1. Isocratic Resolution Experiments
Benzyl alcohol Benzaldehyde Methyl benzoate Toluene
1.1 µg/µL 80 ng/µL 1.2 µg/µL 0.80 µg/µL
Stationary phase: C-18 Column, 100 x 4.6 mm
Mobile phase: 70% H 2O : 30% MeCN
Flow rate: 1.5 mL/min, ~ 1200 psi
Detector: 254 nm
A sample chromatogram of a mixture of benzyl alcohol, benzaldehyde, methyl benzoate and toluene is given below, under isocratic elution conditions:
Note that each component was resolved into a distinct peak. Benzyl alcohol had a retention time of 0.895 minutes while toluene had a retention time of 1.772 minutes. The most polar compound in the series, benzyl alcohol, had the greatest solubility in the polar mobile phase and the lowest affinity for the non-polar octadecylsiloxane, C-18, stationary phase. Therefore, it was the first to arrive at the detector. By comparison, the least polar compound in the series, toluene, had the lowest solubility in the polar mobile phase and the greatest affinity for the non-polar stationary phase. Hence, it was the last to arrive at the detector.
a) Standard chromatograms
In order for students to practice qualitative and quantitative analyses, the HPLC method must be calibrated by recording the chromatogram of a standard universal mixture that contains all the possible components in an unknown.
The following standard chromatograms were recorded using a universal mixture. It contained benzyl alcohol, benzaldehyde, methyl benzoate and toluene, at three different levels: the first standard corresponds to the undiluted mixture; the second is a two- and the third a three-fold dilution of the reference mixture, respectively.
b) Calibration curves
The calibration of the HPLC method for quantitative analysis was based on relative concentrations at three distinct “levels”: the undiluted standard was assigned a concentration of 1000, the two-fold dilution 500, and the three-fold dilution 333, respectively. This procedure was adopted for the sake of simplicity; that is, in order to clarify the function of the calibration. In practice, student calibration curves would be constructed based on absolute component concentrations.
Consider for example the case of benzyl alcohol. At a relative concentration of 1000 (undiluted), the area calculated by the Turbochrom software is 1,479,568; at a concentration of 500 (twofold-dilution), the area is 721,996; finally, at a concentration of 333 (three-fold dilution) the area is 476,227. Accordingly, when these areas are plotted against the relative concentration values, a straight line obtains that intercepts the origin.
c) Student unknown: qualitative and quantitative analysis
In qualitative analysis, students compare the retention time of each component peak -- that is, the time required for an analyte to be eluted from the column -- versus the retention times in the standard. A match in the retention times constitutes positive identification for each of the components present in the unknown mixture.
In quantitative analysis students use the fact that the area under each peak is directly proportional to the concentration of that component. By measuring the areas in the standard chomatograms, at various concentration levels, a calibration curve can be constructed. Working with the calibration curves, students are able to determine the amount (in the microgram to nanogram range) of each component that is present in the unknown sample.
A representative student unknown chromatogram is presented below. For the sake of simplicity, the unknown was chosen to contain only one component. In practice, a student chromatogram will contain at least two peaks and therefore at least two distinct components.
By comparing the peak retention time, in this case 1.17 minutes, against the standard retention times, a student would determine that the unknown sample contained methyl benzoate. Thereafter, using the area under the peak provided by the Turbochrom software at 699,996, the corresponding calibration curve for methyl benzoate yields a (relative) concentration of about 1020.
2. Gradient Resolution Experiments
The second protocol involved gradient elution of a multicomponent mixture of six analgesic drugs commonly found in over-the-counter pain-relieving formulations: Aspirin, Acetaminophen, caffeine, Naproxen, Ibuprofen and salicylamide.
As stated earlier, HPLC analysis is a prominent analytical tool in pharmaceutical laboratories. Unlike the isocratic elution of benzyl alcohol, benzaldehyde, methyl benzoate and toluene, HPLC analysis of analgesics provides a more meaningful laboratory separation that illustrates a practical application. Once a method for the separation is developed, students can conduct both qualitative and quantitative analysis of an analgesic mixture.
The creation of a successful protocol for the separation of the compounds above represents a formidable problem in HPLC methods development. In order to develop the gradient elution analysis, it was first necessary to conduct many preliminary trial-and-error runs. These experiments indicated that the analgesic compounds could be divided into two groups of polar and non-polar molecules.
a) Isocratic elution of polar analgesics
The group of polar molecules included Aspirin, Acetaminophen and caffeine which could be eluted on an octylsiloxane, C-8, column. Furthermore, the mobile phase was composed of 67% H 2O, 3% 5 mM KH 2PO 4 (pH 6.1) and 30% methanol (volume by volume). Note that the presence of the potassium phosphate buffer was necessary in order to obtain reproducible results, particularly with respect to each component’s retention time. The optimized experimental conditions were as follows:
The following HPLC chromatogram was obtained by elution of a mixture of the three polar analgesics according to the conditions specified above:
b) Isocratic elution of non-polar analgesics
The remaining compounds, namely, Naproxen, salicylamide, and Ibuprofen are relatively non-polar and therefore could be eluted from a C-8 column using a mobile phase (solvent) that was predominantly made up of methanol. The optimized conditions for the separation of the non-polar analgesic group were as follows:
The following HPLC chromatogram was obtained by elution of a mixture of non-polar analgesics:
Note that under these conditions, it was possible to resolve Naproxen and Ibuprofen only. When a mixture of all three components was chromatographed, considerable overlap between Ibuprofen and salicylamide took place. The latter was probably due to the relatively polar nature of the octylsiloxane, C-8, stationary phase.
c) Gradient elution of polar and non-polar analgesics
According to the preceding isocratic elution experiments, the polar analgesic compounds Aspirin, Acetaminophen and caffeine required a relatively polar solvent. The mobile phase was made up of 67% H 2O, 3% 5 mM KH 2PO 4 (pH 6.1) and 30% methanol (volume by volume). By contrast, the non-polar analgesics Naproxen and Ibuprofen required a non-polar solvent. The mobile phase was made up of 7% H 2O, 3% 5 mM KH 2PO 4 (pH 6.1) and 90% methanol (volume by volume).
Because of the singular discontinuity in the polarity, the solvent ramp to resolve the analgesic mixture involved two time-dependent steps of decreasing polarity: in step 1 the mobile phase contained 67% H 2O, 3% 5 mM KH 2PO 4 (pH 6.1) and 30% methanol(volume by volume), for a period of two minutes. In step 2 the mobile phase was made up of 100% methyl alcohol.
Thus, in the first transient, the mobile phase is predominantly aqueous and therefore highly polar -- it elutes the polar analgesics. Conversely, in the second transient, the solvent is predominantly methanol which is relatively non-polar -- it elutes the non-polar analgesics.
d) Gradient elution chromatograms
The chromatogram below shows a solvent-only (“blank”) trial run. The temporal ramp in the solvent polarity has been highlighted using a color gradient ranging from blue (polar) to yellow (non-polar). As the white vertical marker indicates, two minutes after the run was initiated, absolute methanol was used as the solvent.
The arrival of methanol at the detector is clearly visible; the baseline shows a positive slope beginning after approximately six minutes into the run. The baseline drift in this case is generated by the fact that methanol has an appreciable absorbance at 240 nm, the wavelength at which the detector was set.
A mixture containing Aspirin, Acetaminophen, caffeine, Naproxen and Ibuprofen was then analyzed under gradient elution conditions. As the chromatogram below shows, all five analgesics were successfully resolved.
The gradient method operated as follows. When the sample was first loaded onto the column, the mobile phase was predominantly water. Consequently, the more polar Aspirin, Acetaminophen and caffeine traversed the stationary phase. The relatively non-polar Naproxen and Ibuprofen remained tightly adsorbed at the column’s gate.
After two minutes, as shown by the vertical white marker, switching to absolute methanol decreased the polarity of the mobile phase. As a result, the non-polar analgesics, Naproxen and Ibuprofen, were successfully dislodged from the non-polar matrix and eluted by the solvent.
3. Conclusion and Future Work
While the simple isocratic elution analysis of non-polar organic compounds is student-ready, the more difficult gradient analgesic resolution still requires additional work. In particular, quantitative calibration curves need to be constructed. In addition, only five of the total six analgesics were successfully resolved. As noted before, Ibuprofen could not be completely separated from salicylamide, using a relatively polar octylsiloxane, C-8, column.
In order to outflank this difficulty and achieve the total resolution of all six compounds, it is necessary to use a more hydrophobic octadecylsiloxane, C-18, stationary phase. The latter would enable students to simultaneously conduct both qualitative and quantitative pharmaceutical analyses.
Turbochrom Software
An important aspect in conducting liquid chromatography experiments using state-of-the-art analytical instrumentation is the fact an operator has to be thoroughly familiar with the software required to “instruct”, or program, the chromatograph. In the case of our Perkin-Elmer Series 200 HPLC, the software is a multidimensional cybernetic interface known as Turbochrom.
Basically, Turbochrom is used to create methods for analysis, to program the instrument’s autosampler, and to process the resulting chromatographic data. Because of its complexity, and in the interest of brevity, a discussion of Turbochrom has been omitted in this report. Suffice it to say that mastery of Turbochrom is a convoluted task that is extremely labor intensive in its own right, and currently very much in progress.
Epilog
In his book The Black Riders and Other Lines, no. XXIV, which was published posthumously in 1905, Stephen Crane -- arguably the greatest American poet, writer, and student of social and psychological reality of the 20 th century -- wrote:
I saw a man pursuing the horizon;
Round and round they sped.
I was disturbed at this;
“It is futile,” I said,
“You can never --“
“You lie,” he cried,
And ran on.
When the conclusion is true, the only logical alternative left is to reason backward to the evidence. Although he was not a natural philosopher like Albert Einstein, Steve Crane recognized that reality is curved. He who runs far and fast enough will inevitably regress at the Origin. The shortest distance between two points is not always a straight line.In the horizon, even parallel paths converge.
The foregoing experiments represent only the “finished” product that can be used for teaching organic chemistry students. Every successful chromatographic run reported herein was preceded by many unsuccessful trial and error chromatograms until each method was optimized. Thus, many hours were spent working on the project; far in excess of the “release” time that was awarded in the original grant.
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