Gas Chromatography Graph

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					Experiment 11: Gas Chromatography (GC)
PURPOSE:

      Introduce the student to the use and operation of a gas chromatograph. Understand the
concepts of retention time, resolution, optimum flow, and internal standards in gas
chromatography. Separate mixtures of volatile organic compounds. Determine the amount of
pentanol in an unknown sample.

THEORY:

       Harris, D. C. (2003); “Quantitative Chemical Analysis 6th ed.”; pp. 91-92, 555-572,
       579-590, 598-600.

       In this experiment, mixtures of volatile organic compounds will be separated and
analyzed, and a sample containing an unknown percentage of pentanol will be quantitated by
GC analysis. You will work in small groups on this experiment, sharing most of the data.
However, each student will have his/her own unknown and all calculations and analysis done
outside of the lab period are to be done individually.

        In GC, a liquid sample is injected into a separation column as a sharp plug. The GC
column is usually coated with stationary phase of a given overall polarity. The column is
contained within an oven. The sample is vaporized to a degree and at a rate dependent on its
boiling point. It is carried by an inert gas (usually helium) through the column to a detector. The
detector signals a chart recorder, which records the response, ideally in the shape of a
Gaussian peak.

          Several factors determine the rate at which an injected compound travels through the
column and reaches the detector. A compound’s volatility at the column temperature influences
the distribution of the compound between the gaseous mobile phase and the stationary phase.
Other things being equal, a compound’s band will move through the column more quickly if its
distribution favors the mobile phase. Boiling points are often used as a measure of the relative
volatilities of compounds in a mixture. However, volatility alone does not determine the
distribution between the stationary and mobile phase. Specific interactions between the injected
compound and the stationary phase play an important role as well. Recall that “like dissolves
like” so a polar compound will tend to strongly distribute into a polar stationary phase.
Conversely, a polar compound will not have a strong affinity for a non-polar stationary phase
and, thus, will elute relatively quickly. In summary, components in a mixture can be identified by
analyzing the difference in their retention times, which is dependent upon their volatility and
polarity.

         Quantitation is possible in GC methods by analyzing the peak area or peak height. For
the detector you will use in this experiment, peak area will provide better results. A larger peak
area indicates a larger amount of analyte present in the sample. Spiking samples with internal
standards helps to compensate for the imprecision inherent to GC methods (e.g. variable
injection volumes). In this method, the analytical sample and standards are spiked with a fixed
amount of a solute whose retention time is similar to (but resolved from) that of the sample. The
ratio of the area of the analyte peak to that of the internal standard is used as the y-axis variable
to prepare a calibration curve and subsequently used to determine the analyte concentration in




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an unknown mixture. In our experiment, n-butanol is the internal standard, n-pentanol is the
analyte, and ethanol is the solvent.
        In this experiment, you will use a Gow-Mac GC equipped with two columns. Column A
is a polar column with a tradename of Carbowax 20M and column B is a non-polar column with
a tradename of DC 200. Only one column may be used at a time. The direction that the chart
recorder pen will deflect depends upon which column is in use; make certain that you change
the polarity of the pen direction whenever you change columns.

        Several factors affect separation efficiency in GC, including column temperature and
carrier gas flow rate. The temperature for the GC oven has been preset for you; record the
temperature in your lab notebook. The carrier gas flow rate is adjustable for both columns and,
thus, the flow rate will not be the same for both columns. You will use a soap bubble flowmeter
to measure the flow rate of the carrier gas.

         Before starting, the detector and chart recorder must be zeroed. Set the Attenuation to
infinity and position the chart recorder pen to the desired baseline position using the recorder
Zero offset. Slowly turn the Attenuation control towards 1. If the chart recorder pen’s position
moves from the baseline setting, use the Zero control on the GC to reset the pen to its original
position. Once you are able to change the attenuation from infinity to 1 without significantly
changing the pen’s position, the instrument is zeroed. Begin your experiment by setting the
attenuation on 4 and the chart recorder sensitivity to 1 mV. Adjusting these parameters will
change the peak size on your chart paper. Setting the recorder’s sensitivity to a higher setting
will cause the peaks to become smaller; increasing the attenuation to a larger number will cause
the peaks to become dramatically smaller. Set the chart recorder speed to 2 cm/min. The TA
will demonstrate the proper use of the GC injection microsyringes. Treat these expensive
devices with care! Whenever a sample is injected into the GC, it is important to mark the chart
paper at the moment of injection (use pen or pencil). Also, you should inject as much air as
possible with the sample in order to obtain an air peak (this allows determination of adjusted
retention times).

        The following chart lists some important properties of the classes of compounds used in
this experiment:

Compound                     Polarity              Boiling Point

 alcohols                    polar                 boiling point increases with
                                                   molecular weight

 ketones                     polar                 lower boiling point than alcohols            of
                                                   comparable molecular weight

 alkanes                     non-polar             boiling point is lower than ketones of
                                                   comparable molecular weight

 alkyl benzenes              non-polar             high degree of double bonds increases
                                                   stability, thus, relatively high boiling points




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PRELAB EXERCISE (2 pts. /each):
     a. A simple way to estimate the flow rate in a gas chromatograph is by the use of a
        bubble flow meter. The flow rate is determined by measuring the time required for a
        soap bubble to travel a given volume (typically 10.00 mL). Based upon the diagram
        shown on figure 1, calculate the how long will it take for a soap bubble to travel 10
        mL at a flow rate of 60 mL/min.


                                  10.0 mL          10.0 mL         10.0 mL         10.0 mL




                         from
                        column
                                  0.0 mL           0.0 mL          0.0 mL          0.0 mL
                         outlet




                                            t = 0.0 min     t = 0.5 min     Flow = 20 mL/min
Figure 1: Use of the bubble flow to estimate the flow rate in a GC-column.

       b.   Describe a thermal conductivity detector and explain its advantages and limitations.
       c.   Explain the concepts of partition in gas-liquid chromatography.
       d.   Describe how you can determine the optimum flow in GC.
       e.   Mention four factors that affect the resolution in GC.

EXPERIMENTAL:

Procedure: Sample Prep
   1. Each group needs one of each of the following solutions:
      Mix A: equal amounts of n-heptane, 2-butanone, 1-butanol and o-xylene
      pure n-heptane
      pure 2-butanone
      pure 1-butanol (also known as n-butanol)
      pure o-xylene

   2. Each group also needs ethanol, n-butanol, and n-pentanol for the quantitative study.

Each group must also obtain a shared group unknown.




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2. Prepare the following standards in a 10-mL volumetric flask. Fill the flasks to the mark with
ethanol after adding the pentanol and n-butanol, and mix well. (volumes in mL):

          % pentanol (v/v)             vol. n-pentanol*         vol. n-butanol*
          20%                          2.00                     1.00
          40%                          4.00                     1.00
          60%                          6.00                     1.00
          80%                           8.00                    1.00
         *n-butanol is the internal standard, n-pentanol is the analyte.

3. Pipette 5.00 mL of your unknown into a 10-mL volumetric flask. Add 1.00 mL n-butanol and
fill to the mark with ethanol. Mix well.

Procedure: Chromatography
For each set of chromatograms, record the following:
       solution injected
       recorder sensitivity (mV)
       flow rate (mL/min)
       chart speed
       injection volume (µL)
       column temperature (oC)
       type of column
       GC attenuation

4. Inject 1 µL of mix A onto column A.

5. Individually analyze each compound in mix A on column A by individually injecting 1 µL of
each pure substance, obtaining a chromatogram for each one.

6. Repeat steps 4 & 5, using column B instead of column A. REMEMBER: you cannot operate
both columns simultaneously. When you switch columns, remember to switch the detector
polarity switch.

7. Inject 1 µL of the pentanol standards onto column A (do not use column B for this part).
Adjust parameters so that the peak areas can be easily measured.

Hint: Maximize the scale with the most concentrated standard. After carefully cleaning the
syringe with ethanol, inject your samples in an ascending concentration order.

   8. Inject 1 µL of the unknown onto column A; repeat 3 times.


Cleanup: Rinse syringe with methanol or ethanol. Rinse pipets and volumetric flasks with
ethanol. Do not discard your standard compound vials…they will be re-used.




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Calculations:

Useful equations:
Resolution (Rs) = 2(t2 - t1)/(W1 + W2)
       where t1 = retention time of one component
               t2 = retention time of second component
               W1 = width of peak at baseline of first component
               W2 = width of peak at baseline of second component

Efficiency (n) = 5.54 (tr/W1/2)2
        where tr = retention time
                W1/2 = width of peak at half-height
To determine concentrations:

Measure peak areas for the butanol and pentanol peaks. For chart paper with marked squares,
count squares and partial squares under the curve. Choose your baseline to match flat portions
of the recorded curve, where nothing was eluting.

   a. Another method is triangulation, where you estimate the peak as a triangle, calculating
      the area as (½ base x height). Corrections are needed at the top of the peak and on the
      two baseline regions, where a triangle doesn’t match the peak shape.
   b. A third method is computer calculation of peak areas, which is only possible if the data is
      originally collected using computer software instead of on chart paper.
   c. For each standard (20%, 40%, 60%, 80% pentanol), calculate the ratio of peak area of
      pentanol / peak area of butanol.
   d. Create a calibration graph of the peak area ratio versus % pentanol. Fit a line, and show
      the line and the equation on your graph.
   e. Calculate the peak area ratio (pentanol/butanol) for your unknown chromatogram.
      Calculate the %pentanol for that unknown solution from the best fit linear equation on
      the calibration graph.
   f. Finally, calculate the concentration of the original unknown solution you were given. The
      solution tested was diluted (5 mL of original unknown diluted to a total of 10 mL), so you
      need to calculate the original concentration before dilution.
   g. Repeat the unknown calculation for each injection you did, then calculate the average,
      standard deviation, and 95% confidence interval.

Regular Lab Report: You will submit both a regular report analyzing your unknown and a formal
written report for this experiment, which will be graded separately. Both are due one week
following the experiment. Note: Some materials may need to be provided in each of the two
reports. Do not assume the grader will look at both reports at the same time. They will be
graded independently. All lab report work is to be done independently. Members of a group
that worked together should share data, but not work together in any other way. Include the
following in your regular lab report:

1. Your name and your unknown number.
2. The percent pentanol in your unknown sample, the standard deviation, and the 95%
   confidence interval.
3. Sample calculations for the determination of the concentration of your unknown.
4. A copy of your calibration graph (also include a copy in your formal report!)
5. Answer the following questions:



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           a. Injections done without filling the needle of the syringe with air before injection
              sometimes give chromatograms with two peaks for each compound. Explain
              how this might happen.
           b. Explain in your own words how/why the use of an internal standard corrects for
              variations in injection volume.
           c. Packed column GC such as our experiment today isn’t particularly sensitive.
              Capillary column GC is used when high sensitivity is needed. Give a practical
              example of an analysis where packed column GC would be appropriate, and a
              second example of an analysis, this time where capillary column GC should be
              used.
           d. Our replicate analyses of the unknown sample test the precision of only some
              parts of our procedure. Which parts of the procedure are we testing? Which
              parts of the procedure aren’t we testing? What should we do to test the precision
              of all parts of our procedure?

Formal Lab Report:
You will also write a formal report for this laboratory, also due one week after the lab. Although
this experiment was performed in groups, each student must write his/her own report. Be sure
to proofread your report – correct spelling, grammar and overall clarity will be graded.

1. Title page: Include name, title, date, unknown number, and lab partners.
2. Introduction: Discuss principles of GC (including the internal standard method) and the
   purpose of this lab.
3. Experimental: Briefly describe solution preparations and experimental procedures.
4. Data: Neatly organize relevant data into tables. Include GC parameters, elution order and
   retention times. Do not include your chromatograms in this report.
5. Results and Discussion. Include the following points within your discussion:
            a. Explain the elution order for mix A on the two columns.
            b. Calculate the resolution of 2-butanone and 1-butanol on the two columns. Which
               column yields better resolution? Why?
            c. Calculate the efficiency of 1 butanol on the two columns. Which column has
               better efficiency? Why?
            NOTE: equations are given above, or in your textbook.
            d. Prepare a calibration plot (peak area pentanol)/(peak area butanol) vs. %
               pentanol. Remember, do a best-fit line for a calibration plot; do not force the line
               through the origin.
            e. Use your calibration plot to determine the % pentanol in your original unknown
               sample (NOT your diluted aliquot). Include a copy of your calibration plot.
6. Conclusions. Discuss sources of errors and how they could affect your results, along with
   the conclusions drawn from this experiment. Within this section, answer the following
   questions (consulting the Harris textbook is recommended!):
   a. How could you have improved the resolution achieved?
   b. What changes would you predict in retention time and peak shape if the following occurs
        (two separate answers):
        The column temperature is increased between injections?
        The carrier gas flow rate is decreased between injections?
   c. Explain the effect of each of the following three processes on band width:
        Eddy diffusion
        Longitudinal diffusion
        Resistance to mass transfer
7. References


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