Experiment 7: High Performance Liquid Chromatography and Gas Chromatography

 

 

Collaborative Learning

To begin, two students from each group will work on the high-performance liquid chromatography (HPLC) portion of the experiment, while the other two will work on the gas chromatography (GC) portion. Halfway through the lab period, the students who performed HPLC will explain what they did to the students who performed GC, and the students who performed GC will explain what they did to the students who performed HPLC. Then the two students who initially performed HPLC will go on to perform the GC portion of the experiment, and the two students who performed the GC portion of the experiment will go on to perform the HPLC portion of the experiment.

 

Experimental Theory

In general, chromatography is characterized by the separation of a mixture of analytes (components of a mixture to be analyzed by chromatography) into its individual components by two different phases. This separation is based on the differences in each analyte’s affinity for each phase.

The two phases involved in chromatography are known as the stationary phase and the mobile phase. The stationary phase is composed of a substance on a solid support, while the mobile phase is a fluid that passes through the stationary phase. In column chromatography, all the analytes begin in the mobile phase. The analyte mixture (in the mobile phase) is then introduced on to the column (stationary phase), From here, some of the analyte in the mobile phase will transfer to the stationary phase in a process known as partitioning. If we consider a particular analyte "A," partitioning can be thought of as a type of equilibrium between A in the mobile phase and A in the stationary phase. This equilibrium is described by the partition coefficient K.

K gives the relation between the concentration of A in the stationary phase and the concentration of A in the mobile phase. Mobile phase is continually pushed through the stationary phase, and as a result, partitioning occurs repeatedly over the length of the column. If an analyte has a high affinity for the stationary phase (i.e., K is large), it will adhere to the stationary phase more tightly and take a longer time to be carried all the way through the column by the mobile phase. On the other hand, if the analyte has a high affinity for the mobile phase (i.e., K is small), then the mobile phase will carry the analyte through the column relatively quickly. As a result, column chromatography will separate a compound with a relatively high affinity for the stationary phase from a compound with a relatively high affinity for the mobile phase. Although we have only discussed the method of column chromatography specifically, all chromatography is based on this same idea of separation of compounds based on their differences in affinity to two different phases.

 

Experimental Descriptions and Procedures

I. Gas chromatography

 

A. Introduction

Gas chromatography is generally used to separate mixtures of volatile compounds based on differences in the boiling points of these volatile compounds (this explains why you’ll be using GC to separate a mixture of cyclohexane and toluene&emdash;if you’re confused, think back to how you separated cyclohexane and toluene from a mixture of the two in the last experiment). The GC apparatus contains a long stainless steel tube inside a temperature-controlled oven. The stainless steel tube is packed with a liquid phase on a solid support. This acts as the stationary phase. An inert gas (usually He or Ar) acts as the mobile phase by passing through the stationary phase.

A tiny amount of a liquid sample mixture is injected into the steel tube of the GC apparatus, and the oven heats the various components of the mixture. Components of the sample mixture may vaporize and be moved through the column by the mobile phase, or they may not vaporize and remain fixed on the stationary phase. So, low-boiling compounds will vaporize quickly and pass through the column (and reach the detector) quickly, while high-boiling compounds will remain on the stationary phase for a longer time and require a longer time to pass through the column (and reach the detector). As a result, compounds will be separated in GC based on differences in their boiling points. Under the proper conditions, each peak in a gas chromatogram will correspond to a particular compound in the sample mixture. The area of a peak is proportional to the concentration of the corresponding compound in the sample.

(For a more detailed description of how GC works, see your theory handout for Expts.

6-7, pp. 11-12).

 

B. Purpose

In this experiment, the contents of your fractions obtained from distillation of cyclohexane and toluene (Expt. 5) will be analyzed by gas chromatography. The gas chromatograms that you obtain for these fractions will then be compared to the chromatograms for standard samples of pure cyclohexane and pure toluene.

 

C. Safety

*Wear goggles and aprons at all times

*Be careful. The injection port on the GC is hot.

*The Hamilton syringe needles are extremely delicate and extremely sharp. Handle them

carefully lest you bend the needle or stab yourself.

 

D. Materials

--Gas chromatograph

--10 microliter Hamilton syringe

--toluene

--cyclohexane

 

E. Procedure

In this experiment, you will be running a number of samples. Initially you will perform repeated GC runs on cyclohexane and/or toluene (both obtained from reagent bottles) and vary the GC parameters (flow rate, oven temperature, attenuation) for each run. This will allow you to determine the optimal parameters that you will use to obtain chromatograms for your fractional distillation samples. Also, be sure to obtain at least one chromatogram for both cyclohexane and toluene from the reagent bottles. Samples are run and chromatograms are obtained by following steps 1-10.

1. Rinse the syringe two or three times by drawing the sample solution up into the

syringe and then squirting the solution out on to a paper towel.

2. Draw the sample up into the syringe. Draw up 1 mL if you’re injecting a mixture, and

0.5 mL if you’re injecting a pure compound.

3. Add air to the sample by drawing the bottom of the plunger back to about 5 mL.

4. Put the recorder pen in the down position and make sure that the chart paper is moving.

5. Insert the needle into the septum over the injection port (remember, be careful). Inject

the sample by quickly and smoothly pressing down on the plunger.

6. Remove the needle from the injection port.

7. Note the settings for the attenuation and the column temperature of the GC, and

remember to write down the sample size and the identity of your sample.

8 Wait for the entire sample to elute (leave the column) before you run a new sample.

9. Rinse the syringe with methanol.

10. Remove your chromatogram from the chart recorder.

More specific instructions on how to operate the GC apparatus will be given to you in lab.

 

 

F. Data Analysis

--Determine the retention times and peak widths for all of the peaks in your

chromatograms. Also, calculate the resolution for chromatograms with more than one

peak.

--Determine the area under each peak in the chromatograms of your fractional distillation

samples. Correct these areas using the substance-specific correction factors provided to

you. Using these corrected areas, determine the percent composition of each fractional

distillation sample.

 

II. HPLC

 

A. Introduction

In an HPLC apparatus, a liquid mobile phase is continually pumped through a stationary phase column. When a sample is injected into the HPLC apparatus, it is picked up by the mobile phase and carried to the stationary phase column. At this point, partitioning of the sample components between the mobile and stationary phases begins to occur. The mobile phase will then continue to be pumped through the column, and it will eventually carry each component of the injected sample through the column to a detector. Different compounds in a mixture will be separated based on the affinity of each compound for the mobile and stationary phases (in other words, some compounds will adhere tightly to the stationary phase, and they’ll take a longer time to be moved through the column to the detector, while some compounds will adhere less tightly to the stationary phase, and so they’ll be moved through the column to the detector more quickly). Under the proper conditions, each compound in an HPLC sample will give a distinct peak on an HPLC chromatogram. The area of each peak is proportional to the concentration of the corresponding compound in the sample.

(For a more detailed description of how HPLC works, see your theory handout for

Expts. 6-7, pp. 13-18).

 

B. Purpose

In this experiment, you’ll be using HPLC to separate the components of various soft drinks. You will then use the HPLC data to determine the concentration of caffeine in these different types of soft drinks based on the chromatograms given by standard caffeine solutions.

 

C. Safety

*Be sure to wear goggles and gloves when performing this experiment.

*Again, the Hamilton syringe needle that you will be using is both fragile and sharp. Be

careful not to bend the needle, and also be careful not to stab yourself or anyone else

with the needle.

 

 

 

 

 

 

D. Materials

--HPLC machine --Soft drink sample

--50 microliter Hamilton syringe --Caffeine stock solution

--Plastic tubes w/ caps --Distilled water

--5 mL, 10 mL graduated pipets --Nylon Acrodisc filters

--Pipet bulbs --10 mL volumetric flasks

 

E. Procedure

Note: Work in groups of two. Each group will run two samples (one caffeine standard and one soft drink sample.

 

Preparing HPLC standards and samples

  1. Each group of two will prepare 10 mL of one of the following dilutions of the caffeine stock solution…

    Group 1: 1:10 dilution (volume of caffeine stock: total vol. of dilution)

    Group 2: 1:5 dilution

    Group 3: 3:10 dilution

    Group 4: 1:2 dilution

    Group 5: 3:5 dilution

    Group 6: 4:5 dilution

    These dilutions will be used as standards in calculating the concentration of caffeine in your soft drink samples. Make sure to dilute in distilled water. Also, calculate the concentration of the dilution that your group makes based on the concentration of the caffeine stock solution.

  2. Make 10 mL of a 1:1 dilution of the soft drink sample by adding 5 mL of soft drink to
  • 5 mL of distilled water.
    •

     

    Filtering standards and samples

    1. Obtain a 10 mL plastic syringe with a Luer-LockTM tip. Pull the plunger out of the

      syringe.

    2. Attach an Acrodisc filter to the tip of the syringe.
    3. Fill the syringe with the standard or sample that you’ve prepared.
    4. Use the plunger to push the solution through the syringe filter and into a plastic tube.

     

    Running HPLC standards and samples

    1. Set the parameters of the HPLC apparatus to the appropriate values.

    2. Rinse the 50 microliter syringe twice with your caffeine standard sample.

    3. Draw up 20 microliters of the standard into the syringe. Make sure that there are no

    bubbles in the syringe.

    4. Insert the syringe needle into the injection port and inject your sample.

    5. Turn the injector knob to allow the sample to be carried on to the column.

    6. After an acceptable standard peak has been obtained, obtain a chromatogram of the

    soft drink sample by repeating steps 1-5 using the soft drink sample. A peak

    corresponding to caffeine should appear at the same distance from the injection peak

    as on the caffeine standard chromatogram.

    7. After all your samples have been run, inject 20 microliters of distilled water into the

    HPLC machine and allow it to run. This will clean out and sugar left behind by the

    sample.

    More specific instructions on operating the HPLC apparatus will be given to you in lab.

     

    F. Data Analysis

    --Calculate the retention time and adjusted retention time for each peak in the

    chromatograms that you obtained. In addition, for the caffeine peaks in each

    chromatogram, calculate peak width, number of theoretical plates, and plate height

    (HETP).

    --Calculate the resolution for each soft drink sample chromatogram.

    --Calculate the area under the peak for each caffeine standard dilution. Make a graph of

    peak area vs. concentration of the standard dilution. Find the equation for the best-fit

    line for this graph.

    --Calculate the area under the caffeine peak for each soft drink sample chromatogram.

    Then, using the equation of the line that you obtained in the previous step, calculate the

    concentration of caffeine in each soft drink sample.