Reverse-Phase High-Performance Liquid Chromatography ...

1 UC Berkeley College of ChemistryChemistry 105 Instrumental Methods in Analytical ChemistryReverse-Phase high -PerformanceLiquid Chromatography Analysis ofBiomoleculesShort ReportAuthor:JonathanMelvilleLab Partners:JakePrecht, PhuongTran, and JeremyHsuGraduate Student Instructors:RichardCooper& DanielMortensenApril 23, 20141 TheoryRP-HPLC, or Reverse-Phase High-Performance Liquid Chromatography is a type of chro- matography the features a Liquid mobile phase, higher resolving power than traditionalliquid Chromatography , and a specific combination of stationary- and mobile-phases thatresults in separations that are the opposite of a traditional HPLC.

2 Chromatogra-phy, in general, utilizes the flow of a mobile-phase containing the analyte througha stationary-phase depending on the intermolecular forces of attraction between theindividual analytes and the stationary- and mobile-phases, the various components ofthe sample will exit the chromatographic column at different times[?]. A substancethat experiences greater intermolecular forces of attraction to the stationary-phase willbe slowed, and will exit the column later than a substance without such attraction.

3 Ifthe mobile-phase is a Liquid (as opposed to, say, a gas), the process is considered liquidchromatography ; HPLC improves upon this process by pumping a pressurized liquidmobile-phase through an adsorbant stationary-phase, yielding far greater separation andresolving power than regular gravitationally-run Liquid Chromatography . The labelling Reverse-Phase is far more arbitrary in origin historically, chromatographic columnsutilized a polar stationary-phase and a nonpolar mobile-phase; Reverse-Phase chromatog-raphy , by contrast, uses a nonpolar stationary-phase and a polar mobile-phase, resultingin an inverted separation from normal-phase Chromatography .

4 More polar analytes willtend to eluteearlierdue to decreased interaction with the now-nonpolar of its versatility, ease-of-use, and superior separation, HPLC is one of themost commonly-used classes of Chromatography (itself one of the most common analyticalmethods), along with other methods like gas Chromatography and column Results and DiscussionThis section contains only tabulated results from the Appendix. Derivations can be foundin Appendix A on page 5. Graphs can be found in Appendix B on page 12. Raw data1 Figure 1: Methionine, a nonpolar amino acid and one of our peptidescan be found in Appendix on page ResultsDue to experimental failures that are detailed further in the appendix, we were unableto absolutely and quantitatively determine the concentration of peptides in our unknownsample.

5 However, we were able to determine the ratios of peptides in our sample relativeto the concentration of our calibration samples:Peak 1 + (relative)Peak 2 + ppm (relative) (?!) Accuracy and ErrorOf course, our accuracy in this experiment was exceedingly low. Ignoring the fact that weare missing a crucial piece of data to turn our relative concentration values into absoluteones (due to a failure for the instrument to read our sample, no less), the data wedohaveconspires to give us abysmal fits that lead to nonsensical data. A brief examination ofour calibration curves shows that our error bars are gigantic and ourR2values abysmal(even, in one case, producing a negative slope that directly caused the apparently negativeconcentration of peptide seen above).

6 Because of the vast amounts of missing data pointsin our tables (every N/A was a point not found on the instrument), we are simply notable to calculate anything with any degree of 2: Bradykinin, a blood-vessel-dilating peptide that is comparatively DiscussionWhile we cannot make many quantitative statements about the success of our experiment,we can still state certain facts with a reasonable degree of certainty. We can, for instance,determine the identity of the first and second peaks eluted by looking at the structuresof the two peptides , the first peptide, is a rather small essential amino acid and is largelynonpolar in character due to the presence of a fairly lengthy alkyl chain.

7 Comparedto the much larger bradykinin, enriched with numerous amines, amides, imines, andaromatic groups, we can say with a reasonable amount of certainty that the first peakis that of bradykinin and the second is methionine. Because we are performing Reverse-Phase HPLC, our stationary phase is a rather waxy resin with long alkyl chains thatgive it significant nonpolar character, whereas our mobile phases are comparatively polarin nature. As a result, the more nonpolar methionine is more slowed by intermolecularinteractions with the stationary phase, allowing the more polar bradykinin to elute data was analyzed using the HPLC s built-in light source, an Agilent 1100/1200 Diode Array Detector that filters light from a tungsten-filament bulb through a couplinglens, a deuterium lamp, an achromatic lens, a holmium oxide filter, a support lens, aflow cell.

8 And a spectro lens befor finally passing through a programmable slit and ontoa grating which projects a final spectrum[3]. Because our sample only called for UVirradiation and not visible light (a detail noticeable in the data reports), onlythe deuterium lamp is of the data loss that plagued this entire experiment, we are unable to cal-culate the niceties of our isocratic separations, such as the number of theoretical the exact same reasons, we are unable to determine the amount of peptide in ourunknown sample. We were able to determine the relative concentrations (1690.)

9 Ppm ppm) by using the relative response factor, but even this approach is unhelpful dueto abysmal correlation coefficients (ranging from to ) and simply nonsensical an-swers (negative concentration?!). While it is possible that our poor outputs are the resultof detection limits and exceeding the linear range of the instrument, it is unlikely that oursamples deviated so significantly from reasonable values that virtually none of them wereable to construct a decent fit. Changing the wavelength of the detector would changethe response factors of our analytes; methionine, containing little to no -conjugation,is likely not an exceptional absorber compared to bradykinin, which has large, extended systems that likely absorb electrons of UV/Vis light readily.

10 This also explains thecomparatively lower absorption values of methionine compared to , the many flaws that riddled this experiment were likely a result of poorexperimental preparation and instrumental methods rather than shortcomings of theHPLC instrument itself. Because our group was one of the last groups to complete thislab, we were forced to make do with insufficient quantities of peptide starting material,and whilst coordinating our sample preparation with another group we inadvertentlyscrewed up the preparation of several of our samples.