Chromatography

Question 0

What is chromatography?

Answer 0

Chromatography is a separation method that places a mixture of compounds in a stationary, porous sorbent, resulting in a narrow band that is expected to separate based on each components affinity towards stationary phase or a liquid or gas mobile phase.

Question 1

What is the speed at which molecules travel through the stationary absorbant phase?

Answer 1

The speed at which molecules travel through a stationary absorbent phase is dependent on the fraction of the time that molecules (proteins) traveling through the absorbant interact with the stationary phase. This is dependent on the structure of the compound (include compound structures and hypothesis that they will travel through the column), the structure of the stationary phase, and the structure of the mobile phase.

Question 2

Is chromatography a stationary or dynamic process?

Answer 2

Chromatography is a dynamic process that separates compounds by moving solute through a stationary phase by running a mobile phase to obtain equilibrium distribution. (talk about what the mobile phase is and what the stationary phases are and their compositions/ contributions)

Question 3

What do all chromatrographic systems contain?

Answer 3

All chromatographic systems contain a stationary phase, a mobile phase, and a sample of molecules to be separated. (what are the stationary phase, mobile phase, and sample of molecules that will be separated).

Question 4

What is an impelling force and a retarding force?

Answer 4

An impelling force is one in which the mobile phase uses to carry molecules for which it has infinity. (what is the impelling force in this chromatogram)

A retarding force is the force imposed by the stationary phase that holds back the molecules with which it interacts with. (what is the retarding force?)

(talk about the predicted outcome for the proteins based on the observed composition of the mobile and stationary phase, do we expect the impelling force or retarding force to impact the compound to a particular degree relative to others?)

Question 5

What is the formation or development of the chromatogram?

Answer 5

The formation or development of the chromatogram is the process of impelling forces and retarding forces acting on analyte to form a separation of molecules as the mobile phase runs through the stationary phase.

Question 6

What are the three forces responsible for the directional movement of the solvent (mobile phase through the chromatograph?

Answer 6

The three forces that can be responsible for the movement of solvent through stationary phase can be gravitational force (often used in low-pressure liquid chromatography), pump-generated hydrostatic force (as in high-pressure liquid chromatography) or capillary force (as in paper and thin layer chromatography)

The force responsible for the movement of solvent through the chromatography column is a pump-generated hydrostatic force (note the functionality of the system generating the force), this is high pressure liquid chromatography.

Question 7

What are chromatographic methods employed for? Give an example of each.

Answer 7

Chromatographic methods are employed for analysis, purification, or characterization of a wide range of compounds.

Analytical chromatograph example is a gas chromatographic screening of foods for cancer-causing nitrosoamines.

Preparative chromatograph example is purifying individual proteins from complex biological mixtures

This is a preparative chromatography technique and the property we are using chromatography to determine in this experiment is native molecular weight of oligomeric proteins.

Question 8

What are the various types of equilibration processes?

Answer 8

The various types of equilibrium processes include:

1. Partition: the stationary phase is a liquid supported on an inert solid
2. Adsoption: the stationary phase is a sold on which the sample components are absorbed
3. Size-exclusion, or pore penetration: molecules are separated based on their ability to penetrate a sieve-like structure
4. Ion exchange: the mechanism of separation is based on ion exchange equilibria
5. Afinity Chromatrography: highly specified interactions cause binding of solute molecules

Size exclusion chromatography was performed. Molecules were separated on their ability to penetrate a sieve like structure in toyopearl resin (look at dimensions of Toyopearl resin to see what molecular wieghts are likely to penetrate, etc.)

Question 9

What are the main modern chromatographic method techinques?

Answer 9

The main modern chromatographic method techniques are
paper chromatography, thin layer chromatography, ion exchange chromatography, gel permeation chromatography (size Exclusion), affinity chromatography, gas chromatography, super critical fluid chromatography, high performance liquid chromatography, capillary (zone) electrophoresis

Question 10

How is preparative chromatography usually carried out in regards to separating a protein of interest?

Answer 10

preparative chromatography used to separate a protein of interest is typically carried out in the form of column chromatography.

Question 11

Describe column chromatography.

Answer 11

Column chromatography wet stationary phase is packed into a column , the column is equilibrated with buffer, and mixture of macro molecules is loaded as a narrow zone at the top of the column.

Describe the wet stationary phase that is packed, the buffer that is equilibriated and how it is equilibriated, and name the mixture of macromolecules that are loaded at the narrow zone at the top of the column.

Question 12

Fresh buffer is continuously pumped through the column, the solute molecules in mobile phase are carried along with the flow toward the end of the column.

Answer 12

molecules in the stationary phase remain immobile. Molecules that partition into the mobile phase are mobile.

Question 13

What is the retention time or elution time?

Answer 13

The retention time or elution time is the net time a particular molecule spends in the column.

What are the retention times of each species, what does this say about their size or composition?

Question 14

Why do molecules exit as gausian shapped peaks and not sharp peaks?

Answer 14

The reason that molecules exit as gaussian peaks and not as sharp bands, is due to diffusion. The maximum position of the peak corresponds to the retention time or elution volume.

Describe the degree of diffusion of each species?

Question 15

Describe what the dimensions of the gaussian curves are for column chromatography.

Answer 15

The dimensions of the gaussian curves for column chromatography,
The maximum peak of the curve corresponds to the retention time or elution volume
The width of the peak corresponds with the efficiency of separation in the given column.
The area of the peak gives the total amount of that solute, allowing quantitative determination of the sample composition

What is the sample composition of the mixture that was given based on the areas of the gaussian shaped peaks.

Question 16

What is the role of the buffer in the chromatography experiment?

Answer 16

The role of the buffer in the chromatography experiment is to ensure good stability and solubility of the molecules that are being separated. Denaturation and precipitation of the protein of interests in the column not only ruins the purification of the active protein by clogging pathways that can leads to problems with resoluton and increased diffusion.
Buffer ensures that only desired kinds of interactions between the solute molecules and the station phase take place during separation.

Buffers in size exclusion chromatography typically contain 150 mM NaCl to minimize binding of macromolecules to the stationary phase such that the separation only depends on the ability of solute molecules to penetrates pores of the stationary phase.

Question 17

What kind of buffers would you want to pump through a affinity chromatographic column?

Answer 17

The kind of buffers you want to pump through an affinity chromatographic column are a low salt buffer during sample loading to cause ligands to bind to the stationary phase, followed by a high salt buffer to elute the remaining portion of the sample that we don’t want / didn’t bind to the stationary phase.

Question 18

Describe the shapes and sizes of chromatography columns.

Answer 18

long skinny columns are used to achieve good separation of similar molecules during elution in applications such as size exclusion chromatography.

short, fat columns are used for applications such as affinity chromatography because the molecule of interest is strongly bound to the stationary phase and will remain bound during washing stage while all the impurities leave the column.

Larger diameters allows large sample capacities and thus larger purification of large quatinites of the protein of interest. Columns of small diameters are used for analytical purposes such as checking the purity of the sample, or the determination of native molecule wieght.

Describe why the dimensions were choosen for our sample.

Question 19

what do we use to avoid giving a system dependent retention time?

Answer 19

To make results reproducible to experimentors who may not pocess the same relative views as to what is “well packed” or may not pocess the same tools (column length/ width or resin) as us we use a value called the capacity factor.

The capacity factor, or retention factor K’ is Tr - Tm/ Tm
where Tr is the retention time of the solute and Tm is the time it takes solution to travel through the column if it was only in the mobile phase.

Tm is measured using a solute that doesn’t interact with the stationary phase

Describe how we know solute isn’t interacting with the stationary phase, talk about Tm,

Question 20

What is the void volume and the dead time?

Answer 20

The void volume is the volume of free space around the stationary phase
the Dead time is the amount of time a solute does not interact with the stationary phase
Dead time is dependent on factors such as the geometry of the column, packing of the resin in the column the flow rate, but doesn’t depend on the nature of the solute.

Question 21

Why doesn’t the capacity factor depend on the geometry of the column or the flow rate?

Answer 21

The capacity factod doesn’t depend on the geometry of the column or the flow rate because it is largely a property of the solute and the stationary phase and the quality of packing.

More packing means more interaction between analyte and resin, more resin per cm if packed tighter.

Question 22

What is the selectivity factor?

Answer 22

Selectivity factor is a mixture of two solutes that one tries to separate. For any column geometry and flow rate, the likelihood of separating two species is determined by the ration of their capacity factors.

selectivity factor is alpha: alpha devided by capacity factor Tra/ capacity factor Trb

good separation is expected when the value of the selectivity factor differs significantly from unity.

(look up what values of selectivity factors result in good resolution.

Question 23

What can be done to increase separation in the column?

Answer 23

parameters that influence separation of molecules in column chromatography include: buffer flow rate, temperature, and dimensions of the column

Question 24

How do you measure peak with in column chromatography?

Answer 24

Peak width in column chromatography is measured at the base of the peakm the base us defubed by base intersection of two peak tangent lines with the baseline. Tangent lines are often taken from the half height of the peak.

Question 25

What is resolution defined as in column chromatography?

Answer 25

resolution is defined as the ration of peak different to the average peak width

Rab = (delta Tr)/(w avg) = (Trb-Tra)/(Wa + WB / 2)

peaks are considered well resolved at the base when Rab >1.5

resolution is improved by increasing the peak separation while controlling the peak width.