Question

EXPERIMENT 1: How Much Caffeine Am I Drinking?

INTRODUCTION

In many situations an analyst will want to identify and quantify a compound (or many compounds) that are part of a more complex mixture. Obvious examples of this could be a protein analysis of a foodstuff or an illegal drug in a blood sample. The analyst could extract the compound and then do an analysis on the pure substance but a much more efficient method would be to use some form of chromatography. As you know chromatography is a separation technique, applicable to analysis of organic, inorganic and biological compounds, and all chromatographic techniques have a common feature in that they use two immiscible phases: a stationary phase and a mobile phase.

In CHE240 you did experiments utilising TLC, paper chromatography, electrophoresis, and GC. One of the most widely used chromatographic techniques is high performance liquid chromatography (HPLC). HPLC is particularly useful for analysing compounds that are thermally labile, i.e. degrade upon heating, (such as natural products and biomolecules) and those that are not easily volatilised, e.g. ionic species. A large number of columns are available for HPLC and the technique is regularly used for analysis of pharmaceuticals, drugs of addiction, ionic species, and organic compounds in solution, to name only a few.

HPLC is often used in quality control and regulatory applications in order to identify and quantify substances that may have some harmful/unintended effect if consumed in sufficient quantitity. Quite often these chemicals are highly regulated and manufacturers need to ensure that their products meet these regulations and that the labelling on their products is correct. It is this context that you will be analysing the caffeine content of various common drinks in this experiment. In order to report a quantitative analysis of caffeine you will need to:

• determine the retention time of the caffeine peak in the chromatogram

• construct a standard calibration curve using a series of supplied caffeine standards; and

• prepare and analyse the caffeine content of unknown samples, including tea, coffee, and a soft drink (optional).

BACKGROUND ON THE INSTRUMENT

HPLC is a powerful and versatile technique due in part to the many possible variations of mobile phase, stationary phase and detector systems which are available. The diagram below illustrates a typical HPLC system. Mobile phase from the reservoir passes through a pump and sample injection valve, through an ‘in-line’ filter to protect the column, through the column packed with the stationary phase to the detector, and finally to waste. You will notice that the Shimadzu HPLC you use for this experiment has all of these components in a fairly neat box thus reducing the benchtop ‘footprint’ of the instrument. Also the instrument parameters and data collection and manipulation are computer controlled, and an autosampler is incorporated into the design also.Columns for HPLC are constructed from precision bore stainless steel and are packed with small diameter materials (eg, silica or modified silica, typically 5-10 μm in diameter but can be as small as 3 μm). A large number of different stationary phases are available but the most common packing material used is C18, a reversed phase packing.

High performance pumps are required because of the need to achieve constant flow rates against the high backpressure (500 to 3000 psi, 30-200 atm., 3-20 MPa) developed. These high pressures are created by the need to force the mobile liquid phase through the small-sized, densely-packed stationary phase support material.

Solvents must be degassed prior to use, hence the helium (He) being bubbled through the solvent reservoirs in the diagram, in order to prevent air bubbles entering the column. This can result in uneven distribution of solvent and/or sample in the column as well as possibly producing a hole or crack in the column packing, severely reducing the column efficiency and quality of resulting data. Most modern instruments, including the one you are using today have an online degasser built into the system. Solvent should also be filtered prior to use so as to minimise the possibility of clogging the column and/or the tubing that connects the components with dust.

The mobile phase to be used can be premixed or mixed by the instrument and solvent gradients are often used to optimise analysis time. Today you will be running isocratically (i.e. no gradient) using a mixture of water:methanol:glacial acetic acid.

EXPERIMENTAL

The instrument parameters should be set up for the analysis of caffeine. Check the parameter table below with that displayed on the instrument. Make sure that you can identify the instrument components and see your demonstrator for a run down of the HPLC operation and a demonstration of sample injection technique.

There are basically 4 steps to conducting the experiment – preparing the samples, determining the retention time of the caffeine, calibration, and analysis of the samples.

Due to time constraints the calibration and sample preparation need to be done concurrently. So, while the initial sample above is eluting one of you should begin organising the calibration and the other the sample preparation.

All solutions, mobile phase, standards, samples must be filtered and degassed prior to use.

Before any samples are injected they must be filtered through a 0.45 μm nylon Millipore filter paper. A syringe filter is convenient for this. See your demonstrator for instructions.

Sample Preparation

At least 3 samples from the following list should be prepared.

Coffee

Weigh accurately, approximately 0.6 g of coffee and dissolve (brew) in about 100 mL of boiling water in a 250 mL beaker. Brew for 10 minutes then cool on ice (to room temperature) and make up to 250 mL in a volumetric flask with distilled water. Adding a single drop of ethanol to the solution in the volumetric flask before adding the distilled water will reduce any frothing that may occur.

Boiling water can be obtained from the Environmental and Physical Sciences tea room.

Decaffeinated coffee

As for coffee.

Tea

As for coffee but weigh accurately, approximately 0.8 g of tea. Cool and quantitatively filter using a fluted filter paper (to remove the tea leaves) into a 250 mL volumetric flask and make up to volume with distilled water.

Please answer following questions

1.When making recommendations on the amount of coffee someone should drink, public health officials will often indicate the amount of coffee in a ‘standard’ or ‘normal’ cup.what would be the amount of caffeine in a normal cup of the beverages tested, if a normal cup is defined as 1 g of dried material per 200 mL of water?

2.Once you finished the experiment do you have caffeine peak in your HPLC analysis was overlapping with one from a non target component? What could you do to improve the separation?

Answer 1

1.A normal cup of coffee contains 80–175 mg of caffeine, depending on what "bean" (seed) is used and how it is prepared (e.g. drip, percolation, or espresso)and also if the normal cup is defined as 1g of dried material per 200 ml of water. Thus it requires roughly 50–100 ordinary cups of coffee to reach a lethal dose.

2.Reverse phase HPLC is used to determine the concentration of caffeine in coffee.

Five standard solutions of caffeine are prepared and injected into the HPLC.A calibration curve for peak area against concentration of the caffeine standards can then be employed to determine the concentration of caffeine in the four beverages. The solvent (mobile phase) in this experiment is 47% methanol / 53% water.

inject the prepared laboratory sample of the beverage and note the quality of the separation of the caffeine peak from any other components in the sample (triplicate).

If the caffeine peak is completely resolved, record the peak tr, h, and A. If the sample caffeine peak height from the sample is larger than the 50 ppm standard, the beverage sample is too concentrated. Dilute quantitatively to bring the concentration into the calibration range if necessary and re-inject the diluted sample. (triplicate).