The Modern Student loborotory:
HPLC HPLC Method Development with the Photodiode Array Detector A Laboratory Experiment Vincent T. ~emcho'and Harold M. McNair
Virginia Polytechnic Institute and State University, Blacksburg, VA 24061 Henrik T. Rasmussen
Colgate-Palmolive, 909 River Road, PO Box 1343, Piscataway NJ 08855-1343 The Photodiode Array (PDA) detector is perhaps one of the most versatile and useful HPLC detectors available today. Most PDA's monitor at least two different wavelengths to allow for the selective trace detection of compounds with different UV maxima. Complete UV spectra can be taken at any point during a chromatographic run to allow for comparison with a standard spectrum of a given analyte, which is an aid in qualitative analysis. Monitoring of "peak purity" is also possible. In this technique, several spectra are collected at different times during the elution of a chromatographic peak. These spectra are then compared for similarity. Any differences between spectra indicate possible coelution of two or more components. The PDA is becoming increasingly imoortant in anda basic laboraboth industrial and a~ademiciettin~s, tow experiment illustrating the utility of this t w e of detector should be beneficialto studenti on the d e g e undergraduate level. The experiment we describe demonstrates the utility of a PDA and serves as an introduction to retention theory in reversed-phase HPLC. The use of a sample consisting of common preservatives (parabens) and a plasticizer (a phthalate) can also lead to a discussion of these materials as they are used in cosmetics and packaging applications.
give resolution of the entire mixture. A flow rate of 1.5 m M n is used throughout the experiment. Reagents HPLC-grade water and methanol were obtained from EM Science (Gibbstown, NJ). The parabens are from PolyScience (Niles, IL). The diethyl phthalate is from Fisher Scientific (Fair Lawn, NJ). The methanol and water are degassed with a helium sparge before use. Sample Preparation The solvent 5050 MeOH-H20 (vlv)is used in preparing 100-mg/mL stock solutions, one each of methyl paraben, ethyl paraben, propyl parahen, and butyl paraben, and a 100-mg/mL stock solution of diethyl phthalate. Then 1mL of each of these stock solutions is added to a single 50-mL volumetric flask, and the flask is brought to volume with the 50:50 MeOH-H20 solvent to provide a mixed sample solution. In addition to this mixed solution, two standards are necessary In a second 50-mL volumitric, 1mL of the methyl paraben stock solution is diluted to the mark with the 5050 MeOH-H20. The third solution is prepared as 1mL
Experimental Apparatus and Operating Conditions
The chromatographic apparatus consists of an L-6200 ternary gradient pump (Hitachi; Mito, Japan); an LC-235 PDA detector with GP20 printer (Perkin-Elmer; Norwalk, CT, USA); and a "3 by 3" C-18 column (4.6 mm x 3 cm, 3-bm packing) (Perkin-Elmer). The column eftluent is monitored at two wavelengths. One corresponds to a maximum in the W spectrum of a paraben (255 nm). The other corresponds to a maximum in the spectrum of a phthalate (225 nm). The analysis is normally performed isocratically, with the gradient pump being used only to rapidly change the initial concentrations of methanol and water during the development of the separation. An initial operating condition of 8O:ZO MeOH-H20 (vlv) should give coelntion of two components of the mixture (the desired starting condition). A final operating condition of 4050 MeOH-H20 should
' Author to whom currespondence should be addressed.
Figure 1. Initial chromatogramshowing coeluting compounds. Conditions are described in the text. Volume 69 Number 4 April 1992
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The Modern Student laboratory: HPLC of the diethyl phthalate stock diluted to the mark with 50:50 MeOH-H20 in a 50-mL volumetric. Results and Discussion Some adjustment of initial conditions may be necessary to achieve complete welution of the propyl paraben and diethvl Dhthalate. thoueh the conditions listed in the experin;en'tal section worEed well in this case. The desired end result is a chromatoeram with onlv four svmmetrical peaks, as in Figure 1. At this point, the data provided with the chromatogram should be discussed: spectrum numbers, retention time, wavelength of maximum absorbance, and purity index. An explanation of the retention mechanism in reversed HPLC should also be provided by the instructor. Explaining that analytes with longer hydrophobic moieties are retained preferentially should provide students with enough information to predict an elution order, so long as the phthalate compound is not included in the prediction. Students should be reminded of the structures of parabeus and phtbalates.
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COOR
Paraben
PMhalate
Any deviation from a perfect match results in an index value that is greater than 1. This is a clue to the experimenter that the peak is likely to represent two or more coeluting compounds. By examining the purity index values and explaining the process of comparison of spectra performed by the detector, the class should be able to select the peak that represents the coelution of diethyl phthalate and butyl paraben. Method Development Followine a discussion of the reversed-~hasemechanism, the students are prepared to begin ieveloping the HPLC method for the separation of the mixture. An adjustment in the strength of the mobile phase is made that imDroves the se~arationof the five com~onentsof the mixture. Ultimatelv. aRer several adiustments in the streneth of the mobile phase, all five cokpounds of the mixture are resolved (see F i w e 2). Immediatelv aRer ~erforminethis separation, the spectra taken at pe& maximum for one of the parabens and the phthalate are printed, as in Figures 3 and 4. The similarity of the paraben spectra, and the contrast between the paraben spectra and the phthalate spectrum, can serve as a starting point for a discussion of the UV spectroscopy involved. Under the same chromatographic conditions, a standard is run to further verify the identity of the diethyl phthalate peak. The Absorbance Profile Map Finally, an absorbance profile map is constructed to show the students how the observance of similar UV maxima in the spectra taken during the elution of different
Purify Index Values Purity index values, calculated by the detector electronics. are verv helnful in method develo~ment.These values a& obtaine"d byhybverlayingseveral spectra taken at different points during the elution of a chromatographic peak. This task is readily performed electronically. If the spectra fit perfectly on top of one another (that is, if they are identical), the detector indicates a high likelihood of peak puritv bv a reference number. twicallv 1.0. Figure 3. The UV spectrum of propyl paraben taken at peak maximum. -:.
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