading to a non-Gaussian peak shape. Figure 1c displays a lowfield contour plot of the HPLC/NMR chromatogram, Figure 1b the projection of this plot onto the time axis of the chromatogram. At a time resolution of 50 s row-1, the peak is visible within 15 rows of the NMR chromatogram. The total peak volume can be calculated as 1 mL which is ∼8 times the detection volume of the NMR cell (120 µL). In Figure 2a the signal-to-noise (S/N) ratios of the analyte signal and the signal of the internal standard obtained by stepwise coaddition of an increasing number of rows (beginning with two rows at the peak maximum) are shown in one plot. It is apparent from this figure that coaddition of an increasing number of rows leads to better S/N ratios. A maximum S/N ratio of the analyte signal can be observed after coadding eight rows, while the S/N ratio of the internal standard resonance line follows a square root function. Coaddition of further rows leads to a slight decrease of 592 Analytical Chemistry, Vol. 70, No. 3, February 1, 1998

Figure 1. UV and NMR chromatogram of a 200-µg injection of 2-nitrobenzoic acid. (a) UV trace at 400 nm, (b) projection of spectral points to the chromatographic axis, and (c) low-field contour plot of the NMR chromatogram. For compound identification, refer to Table 1. The numerical value outside the parentheses corresponds to the compound identification number listed in Table 1; the expressions in parentheses assign the signals in the NMR chromatogram to the protons generating these signals: 4-NBA, 4-nitrobenzoic acid.

Figure 2. S/N ratios of the analyte signal and the signal of the internal standard obtained by gradual coaddition of rows (a) and calculated amounts of injected analyte as a function of gradual coaddition of rows (b). Table 2. Calculated Amounts of 2-Nitrobenzoic Acid after Injection of Different Amounts onto the Columna inj amt (µg)

calcd amtb (µg)

deviation (%)

200 125 50

209 122 48

5 -2 -4

a Quantitation using internal standard method I at a flow rate of 0.1 mL min-1 (V˙ in eq 1.2) and a time resolution of 50 s row-1 (R in eq 1.2). b ma in eq 1.2.

the S/N ratio for the analyte signal. Nevertheless, the S/N ratio after 15 rows is still twice as high as it is calculated for two rows. Figure 2b represents the amount of 2-nitrobencoic acid calculated according to eq 1.2 for each 1-D spectrum (obtained by stepwise coaddition of rows). The coaddition of all rows showing the resonance lines of the analyte leads to a calculated amount of 209 µg, close to the known injected amount of 2-nitrobenzoic acid (200 µg). Table 2 compares the calculated and injected amounts of 2-nitrobenzoic acid for 50, 125, and 200 µg injected onto the column under the same chromatographic and spectroscopic conditions. There is a good agreement with the known amounts injected onto the column; the deviations are below 5% in all cases. Internal Standard Method II. Figure 3 shows both the UV trace and the low-field contour plot of the NMR chromatogram

Figure 3. UV trace at 300 nm and NMR contour plot of the first injection of 250 µL of the artificial mixture used for validation of the internal standard method II. For compound identification, refer to Table 1. The numerical value outside the parentheses corresponds to the compound identification number listed in Table 1; the expression in parentheses assign the signals in the NMR chromatogram to the protons generating these signals.

of an injection of 250 µL of the standard solution mentioned in the Experimental Section. In Table 3, the injected amounts are compared with the calculated ones (triplicate injections) at two injection levels. With the exception of RDX and TNT, the calculated and the expected values show differences in the range of 10% (Significant lower values for RDX at the 500 µL injection may be explained by the fact that this compound was not completely dissolved in the mobile phase due to its low solubility in water while TNT is injected at the limit of detection leading to an inaccurate integration area.). The relative standard deviations Analytical Chemistry, Vol. 70, No. 3, February 1, 1998

593

Table 3. Validation of Quantification for Internal Standard Method IIa compound (assignment)

signal for quantif

S/N ratio

inj amt (µg)

3,4-DNBA (7) 2,4-DNBSA (3) 3,5-DNBA (6) 4-NP (5) RDX (4) 2,3-DNT (11) 1,2-DNB (9) 4-A-2,6-DNT (10) 2,4,6-TNT (8)

H5 H6 H 2,6 H 2,6 H 2,2′, 4,4′, 6,6′ methyl H 3,6 methyl methyl

Injection Volume 250 µL 108 554 118 438 222 302 135 165 115 66 108 64 24 18 36 14 10c 3

3,4-DNBA (7) 2,4-DNBSA (3) 3,5-DNBA (6) 4-NP (5) RDX (4) 2,3-DNT (11) 1,2-DNB (9) 4-A-2,6-DNT (10) 2,4,6-TNT (8)

H5 H6 H 2,6 H 2,6 H 2,2′, 4,4′, 6,6′ methyl H 3,6 methyl methyl

146 123 262 167 90 134 18 103 22

Injection Volume 500 µL 1108 876 604 329 132 127 36 28 6

calcd amtb (µg)

deviation (%)

RSD (%) (n ) 3)

611 476 305 185 64 66 18 13 5

10 9 1% 12 -3 4 -2 -5 54

5 4 3 4 14 3 6 16 37

1215 925 589 351 101 130 34 30 13

10 6 -2 7 -24 2 -4 9 112

3 2 3 2 7 4 12 18 39

a For every injection volume, three injections were performed. Internal standard was 2,6-diaminotoluene, which was dissolved in the mobile phase with the other analytes in a concentration of 317 µg mL-1 leading to an injected amount (ms in eq 1.3) of 79 µg for the 250-µL injection and 158 µg for the 500-µL injection. b ma in eq 1.3. c Estimated value.

Figure 4. Low-field part of the 1-D spectrum as a result of the coaddition of the rows 10-14 (projection of the NMR chromatogram to the spectroscopic axis) in Figure 3. 3,4-DNBA (7), 2,4,6-TNT (8), and 1,2-DNB (9) are completely measured within these rows. After coaddition, the resulting 1-D spectrum contains the quantitative information of the injected amount of analyte. Integral values are normalized to the integral of the standard 2,6-diaminotoluene (methyl group) appearing in rows 1-6, which was set to the value 1.

of the calculated amounts are below 3% for strong signals (S/N >100) but increase drastically with decreasing S/N ratios. Figure 4 shows the resulting 1-D spectrum after coaddition of the 10-14 as marked in Figure 3 by perpendicular lines. The 594

Analytical Chemistry, Vol. 70, No. 3, February 1, 1998

signals shown belong to the fraction of analytes that are in the detection volume within the time interval marked in Figure 3. The validation of method II demonstrates that it is possible to get quantitative results from the spectral data of the NMR

chromatogram with sufficient accuracy, if the injected amount of the internal standard is known exactly. Equation 1.3 is strictly valid for isocratic conditions, where the composition of the mobile phase does not change. For gradient runs, method I should be applied because the internal standard in the mobile phase and the analyte will be measured at exactly the same spectroscopic conditions. For both methods there are some limitations that have to be considered: (a) The integration of overlapping signals that belong to coeluting compounds leads to erroneous quantification results. Due to the high selectivity of this coupling technique, it is very unlikely that coelution of two or more compounds with NMR signals that show identical chemical shift values occur. (b) Relaxation times for the protons used for quantification have to be determined prior to quantitative measurements. If the protons of the analytes have varying relaxation times, the relaxation delay of the NMR measurement has to be set to a sufficiently high value so that the proton with the longest relaxation time is completely relaxed between successive scans. Therefore, the proton with the longest relaxation time which will be used for quantification, determines the required relaxation delay of the continuous-flow measurement. (c) The precision of the method decreases drastically with decreasing S/N ratios (refer to Table 3). CONCLUSIONS Quantification of complex mixtures of organic compounds such as found in environmental samples by on-flow HPLC/NMR is possible if an internal standard is added to the sample. This

internal standard may either be mixed to the mobile phase or be added separately during any time (preferentially toward the end) of the chromatogram. While the former method is particularly suited for gradient runs, the latter is restricted for isocratic elution. Necessary requirements for such a quantitation are as follows: (a) the HPLC pump must provide a constant flow; (b) the solvent suppression technique should not influence the intensities of the signals of the analytes and the internal standard; (c) the protons used for quantification must be completely relaxed. Such quantification may be carried out with an error of