Determination of Aromatic Ethyl Esters in Whiskey

Experiment 7: Determination of Aromatic Ethyl Esters in Whiskey

Asia Nakamura; Partner: Yuchen Li

Intro:

        The aromatic compounds in whiskey are made during fermentation, the breaking down of sugar to make alcohols. Short chain ethyl esters are formed early in the process, longer chains are formed later. Use of Solid Phase Extraction and gas chromatography are used to separate these small amounts of mixed chemicals for analysis.  

        SPE is the process of selectively concentrating and purifying a sample based on the  stationary phase used. It utilizes a syringe shaped column with a small resin bed(stationary phase) where the liquid sample (mobile phase) is passed through. The stationary phase used, LiChrolut EN, provides a good retention time of the aromatic ethyl esters of interest. The analytes of interest will initially remain on the resin. A suitable solvent will later be used to elute the analytes of interest.

        GC is used for separation of analytes. The analyte of interest is analyzed using a mass spectrometer detector. The retention time is detected at the end of the column, and internal standards are run so relative retention times can help identify the unknown species because of slight changes in conditions. The column used is nonpolar (stationary phase: (5%--phenyl)-methylpolysiloxane bonded to a .32mm diameter tube 30 m long). We will receive a total ion chromatogram, individual mass spectrum of each peak and m/z extractions. This contains a molecular ion (M+) which corresponds to the molecular mass of the compound and essentially the entire compound with a positive charge. The molecular ion is the largest m/z in the mass spectrum the second most important peak is the tallest peak = base peak (most common fragment to be formed) and given an arbitrary height of 100. All other peak heights are relative to the base peak. Every peak that has a certain m/z, one or more peaks may be shown.

Methods:

Part 1: SPE COLUMN PREP

        Rinse column with 10mL methylene chloride, 10mL methanol, 10mL 15% ethanol in water

Part 2: SAMPLE CONCENTRATION AND PURIFICATION        

        Load 25 mL sample onto SPE column

        Rinse SPE column with 25 mL of solution A (50% methanol and 1% sodium bicarbonate in water)

        Dry SPE column through vacuum for 10 minutes.

        Elute analytes with 1.5mL methylene chloride and collect into GC vial

        Submit for GC-MS analysis.

Data:

• See printed chromatograms and sheets

Results:

1. *see graphs and sheets***

1. Ion 144.21 = 168 C Ethyl Hexanoate
2. Ion 172.27 = 207 C Ethyl Octanoate
3. Ion 200.31 = 251 C Ethyl Caprate
4. Ion 228.38 = 269 C Ethyl Laurate
5. Ion 256.42 = 295 C Ethyl Myristate
6. Ion 284.48 = 377.5 C Ethyl Palmitate
7. An increase in molecular weight is correlated with an increase in boiling point. The boiling point effects how long the compound stays in the column, the higher the boiling point, the longer it stayed in the column. For alcohols, they’re polar molecules and their boiling point increases with molecular weight. Volatility of a compound at the column temperature influences the distribution of the compound between the gaseous mobile phase and the stationary phase. Boiling points measure the relative volatilities of compounds in a mixture. It’s the transition from the liquid phase to gas and back that is responsible for the separation using this technique. Very volatile compounds will not interact with the column if the temperature is too high above the boiling point, and too low of a temperature will cause the entire amount of compound to be condensed. The more transfers, the better efficiency.

1. See sheet for identification of compounds

1. The retention times of the standards and unknowns are very similar. However, the slight discrepancies in the retention times can be responsible for the negative concentration observed in the sample. From identifying all the peaks on the graph, it is clear that Ethyl Laurate should have been visible, being a major peak; however, the data does not support that determination. It is from looking at the graphs of the ethyl ester standards that I was able to correlate the peaks with the identity of the compound within the sample.

1. From my sample, I was able to determine almost all of the compounds. I was able to determine Ethyl Hexanoate (.0689 𝞵g/mL), Octanoate (.2469𝞵g/mL), Caprate (.3109 𝞵g/mL), Myristate (.0322 𝞵g/mL) and Palmitate (.0124 𝞵g/mL), the one not identified being Laurate. This absence was mainly due to a negative concentration value which is impossible. Originally, there was a lot of negative concentrations if the calibrated curve was not fit to zero as the points did not lie on the curve. After calibrating the standard curve to fit through zero, there is still a negative concentration value, but far less than calculated previously. This is attributed to error in the standards given and the data from them. Based on the data, there are more fruity ethyl esters than waxy-oily ethyl esters. Comparing to Roxana’s group, they were able to identify all of the compounds in their Whiskey sample. They had a greater amount of waxy-oily ethyl esters than fruity. Due to spacial limits of the bottle, the more concentrated solution could be towards the bottom if allowed to settle, which could have led to the differences in data collected and analysis.

1. SPE is a good technique for isolating caffeine because the mixture is run through rapid and undergoes sample distinction which involves a partitioning of solutes. GC separates and measures the retention time the sample is in the column. A calibration curve of internal standards should be constructed first in order to have a method of comparison in order to quantify unknown data. Optimization of conditions must be determined prior to determine best separation. After, an unknown amount of caffeine can be sampled and run through SPE and then Gas Chromatography. Caffeine has a relatively low boiling point 178 C, but is polar, and would elute first on a chromatogram under the same conditions as the previous experiment.  For SPE, the mechanism is to extract and concentrate low levels of caffeine in water using methanol, water, load sample, dry vacuum, add methanol and collect eluant, and submit for GC. GC should be set relatively at 70 C for 2 min and 250 C for 5 min for the duration of the run for optimal extraction. Standards should be small to be readable, 𝞵g/mL. Caffeine is a polar molecule, hence the stationary phase and solvents used in the experiment would not be useful as the nonpolar column would elute the polar compounds first. A polar column should be used in order to increase retention time of the polar caffeine, and it should be the last peak on the chromatogram. Using the calibration curve, the unknown caffeine can be determined using retention times and correct areas.