Chromatography Lab- Background
Chromatography is a means of separating mixtures based on their solubility in a
particular solvent. It is widely used in the biological sciences to separate amino
acids in a given sample of tissue or cells. Another use of chromatography is called
gel electrophoresis, and it is a chromatography method that separates DNA base
pairs. Different base pairs each have a different weight, and when an electric
charge is passed through them, they migrate a different distance in the medium.
This is how DNA testing is done for legal identification.
Two Examples of Chromatography
High Pressure Liquid Chromatography-HPLC
HPLC aids in the identification, quantification, and resolution of a compound.
Different types of HPLCs can also be used for isolation and purification of
compounds.
Although HPLC is widely considered to be a technique mainly for biotechnological,
biomedical, and biochemical research as well as for the pharmaceutical industry,
these fields currently comprise only about 50% of HPLC users. Currently HPLC is
used by a variety of fields including cosmetics, energy, food, and environmental
industries.
Gas Chromatography/Mass Spectrometer (GC/MS)
Many GC instruments are coupled with a mass spectrometer, which is a very good
combination. The GC separates the compounds from each other, while the mass
spectrometer helps to identify them based on their fragmentation pattern.
Time spent in mobile phase (gas) depends on vapor pressure of components in mixture
and not their solubility. Rate of elution in gas chromatography is partly dependent on
differences between b.p. of compounds in mixture.
Because molecules in ink and other mixtures have different characteristics (mass
and solubility), they travel at different speeds when pulled along a piece of paper
by solvent (in this case, ethanol and water). For example, black ink contains several
colors. When the solvent flows through a word written in black, the molecules of
each one of the colors behave differently, resulting in a “rainbow” effect.
Chromatography Lab
A note has been given to Mr. McCreary, excusing a student for being absent. It has
a number of clues suggesting that it might not be real. Poor spelling, the
penmanship looks like that of a student, the signature is illegible, and the note is
written on strange looking paper. The note only gives a first name of which there
are several in the eleventh grade. After eliminating some students because they
were in school, Mr. McCreary calls in the others, and asks them for any black
writing utensils that they may have on their person. Mr. McCreary finds three,
numbers them as evidence (to protect the individuals involved), and sends them to
the school “crime lab” (our class) for testing.
It is your job to determine which pen wrote the suspicious excuse.
Materials
2 beakers (150 mL)
2 glass stirring rods
Graduated cylinder
2 piece of chromatography paper
1 piece of the evidence (obtained from the instructor)
3 evidence pens for marking (obtained from the instructor)
Ruler
Pencil
Tape
Solvent (50/50 Isopropanol and Water) (approximately 15 mL)
Chromatography-Procedure
1.
2.
3.
4.
Using pencil, draw a line ¾ of an inch from the bottom of the paper.
Evenly mark three Xs across the line.
Obtain the three evidence pens labeled A, B, and C.
Label your chromatography paper either at the top or the bottom with pencil
where you plan to place each pen.
5. Take each pen and put a dot on the corresponding X. (the pen should touch
the paper for no longer than 2 seconds) Be careful that each ink dot is
separate from the other
Chromatography-Procedure
1. Obtain your evidence sample
2. Place both of your chromatographs, the evidence sample and the created
sample with the three different pens dots, in the two different beakers.
Using tape, attach each chromatograph to a glass stirring rod that is placed
on the lid of the beaker. Place the chromatograph so it is only slightly
touching the bottom of the beaker.
3. Take the chromatographs out of the beakers for the moment. Using a
graduated cylinder, measure out 15 mL of your solvent (50/50 Ethyl alcohol
and water) and pour this amount into each of your beakers.
4. Take your attached chromatographs and lower them into the solvent, taking
care not to touch any part of the chromatograph to the side of the beaker.
5. Allow the solvent to travel to within ½ inch of the stirring rod.
6. Take the chromatograph out of the beaker and draw a line to where the
solvent had reached. Allow the chromatograph to dry.
Chromatography-Procedure
Ds
Rf = ___
Df
Rf = Retention Factor
Df = Distance solvent front travels from original color
Ds = Distance each color spot travels from the origin
dot
1. To determine the Rf values for each of the spots, use a ruler to measure the
Df and Ds for each dot. Calculate Rf for each dot.
2. Compare the Rf value of the evidence sample to the Rf values determined for
the three pens and conclude which pen was used to write the suspicious
excuse.
Raft Analogy to Chromatography
Raft = Mixtures (inks)
Current = Solvent/Mobile Phase
Riverbed and Stumps = Stationary Phase (chromatography paper)
Snagging on the Rocks and Stumps = Selective Absorption: colors, i.e. compounds, stop at different distances
Chromatography Lab-Questions
(Your formal lab write up should include the answers to the following questions as well as
any requested data and calculations that is stated below. The questions must be submitted
in number form in the Conclusion section of the lab report. This does not exclude you
from fulfilling the other requirements for the conclusion section. The requested data
must be submitted in the Data and Observations section, while any calculations can be
submitted in the Analysis and Calculations section of the lab report.
Every formal lab report is due a week from the day of completion.
Data
1. Df and Ds values for the three evidence pens and the evidence sample (total
of 4). Include a picture of all of your chromatographs (your original should
be in your lab notebook)
Analysis and Conclusions
1. Rf values for the three evidence pens and the evidence sample (total of 4).
Calculations must be shown.
Questions
1. Based on your data, determine if any of the inks are mixtures of dyes.
Explain your reasoning.
2. Describe the appearance of a chromatograph for a mixture.
3. Based on your data, determine if any of the inks contain only one type of dye
molecule. Explain your decision.
4. Describe the appearance of a chromatograph for a substance with only one
dye component.
5. Based on your data and observations, which pen was used to write the
suspicious excuse? How do the Rf values relate? How do the observations
relate?