Gas Chromatography - Mass Spectroscopy

Purpose:

The Purpose of this experiment is to determine the quantity of caffeine in a sample using an instrument called Gas Chromatography-Mass Spectroscopy (GC-MS). The caffeine samples that are evaluated are coffee, espresso and a deuterated caffeine sample (d3), given by the T.A., of unknown concentration was used as an internal standard.

Theory:

The GC-MS instrument combines two different techniques to form one single method of analyzing mixtures. Gas chromatography performs the separation of components in a mixture, and the mass spectroscopy distinguishes the individual constituent.

A mixture of different specimens can be separated by GC. A sample is injected into the instrument through an injection port. The injection port is maintained at certain mixture, which is called its' mobile phase, to the stationary phase of the instrument. The stationary phase is the GC column, where each compound in the mixture interacts at different rates, and therefore separated. The different molar characteristic determines how compounds will relate with the column. The substances that do not stick to the column elutes faster from the column than the ones that do. The oven temperature in the GC is ramped to make further separations. The ones with a higher point boiling point elutes at a quicker rate.

After separation is achieved, the components enter a detector where an electronic signal can be created whenever a component is detected. The signal size is dependent on tcomponent's concentration. A retention time, which is the time from the injection is made to when elution occurs, is calculated by the instrument computer. The retention time is nearly the same for a compound, as long as GC conditions are the unchanged. Assumptions can be made about a compound's identity if its' retention time is known.

The eluted samples from the GC enter the Mass Spectrometer's electron ionization detector. The compounds are electrically charged, by accelerating them through a magnetic field. The molecules are broken apart into charged fragments. The different charges are detected, and the mass of each fragment of the compound is plotted on the spectrum. A qualitative identification can be made using such spectrum.

By GC-MS, solution's content can be separated into individual components, and be identified, which makes it a powerful tool. The Gas Chromatography and Mass Spectroscopy are two useful techniques, especially when used together.

Experimental:

An organic extraction was performed in order to separate the caffeine and the deuterated caffeine from the solution. The solvent used for the extraction was Methylene Chloride. Methylene Chloride is very suitable for extraction because if is not of high polarity, and it has the ability to extract the caffeine from the carbohydrates it is combined with. Pure caffeine of different concentrations was prepared as standards. The same concentration of caffeine-d3 was added to each of the prepared pure caffeine solutions. A calibration curve will be determined using the standards, and this will provide a way to obtain the caffeine-d3 concentration. Regular coffee and espresso were used as samples, and its' caffeine concentrations were determined.

A solution of 8ng/µL pure caffeine sample was prepared. Dilution was performed to give samples of 2ng/µL, 4ng/µL, 6ng/µL, and 10ng/µL concentrations. For the deuterated caffeine, 2mg of the sample was diluted into a 50mL volumetric flask of methylene chloride (40 ng/µL). In each of the standards, 2mL of the diluted caffeine-d3 was added.

Table 1: Standards (Methylene Chloride was used for extraction in all of the standards)

STANDARD PURE CAFFEINE

CONC. (NG/µL) D3

(ML) P.C.: D3

RATIO

1 2 2 1:1

2 4 2 2:1

3 6 2 3:1

4 10 2 5:1

The samples, regular coffee and espresso, were prepared by using methylene chloride to extract the caffeine.

Table 2: Samples

SAMPLE WEIGHT

(GRAMS) VOLUME OF METHYLENE CHLORIDE (ML)

D3 CONC.

(ML)

Regular Coffee 8 50 2

Espresso 8 50 2

The GC oven was maintained at 100 oC for three minutes and was ramped up to 300 oC after this. The whole run per sample lasted a total of about nine minutes. The standards were analyzed first by GC-MS to set a calibration curve, and then followed by the samples. The integrated areas from the extracted mass of the chromatogram of the ions, 194 for pure caffeine and 197 for the deuterated caffeine, was used.

Data:

See Appendix A

Results and Discussion:

A calibration curve was acquired from the set of aqueous standards of pure caffeine. The concentrations of the standards were 2 ng/µL, 4 ng/µL, 6 ng/µL, 10 ng/µL. Each standard was spiked with 2 mL of the caffeine d3, and this served as an internal standard.

Figure 1: A standard calibration curve for the caffeine determination using the GC/MS.

A plot of the area ratio of