Basic Chromatography Calculations

1. Total or empty column volume

Ve = π • R2 • L = 1/4 π • D2 • L Eq. 1

Ve = empty column volume

R = column radius
L = length of the column (or packed bed)
D = column diameter
If R (or D) and L are expressed in mm, Ve will be in µL. If in cm, then Ve in mL.

2. Linear velocity:

The efficiency of an HPLC column varies with flow rate. Instead of expressing efficiency
as a function of flow rate, it is easier to use linear velocity when comparing the
efficiencies of columns with different internal diameter, Simply stated, linear velocity is the
speed at which the solvent front travels the length of the column (L), and is calculated by
dividing the column length by the retention time (t0) of an unretained component. By
definition, an unretained compound has complete access to the volume of mobile phase
between the particles and to the volume of mobile phase in the pores of the particles,
while not interacting with the external or internal surface of the particles.

v = L / t0 Eq. 2

v (or <v>) = linear velocity (cm/min or cm/hr or mm/s)

L = length of the column or packed bed (cm or mm)
t0 = retention time of an unretained compound (in min, hr or s)

Note: see also the Eq. 4 below for an expression of the empty column linear flow rate or
superficial velocity, which is commonly used to describe transport phenomena in open
tubes, and is also used in process chromatography.

3. Mobile phase porosity:

 εt = F • t0 / 1/4 π • D2 • L Eq. 3

εt = mobile phase porosity (fraction of the column occupied by mobile phase between the
particles and in the pores); dimensionless parameter.
D and L are defined as in Eq. 1 above; t0 defined as in Eq. 2.
F = volumetric flow rate (mL/min)

4. Calculation of empty column linear flow rate from volumetric flow rate:

u = F / π • R2 Eq. 4

u = empty column linear flow rate or superficial velocity (cm/min or cm/hr)

F = volumetric flow rate (mL/min or mL/hr)
R as defined in Eq. 1 above.

5. Calculation of volumetric flow rate from empty column linear flow rate:

Using Eq. 4, the volumetric flow rate can be obtained by multiplying the linear flow rate by
the cross-sectional area of the column.

F = u • π • R2 Eq. 5

6. Relationship between linear velocity and empty column linear flow rate:

Empty column linear flow rate or superficial velocity (u) relates to the linear velocity (v in
Eq. 2 above) as:

u = v / εt or, v = εt • u Eq. 6

u = empty column linear flow rate, or superficial velocity (see Eq. 4)

v = linear velocity (see Eq. 2)
εt = mobile phase porosity (see Eq. 3)

Experiments: Identification of Unknowns Lab (Chem 3341, 3381)

In this experiment, your goal is to separate and identify two solid organic compounds - an
alcohol and a ketone.When you come to lab, you will be given a vial which contains a 1:1
mix of a solid alcohol and a solid ketone. Column chromatography is the method that you
will use to separate them. Before you can do the column, you need to determine which
solvent system will give optimal column separation; this solvent system is determined by
running a series of TLCs in different solvent systems. Once separated, each compound is
identified by matching its melting point, IR, and NMR with that of known compounds.
This experiment hones your techniques in column and thin-layer chromatography: if you
do not do these procedures properly, the compounds will not be separated. Once you have
separated the compounds, identifying them is a puzzle for you to solve. The melting
point, IR, NMR, and even appearance of each compound are clues about their identity.
We provide you with a list of possible unknown compounds. (This situation reflects some
real-world organic laboratory synthetic situations, since often a chemist is working in a
scheme and has a pretty good idea of the identity of an isolated compound.) When you
believe that you have properly identified each unknown compound, you will write
supporting arguments for your compound assignments in a 1-2 page paper. Write the
paper as if you are "convincing" someone else that the compounds are indeed the ones
you state that they are.

Two lab periods are scheduled for this experiment. During the first lab period, run TLCs
of the mixture to determine the ideal solvent for separation. During the second lab period,
separate the compounds by microscale flash chromatography (this procedure is illustrated
below; also review the section on column chromatography). If time allows during the
second lab period, take melting points and run IRs of the separated compounds; if not,
you can finish gathering the data during spare time in subsequent labs. Note: The
unknowns are solid compounds, so you need to use the thin-solid film method of sample
preparation for IR.

Thin Layer Chromatography (TLC) for ID Unknowns Lab

• The first step is to run a series of TLCs of the mixture, each in a different-polarity
solvent.

Microscale Flash Column Chromatography for ID Unknowns Lab

• The second step in this experiment is to run a microscale flash chromatography

column.

Melting points, IRs, and NMRs

For each separated unknown:

• Take the melting point.

• Run an IR. (See below.)
• Obtain the NMR. If you are in 3341, your TA will give you the NMR. If you are
in 3381, you will submit a sample for NMR analysis and work up your NMR
spectrum at one of the computers in the lab area.

FT-IR Analysis

Prepare the sample as a thin-solid film. Briefly: dissolve a few crystals in a few drops of
acetone, pipet the solution onto a single IR plate, allow the solvent to evaporate, and then
place this single IR plate in the spectrometer and run the spectrum. Please see this
spectroscopy page for a detailed procedure.

Print a copy of your spectrum. Then, click on the "search" button and the computer will
search the database for potential matches for your compound. Print the information from
the search.

Using all or some of this data, determine the identity of each of your unknown
compounds, and argue your assignments in a 1-2 page paper.

Column Chromatography Technique

In column chromatography, the stationary phase, a solid adsorbent, is placed in a vertical glass
(usually) column and the mobile phase, a liquid, is added to the top and flows down through the
column (by either gravity or external pressure). Column chromatography is generally used as a
purification technique: it isolates desired compounds from a mixture.

The mixture to be analyzed by column chromatrography is applied to the top of the column. The
liquid solvent (the eluent) is passed through the column by gravity or by the application of air
pressure. An equilibrium is established between the solute adsorbed on the adsorbent and the
eluting solvent flowing down through the column. Because the different components in the mixture
have different interactions with the stationary and mobile phases, they will be carried along with
the mobile phase to varying degrees and a separation will be achieved. The individual components,
or elutants, are collected as the solvent drips from the bottom of the column.

Column chromatography is separated into two categories, depending on how the solvent flows
down the column. If the solvent is allowed to flow down the column by gravity, or percolation, it is
called gravity column chromatography. If the solvent is forced down the column by positive air
pressure, it is called flash chromatography, a "state of the art" method currently used in organic
chemistry research laboratories.

The Adsorbent

Silica gel (SiO2) and alumina (Al2O3) are two adsorbents commonly used by the organic chemist
for column chromatography. These adsorbents are sold in different mesh sizes, as indicated by a
number on the bottle label: “silica gel 60” or “silica gel 230-400” are examples. This number refers
to the mesh of the sieve used to size the silica, specifically, the number of holes in the mesh or
sieve through which the crude silica particle mixture is passed in the manufacturing process. If
there are more holes per unit area, those holes are smaller, thus allowing only smaller silica
particles go through the sieve. The relationship is: the larger the mesh size, the smaller the
adsorbent particles.

Adsorbent particle size affects how the solvent flows through the column. Smaller particles (higher
mesh values) are used for flash chromatography, larger particles (lower mesh values) are used for
gravity chromatography. For example, 70–230 silica gel is used for gravity columns and 230–400
mesh for flash columns.

Results were less than acceptable when large 60-200 mesh material was used, but remarkably
improved when a 200-400 mesh material was in the column. Equally important: particle sizes less
than 40 microns offered no significant improvement in resolution in this system.
The Solvent

The polarity of the solvent which is passed through the column affects the relative rates at which
compounds move through the column. Polar solvents can more effectively compete with the polar
molecules of a mixture for the polar sites on the adsorbent surface and will also better solvate the
polar constituents. Consequently, a highly polar solvent will move even highly polar molecules
rapidly through the column. If a solvent is too polar, movement becomes too rapid, and little or no
separation of the components of a mixture will result. If a solvent is not polar enough, no
compounds will elute from the column. Proper choice of an eluting solvent is thus crucial to the
successful application of column chromatography as a separation technique.

Often a series of increasingly polar solvent systems are used to elute a column. A non-polar solvent
is first used to elute a less-polar compound. Once the less-polar compound is off the column, a
more-polar solvent is added to the column to elute the more-polar compound.

Column Chromatography Procedure

Packing a (silica gel) column:

1. Use a piece of wire to add a plug of cotton to the bottom of the column. There should be
just enough cotton that the sand and silica will not fall out of the column.
2. Clamp the column to a ring stand and add enough sand to fill the curved portion of the
column.
3. Place a pinch clamp on the tubing, then fill the column 1/4 to 1/3 full with the initial
eluent.
4. Prepare a slurry of silica in the initial eluent by pouring dry silica into a beaker of eluent.
(Add a volume of silica gel, such as 20 mL, to approximately double the volume of
eluent, 40 mL.) CAUTION: keep the dry silica in your hood and be careful not to inhale
the lightweight substance.

5. Quickly but carefully pour the slurry into the column. Stir and pour immediately to
maximize the amount of silica that goes into the column instead of remaining behind in
the beaker. You may find a clean spatula or glass rod helpful in transferring the silica.

6. Remove the pinch clamp to allow solvent to drip into a clean flask. Tap on the side of the
column with a rubber stopper or tubing to help the silica settle uniformly.
7. Use a Pasteur pipet to rinse any silica that is sticking to the sides of the column. Allow the
silica to settle while eluent continues to drip into the flask.
8. Once the silica has settled, carefully add sand to the top of the column. Sand is heavier
than silica. If the silica has not settled, the sand may sink into the silica instead of forming
a layer on top of it. (You may need to rinse down sand that sticks to the side of the
column.

Loading a sample onto the column:

9. Drain eluent from the column until no solvent remains above the surface of the sand.
10. Using a long Pasteur pipet, carefully add your sample to the column.
11. Drain eluent from the column until no sample remains above the surface of the sand.
12. Use ~ 1 mL of eluent to rinse your container and pipet. Add this milliliter of sample to the
sand. Drain eluent from the column until no liquid remains above the surface of the sand.
13. Repeat step 12 two or three times to completely transfer your sample onto the silica gel. If
 you do not do and repeat step 12, your sample will remain in the sand instead of on the
silica. Sample remaining in the sand will dissolve in the eluent that you add in step 14,
ruining the possibility of good separation of components.

Eluting the sample:

14. Once you have rinsed your sample onto the silica, carefully add eluent to the top of the
column. To avoid disturbing the top of the column, it's a good idea to carefully pipet an
inch or two of solvent onto the column instead of pouring solvent directly onto the sand.
15. Add more eluent as necessary. The eluent collected prior to the elution of sample can be
recycled. The composition of the eluent can be changed as the column progresses. If the
eluent composition is to be changed, ALWAYS start with least polar solvent/mixture and
change to the more polar solvent/mixture.

Analyzing the fractions:

16. Analyze the fractions by thin-layer chromatography to determine a) if the fraction

contains more than one component and b) if fractions can be combined without affecting
the purity of those fractions.

Other Tips:

• The success of your separation will be dependant on how well you pack and load the
column. It is important to have level sand and silica. It is also important to carefully and
evenly add your sample to the packed column.
• Do not allow the silica to dry out as the column progresses. Cracks will form within the
silica column if it dries, and compounds can fall down the cracks instead of partitioning
between mobile and stationary phases.
• Compounds pass through sand quickly and do not stick to it. Sand is used at the bottom of
the column to help ensure a level silica gel line. The bottom of the column is typically
cone shaped. If no sand were present at the bottom of the column, molecules traveling
down the center of the column would encounter less silica gel than molecules traveling
down the edge, closer to the glass. As a result, a particular component would elute as a
broader band which is undesirable.

• Sand is used at the top of the column to aid even loading of the sample. Sample diffuses
evenly through the sand. Once the pinch clamp is removed from the bottom of the
column, sample loads evenly onto the silica. Without sand, the sample would be added
directly to the silica and would stick wherever it is added, not evenly across the surface of
the silica.