Organic Chemistry 3510, High Performance Liquid Chromatography

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

The Quantitative Analysis of Caffeine in a Beverage

DR. SUNDIN

Organic Chemistry 3510

Introduction: The purpose of this experiment is to quantitatively determine the amount of caffeine in a beverage by high performance liquid chromatography. Due to the nature of the apparatus and the time available, the analysis will be done as a demonstration. Nonetheless, your lab notebook should be prepared and completed as if you did the experiment.
Read pages 85-86.
Background
Column Chromatography: In a typical column chromatography separation, the mixture of compounds to be separated is introduced onto the top of a cylindrical glass column uniformly packed with fine alumina or silica gel particles. Initially, the components of the mixture form an equilibrium with some solute molecules adsorbed on the particles at the top of the column and some solute molecules dissolved in the solvent. The continuous flow of fresh solvent (eluent) through the column sweeps the solvent dissolved solute molecules down the column. As the experiment continues, the solution at any point on the column is continually replaced by new solution resulting in solute molecules re-equilibrating between the solvent and the solid particles. This constant re-equilibration of the solutes between the packing and the solvent results in the solutes moving down the column at rates dependent upon the solutes affinity for the packing and solubility in the solvent. If the affinities and solubilities vary significantly from solute to solute, the solutes will move down the column at different rates often resulting in the formation of moving bands containing a single component separated from other bands by pure solvent. When this happens, the components are said to be resolved. Resolution is a function of the components themselves, adsorbent, particle size, solvent, column length, column diameter, flow rate. and even sample size. Generally, nonpolar molecules pass through faster than polar molecules. If the adsorbent is too "active', the solute will not travel down the column at all. If the solvent is too polar, all the components will be simply washed down the column without any separation. It should also be emphasized that the proper packing or filling of the column can affect the quality of the separation.
Resolution in column chromatography can be greatly improved by decreasing the adsorbant particle size. This results in a larger surface area for a given mass of adsorbant. Additionally, the spaces between the particles is reduced. The net effect is that the small solvent volumes permits a more rapid equilibration between solvent and adsorbant with a resulting increase in separation with even shorter columns. The major drawback is that the solvent flow rate is greatly reduced, or sometimes stops, in these tightly packed columns. Gravity often is simply not strong enough to pull the solvent through.
High Performance Liquid Chromatography: One way to obtain better separations with tightly packed columns is to use a pump to force the solvent through the column packing. This technique is called high performance liquid chromatography (HPLC). Since the pump operates at high pressures (1000+ psi), the technique originally was called high pressure liquid chromatography. Particle size is generally in the range of 5-20 micrometers as compared to 100 micrometers for ordinary column chromatography. Since such tightly packed columns are susceptible to plugging, high quality solvents are used with special filtration of the solvent before it enters the pump and often a guard column (slightly larger diameter packing particles) before the main column. The column and tubing is generally made of stainless steel to withstand the high pressures. Injection of sample under such high pressures uses a special six port valve (see below). A narrated description of the nature of sample injection is available on the Web. A nonnarrated animation of just the injection valve is also available. Detection of the eluates usually occurs in a flow through detector which detects either a change in the index of refraction or the absorption of ultraviolet light by the eluate. The signal generated by the detector is then sent to either a strip chart recorder or a computer to give the typical chromatogram.
A variation unique to HPLC is to use a nonpolar column packing with a polar solvent, so-called reversed-phase chromatography. It is difficult to get nonpolar materials to physically stick to solid supports. Instead, this is accomplished chemically by having alkyl silane groups chemically bonded to small (5-20 micrometers) glass beads. Another advantage of this method is that it avoids column bleed, a problem in gas-liquid chromatography. The nonpolar alkyl groups essentially creates liquid-liquid chromatography as compared to the liquid-solid chromatography discussed earlier. When a polar solvent is used, polar solutes are more strongly attracted to the mobile (solvent) phase then they are to the stationary (absorbant) phase. The order of elution is reversed with the more polar compounds coming off first. The solvent used may be a single solvent or a solvent mixture. With the electronic control of several pumps, the solvent polarity may reproducibly be changed during the analysis. This is called gradient elution. A detailed Separation Science Course is available on the Web.
Experiment
2. Quantitative analysis involves calibrating the detector response. Prepare a working curve of detector response vs. concentration (and see if it is a straight line which follows Beer's Law) and then use the detector response of the unknown sample to obtain its concentration. Usually, our HPLC uses a 20 microliter sample for each analysis. The flow will be about 1 mL/minute at an approximate pressure of 2000 psi. The solvent is usually acetonitrile/methanol/water with a small amount of acetic acid added to keep all organic bases in the protonated form. (This solvent system gives a reasonable separation of caffeine with relatively small retention times.) The column is an alkyl silane bonded to 5 or 10 micrometer glass beads. The detector measures the absorption of ultraviolet light. Exact reactions conditions should be obtained at the time of analysis.
The pump is turned on and the flow adjusted to the proper conditions. The detector is turned on and the system is allowed to run until a flat base line is obtained. The base line is adjusted to zero. An appropriate detector setting (AUFS {absorbance units full scale}) is selected. Samples of known concentration of caffeine in water are prepared and loaded on the 20 microliter loop using a square ended needle! The valve is turned and the contents of the loop are sent to the column. Since the sample is in a different solvent than the eluting solvent, the detector may show a response (often negative) when the sample solvent comes through due to differences in refraction. When the caffeine peak comes through, record its retention time, peak height, and peak area. Run the remaining standard solutions in a similar manner.
The beverage sample must be "prepared" before analysis. If soda is used it is imperative to degas the sample so that gas bubbles do not form in the detector which would give erroneous readings. If coffee or tea is used, the sample must be carefully filtered or the suspended solids can plug up the guard column. Measure the height and area of the caffeine peak (you may have to correct for overlapping peaks).
Using a graphing program such as Quattro or Excel, prepare properly labeled graphs of the results obtained using the standard solutions. Determine if caffeine follows Beer's Law in this concentration range. From your data determine: a) the concentration (ppm) of caffeine in the beverage; and b) the total amount (mg) of caffeine in one serving of the beverage (12 fluid ounces of soda). If the data does not follow Beer's Law, use your best graph. If the data follows Beer's Law, you may use the equation of the line (obtained by regression analysis). Be sure that your graph and all calculations are clearly presented in your notebook.
3. Be able to answer the following:: a. Compare the principles of gas-liquid chromatography, liquid-solid chromatography, and liquid-liquid chromatography.; b. What is Beer's Law? Explain how an analysis can be made even if the compound being measured doesn't follow Beer's Law.; c. Give a block diagram of a typical HPLC apparatus.; d. Explain why a square ended needle is used instead of a pointed needle in HPLC.; e. Explain how exactly the same volume of sample is placed on the column each time.; f. How long is the separation column? What are guard columns made of?; g. Why is acetic acid added to the eluting solvent?
4. The injection valve in the two operation positions:: "Load" Position:; "Inject" Position: