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Lab 3: Chromatography
Terms in this set (77)
Chromatography is a large and diverse collection of techniques that can be used to
separate different components of chemical mixtures
All types of chromatography involve
the transport of sample molecules by a gas or liquid phase (mobile phase) through an immobile solid or liquid phase (stationary phase).
Separation is based on the differing tendencies of the molecules being separated to distribute themselves between the ______ of the chromatographic system.
mobile and stationary phases
Separation can be due to
differences of size, ionic charge, adsorption or solubility.
Types of chromatography
gel filtration, paper chromatography, thin layer chromatography, gas chromatography, high performance liquid chromatography
____ (properly called size exclusion chromatography) is often used as a quick "clean up" method to remove contaminants or small molecules from samples containing protein.
While less frequently used now than in the past, ___ and ____ remain cheap and simple methods for separating small molecules such as phenolics, lipids, sugars and amino acids.
thin layer chromatography
-To use gel filtration chromatography to separate 3 proteins of different molecular weight
• To use paper chromatography to determine the Rf values of a selection of amino acids,
and to use these values to determine the amino acids found in your unknown sample.
• To make an educated guess on the identity of a protein based on elution volumes and
absorbance values provided in the lab. The Bradford/Coomassie method would have been applied to the unknown colourless eluates to detect the presence of a protein.
Paper chromatography is used to
separate and identify the individual components in a mixture containing several unknown amino acids.
The Retardation factor (Rf) describes
the ratio of the distance travelled by the sample (amino acid) to the distance travelled by the solvent (solvent front)
By comparing Rf values of knowns and unknowns, it is possible to
tentatively identify your unknown amino acids
it is important to remember that different compounds may have identical Rf values in a given solvent system. Therefore, rigorous identification by chromatography requires
separation by several different solvent systems.
The solvent that you will be using today consists of
n-butanol, glacial acetic acid and distilled water (2:1:1 v/v)
Is the solvent polar or non-polar
A second complication that must be considered involves the detection of the compounds of interest on __ or _____
paper or thin layer chromatograms
But for colourless compounds, physical or chemical properties of the compounds must be used to locate them. These include detection by:
• Absorption of UV light
• Chemical conversion to coloured derivatives
• Presence of radioactive tracers in the compounds of interest
In this part of today's lab the essentially invisible amino acids on the paper chromatograms will be converted to a coloured complex by reaction with ____.
Amino acids react with ninhydrin how?
Amino acids with a free α- amino group react with ninhydrin, which deaminates the amino acid (liberating ammonia) and forms hydrindantin
At pH values above 5, hydrindantin and the ammonia formed, condense with additional ninhydrin to form a ___-coloured product.
Such compounds as proline and hydroxyproline, do not react the same; why?
having no primary amine group,instead they form a yellow to reddish-coloured condensation product.
such compound such as ___ and ___ do not react the same
What was the name of the paper in which the amino acids were applied?
using___ to mark on the paper, as ink will run.
Handle the sheet of paper by its edges why?
amino acids from fingers will provide ninhydrin positive marks
What is the tube called that picks up the amino acid?
What do you do with the tube to get the amino acids?
put finger on top
How many times do you apply the amino acids
total of 3 times
paper:Allow the spot to ___ between applications
Once the amino acids were dry on the paper, what happens?
it was places in a mason jar with solvent and a petri dish cover to allow the vapours to saturate the paper
What was the solvent in the mason jar for the paper?
The solvent in the jar consists of n-butanol, glacial acetic acid and distilled water (2:1:1 v/v).
The solving forms the
proper stationary phase
After 2 hours, or when the solvent has nearly reached the top of the paper, you will be notified to remove the paper from the jar, unstaple it and mark the position of the
solvent front with pencil (handling by the top edge)
After mason jar: When the paper is thoroughly dry, after approx. 15 min., spray it with the ______ while wearing gloves.
Gel filtration chromatography is a very mild method for purifying (4)
enzymes, polysaccharides, nucleic acids, and proteins
Gel filtration: The method separates molecules according to their
Two types of chromatography done
gel filtration chromatography (proteins) and paper chromatography (amino acids)
Gel particles form the ___ phase of this type of chromatography
the mobile phase is
the solution of molecules to be separated and the eluting solvent, which most frequently is water or a dilute buffer.
The sample is applied to the top of the gel bed; if the sample molecules are larger than the pores in the gel particles,
they never enter the gel and move outside the gel particles with the eluting solvent.
Thus, the very large molecules in a mixture move the___ through the gel bed and the smaller molecules, which can enter the ___, are ___ and move more ___ through the gel bed.
In gel chromatography, molecules are thus eluted in order of ______, that is, the smallest molecules elute last
decreasing molecular size
Describe the gel shape and pores
The gels are relatively inert, cross-linked polymers, and the amount of cross-linking determines the average pore size of a gel.Different degrees of cross-linking, thus, give different pore sizes and different molecular sieving or separation ranges.
Sephadex G-50 is used for separations up to a molecular weight of approx. ____
Name of gel
Sephadex G-50 gel
pink protein and relative size
rhodamine B smallest
orange protein and relative size
cytochrome c middlest
blue protein and relative size
blue dextran largest
blue dextran will not be retarded by the _____
The sephadex g-50 gel is in a buffer of
0.05 M potassium phosphate
Gel: The level of the buffer must not be allowed to_____________. Why?
sink below the surface of the gel as introducing air into the gel will interfere with the buffer/sample flow and may ruin the column.
Gel: what was the purpose of adding sucrose to all the coloured proteins?
to give the solution enough density so that the other compounds present do not diffuse upward into the buffer used for eluting them
What gels could enter the pores?
only cytochrome c and rhodamine B
the blue dextran movement was ___ and was used to calculate
column void volume (Vo)
Running the sample through the column: Never allow
the meniscus of the buffer to fall below the top layer of the beads.
gel step 2. Using a ____ and rubber bulb, gently pipette the entire sample (0.5 ml) into the narrow layer of buffer solution. Press bulb prior to going into the sample to prevent from making any bubbles.
gel step 3: Carefully open the lower stopcock, which permits ______. When there is a narrow layer of sample (0.5-1mm) remaining above the gel, close the stopcock.
the sample to enter into the gel
gel step 4: Carefully add approximately 0.25 mL buffer from your flask (~2 mm layer) to the top of the column by ____________ (Take care not to stir up the sample or gel).
slowly releasing buffer from a Pasteur pipette against the inside wall of the glass column.
Place a __ below the column to collect the buffer solution to begin. You are not concerned with recording the volume of eluate just yet.
The buffer layer above the gel should be ___ now.
Gel 10. Ensure that your stopcock is closed. Slowly begin to add buffer to the column. Is it still
clear (and not pink)?
• If it is clear, slowly add __ using your pipette until the buffer level has
reached 1 cm from the top, careful not to disturb the beads.
If it is not clear, but rather a light pink, stop adding ____ and allow more of the sample to enter the beads by opening the stopcock. Allow the eluate to drip into your graduated cylinder. Once you are certain it has fully entered the gel, slowly add buffer until the buffer level has reached about 1 cm from the top.
The _____ is suspended on a ring stand so that the buffer is gravity fed into the column.
The separatory funnel is suspended on a ____ so that the buffer is ___ fed into the column.
11 gel: The separatory funnel is suspended on a ring stand so that the buffer is gravity fed into the column. Connect the _____ of the funnel to the top of the glass column. It is sometimes possible to get a very good seal with the rubber stoppers, and thus be able to open the stopcock and let it flow continuously.
Continue measuring the total volume of the ___ as it flows into the graduated cylinder provided. If your cylinder gets full, take note of the volume and pour down the sink. You will see the separation of the sample as it moves through the sample into blue dextran, cytochrome C and rhodamine B.
Tip: If you place a ____ behind the column tip, this will allow you to see the first sign of colour. Record this volume.
white sheet of paper
Gel : The _____ is your void volume (Vo)
midpoint or median volume
The volume required to elute an enzyme, or other partially restricted molecule, whose movement is retarded by the gel is known as the ____
elution volume (Ve)
Sephadex gel filtration of unknown, colourless proteins (you will be given data based on these methods): Start collecting the____ of the eluate in _____. These tubes have 1ml-etched markings.
Collect a total of _____. This is equivalent to 25 mL of eluate.
25 microcentrifuge tubes
Visualize (or roughly plot on paper) a graph showing the ____(measurement) of
protein in each of your 25, 1ml aliquots
Graph of 25ml unknown protein:At least one peak should be apparent. A peak indicates ______. The volume of eluate collected up-to and including where the peak occurs is the ____.
that particular aliquot containing a large amount of protein.
elution volume (Ve)
The proteins added to your sample are colourless and so, they are ___ the same as those in your first elution.
Remember, elution volumes relate to _____ of proteins!
Paper chromatography: The further the amino acid runs, the greater its affinity for the ___. Since the paper had polar properties due to being made of cellulose (Krupadanam et al. 2001), ____ mixtures will travel further due to their reduced attractions.
____ are non-polar and therefore travelled farther up the paper
d and l proline
Ninhydrin is also used for ___ since human perspiration contains minuscule amounts of amino acids that have been freed from peptide terminals
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