chromatography methods

Question 1

THIN LAYER CHROMATOGRAPHY (TLC)

Answer 1

the stationary phase (adsorbent) consists of a very thin layer of small particles attached to a flat plate (which can be glass, plastic or metal foil). This is called a TLC plate
The adsorbent in TLC is commonly silica (silicon dioxide, SiO2) gel, a highly absorbent silica powder.
a small amount of the reaction mixture to be separated is dissolved in the minimum
amount of a solvent needed to dissolve all components of the mixture
A horizontal line, called the starting line, is drawn in pencil
A small spot of the mixture is then applied to the plate on this line
The plate is then placed in a covered glass container containing a different solvent which is called the eluant; this acts as the
mobile phase. This eluant slowly rises up the silica gel (a process called elution)

Question 2

TLC RESULTS

Answer 2

If a suitable eluant has been chosen, the compounds move up the plate at different rates, and the
mixture will begin to separate on the plate as they gradually move up it

When the solvent has moved about three-quarters of the way up the plate, the plate is removed from the eluant and the position of the solvent front is marked quickly, before the plate dries. The distance from
the starting line to the solvent front can be measured, as can the distance from the starting line to the centre of each spot. A retardation factor (Rf) can then be calculated for each spot from the following equation:
Rf = distance of spot from origin / distance of solvent front from origin

The Rf values relate to a particular compound separated using a specific eluant and stationary phase,
i.e. a compound will have a different Rf if, for example, a different eluant is used.

This process is easy to use for coloured compounds, however often the compounds are not
coloured. In this case a stain (a chemical that will react with the compounds of interest and generate
a colour – chromophore) is used. Alternatively, in some cases, UV light can be used to see the
compounds if they absorb in that range.

Question 3

Solvent (or Eluant) selection

Answer 3

Analytical scientists
decide by a combination of experience and trial-and-error; they would normally run several TLC
plates, each using a different solvent or mixture of solvents, and find which gives the best
separation.
If methanol, which is very polar, is used instead, the solvent also adsorbs strongly to the adsorbent.
Thus, it would displace almost every molecule that it encounters and everything in the reaction
mixture moves together at the solvent front. In this case, neither of the two extreme solvents –
hexane or methanol – would effect a good separation

Question 4

the elutropic series

Answer 4

a list of solvents in order of their power to carry a given compound through the stationary
phase (e.g. silica gel).
The order of solvents runs in the order of
their polarity.
The series is useful when selecting a solvent for a particular separation where more
polar compounds will normally require a more polar solvent. A good starting point for a new
separation is one with medium polarity such as dichloromethane or acetone.

The Elutropic series. Remember that so long as these are miscible (can be mixed together)
then you can use a combination to obtain a greater range

Question 5

TLC USES

Answer 5

TLC is a simple but effective technique for determining the purity of a crude mixture. A simple spot
check of the starting reaction mixture and the product can tell if a reaction has happened.
It can also be used to separate, purify and recover a compound in a mixture; the silica in the plate at
the point where the compound of interest has migrated can be scraped off, and the purified
compound then extracted

Question 6

COLLUMN CHROMATOGRAPGHY

Answer 6

In preparative chromatography, where a compound needs to be purified for use elsewhere, column
chromatography is more suitable
the solid stationary phase particles (often silica
based with various chemical groups attached, depending on the column) are packed into a column,
and the solvent flows down through the particles by gravity, or it can be pumped.
The mixture is introduced at the top of the column and, as the continually flowing solvent (mobile
phase) flows through the column, the different components move down (with the solvent) at different
rates. Each component flows out of the other end of the column at a different time in the mobile
phase, as they are separated by the chromatographic process. By collecting the solvent in portions
called fractions, each component of the mixture can be isolated as it leaves the column.
If we continually change the solvent running through the column, we can gradually increase the
polarity of the mobile phase, thus removing the more polar components in turn from the column.
This continually changing solvent is called a solvent gradient and is usually achieved by having two or
more reservoirs of mobile phase solvents and two pumps. The pumps can be programmed to change
the proportion of each solvent pumped over time

Question 7

analytical chromatography

Answer 7

Determining the purity, concentration or identity of a substance is the domain of analytical column
chromatography
analytical chromatography is designed to determine how much of
something is present, or to identify what it is. This requires very small quantities of sample and column
material. Imagine the difference in the size of the columns

Question 8

preparative chromatography

Answer 8

Preparative chromatography is used to purify a compound in a complex mixture for
later use. It is mostly a question of scale. Preparative chromatography may require the purification
and recovery of large amounts of material; in some processes, the packing material may weigh
hundreds of kilograms

Question 9

GAS CHROMATOGRAPHY

analytical chromatography

Answer 9

GC is column chromatography in which the mobile phase is a gas. The stationary phase can be in either a packed column or a capillary column
A packed column will contain material that can itself be used as the stationary phase or be coated with a liquid layer as the stationary phase.
A capillary column, also known as an open tubular column, are generally made of fused silica glass (SiO2) but with a coating to protect the fragile silica
This enables the long columns to be coiled without damaging them
Capillary columns have become common with packed columns rarer.

Question 10

GLC

Answer 10

If the stationary phase is a liquid, it is gas–liquid chromatography (GLC), and this is the most common
technique because of its speed and efficiency. In GLC different separations are achieved through
changing the nature of the liquid stationary phase; the gas mobile phase is invariably an inert gas such
as helium.
GC is an extremely powerful technique capable of separating very complex mixtures with very high
resolution. However, to be able to use it, the sample should be either a gas or a volatile liquid capable
of being heated to produce a gas. This is why it has been possible to use this to analyse a very wide
range of organic compounds or gases, as long as they are volatile and stable at the temperature range
used (typically between 50 and 300 °C)

Question 11

Carrier gas (mobile phase)

Answer 11

The carrier gas is the mobile phase used to carry the sample down the column. It needs to be an inert
(unreactive) gas that does not interfere with the type of detector used. The most common gases used
are helium and nitrogen but carbon dioxide and argon are also used.
The flow rate is governed by the gas pressure which is typically controlled by a valve and monitored by a device called a flow meter.
This can accurately measure the gas flow rate through the column. To obtain a consistent and
reproducible chromatographic separation, the flow rate should be constant throughout. The pressure
is usually kept between 25 to 150 mL min−1 of gas through the column.
If the sample is gaseous, the sample volume may range from 1 ml up to 10 mL of gas. Volatile liquids
will have much smaller sample volumes, as little as 1–100 μL. The samples are often injected by syringe
into a heated injector.
Some of the small capillary columns have very low capacities and it is difficult to accurately produce
and inject a small enough sample. This is why many GC systems can split the sample automatically, so
that only a small fraction of the sample goes into the column; the rest being discarded.

Question 12

Carrier gas (mobile phase):
Separation

Answer 12

Separation occurs as the sample is partitioned between the inert mobile phase (carrier gas) and the
stationary phase of the column. The relative partitioning between mobile and stationary phases can
be achieved by altering the temperature of the column during the chromatography run. I
In a typical GC run, the sample is introduced to the column at a particular temperature (e.g. 50 °C).
The temperature may be held constant for a few minutes and then a temperature ramp (gradient)
will start which may enable a gradual temperature increase or several step changes. By the end of
the run, the temperature may reach 250–300 °C.

The most volatile compounds will elute first and
the least volatile will only leave the column when the higher temperature has enabled them to be
partitioned more easily in the gaseous mobile phase.

Question 13

Compromise between speed and efficiency

Answer 13

A faster temperature ramp will increase the
speed of the chromatographic process but the separation may be less efficient. So there is a
compromise between speed and separation efficiency.
Although separation is achieved by means of a temperature gradient, the choice of stationary phase
is the most important factor in determining whether the components of a sample can be separated.
You have already seen that columns can be either packed or capillary Ideally, the packing material should be regularly shaped and uniform in size

Question 14

separation of the samples via polarity

Answer 14

The stationary phases achieve separation by polarity, which means that separation will depend on both
the polarity of the compound and the temperature of the column. Polysiloxanes and polyethylene
glycols molecules of different lengths, functional groups and polarities are the most commonly
available stationary phases

Question 15

Gas chromatography detectors

Answer 15

After passing through the column, the mobile-phase gas containing the separated eluates
(compounds that are separated after elution) then passes through a detector. The detector will also
be plumbed to enable clean mobile phase to pass through a reference cell for a signal comparison to
be made. There are various detectors available for use with gas chromatography.
The most commonly used detectors are the thermal conductivity detector, the flame ionisation
detector and the mass detector (mass spectrometer). Each system has different
advantages and disadvantages. Sometimes more than one detector can be used. However, in these
cases, the exit gas flow is split and the mobile phase does not flow through each detector
sequentially. This is because GC detectors often change the sample in some wa

Question 16

Gas chromatography detectors

Two common detectors:

Answer 16

(a) A flame ionisation detector. Eluted compounds and mobile
phase pass from the column into the detector. The flame ionises the organic compounds present,
producing ions and a current flows, which may be amplified and recorded. (b) One part of a
thermal conductivity detector. The mobile phase from the GC column or the clean mobile phase is
passed across the electrically heated wire and the thermal conductivity is measured

Question 17

Qualitative and Quantitative analysis

Answer 17

A series of standards containing the
specific compounds of interest at known concentrations are prepared and injected into the GC. From this a calibration curve is generated based on the size of the response observed. The response from
a sample is compared with this curve to calculate the concentration of the sample.
A chromatographic peak has two factors that are commonly used to determine a value from which a curve can be plotted. These are peak height and peak area; the latter being preferred as it can give a more accurate value for concentration

Question 18

Qualitative and Quantitative analysis

internal standards

Answer 18

An internal standard is a set amount of a known substance that is not
otherwise present in the sample but has a retention time close to (but not overlapping with)
compounds of interest in the sample. As the amount of the internal standard is known, it can be
compared against a calibration curve of its own. Any differences between what was injected and
what was measured can be considered when the calculation is done for the sample of interest. This
allows for the possibility of some of the sample being lost in the injection process to be considered

Question 19

High-performance liquid chromatography (HPLC)

Answer 19

is very widely used to analyse many classes of
compounds from ions through to proteins. It is a powerful technique as it can fairly rapidly achieve
excellent separations of complex mixtures by a very wide range of columns and detectors.
It can separate :
according to polarity using normal or reverse-phase chromatography,
size by size-exclusion chromatography
charge by ion-exchange chromatography.
In addition, a similar technique :
capillary electrophoresis – can separate compounds by applying an electric field.

Question 20

HPLC

separation

Answer 20

Separation is achieved using columns packed with small particles and pumping the mobile phase
through under high pressure. In fact, HPLC was originally called high-pressure liquid chromatography
because of this. The improvements in speed and separation efficiency in HPLC over traditional liquid
column chromatography are brought about by the ability to pump the mobile phase through the
column at high pressure, plus the improved quality of the columns themselves. HPLC columns
contain very regular sized spheres of high purity silica which are small and well packed into a dense
matrix.
The pump enables the mobile phase to be forced through the column at a reasonable speed. The
stationary phase contains minimal areas of dead space, reducing the potential for stagnant mobile
phase to collect. These factors reduce the values of A (eddy diffusion) and C (mass transfer) in the
van Deemter equation thus effectively reducing the plate height, leading to a better separation
efficiency.

Question 21

the main components of a HPLC system :

Answer 21

The main components of an HPLC system with mobile-phase solvent reservoirs pump, sample injection loop, column and detector.
A mobile phase is pumped from a reservoir into an HLPC column, which may be heated if necessary.
The mobile phase will be selected for its suitability in providing sufficient separation of the
components of the sample. In some separations, a mobile phase may be changed throughout the
chromatography run by pumping different proportions of various solvents from more than one
reservoir as the run progresses.

Question 22

The solvents used should be carefully degassed before being used:

Answer 22

the dissolved air in the solvents should be minimised. It is not necessary to remove all the dissolved air, but enough to prevent bubbles from forming- when the mobile phase is pumped through the system; bubbles can produce spurious peaks in the detector and can also interfere with the correct operation of valves. Solvents can be degassed by placing solvent reservoirs in a sonic bath (in which sound waves are passed through the containers, removing some of the
dissolved air).
Another way of degassing is by bubbling helium through the solvent, or degassing under a vacuum. Modern HPLCs often have inbuilt degassing systems.

Question 23

Adding a known volume of sample to the HPLC column

Answer 23

A calibrated sample loop of typically a few microlitres (μL) is positioned just before the column so that an
appropriate volume of sample can be injected into the column for separation into its components. The sample loop is attached to an injection system which typically is a stainless-steel ring with six separate ports; this enables it to be connected to the mobile phase and the column, as required The sample is injected into the loop (at this point the loop is isolated from the
pump and column). Then the apparatus is turned so that the sample loop is now connected to the
column and the mobile phase is pumped through the loop and the sample loaded into the column.The sample moves from the sample loop directly into the column

Question 24

several different types of packing material for HPLC columns

Answer 24

the most common is the micro-porous particles, which allow the mobile phase to flow through the particle, increasing the surface area.
The stationary phase is coated onto the surface of the particles, inside the pores (where the surface area is largest) and on the outside; it may also be chemically bonded to the particles.
Silica is the most widely used material for column packing as it has excellent adsorbent properties. The Si–OH groups on the surface are well suited for chemical bonding of the stationary
phase, and many different bonded phases can be added.

Question 25

A suitable detector

Answer 25

A suitable detector is placed at the end of the column so that the flow of mobile phase from the end
of the column should pass through the detector. This will enable the compounds to be detected as
they elute from the end of the column. The size of the signal produced by the detector is usually
directly proportional to the amount of the substance present and this enables the system to make a
quantitative measurement. The detector should be carefully chosen to detect the compounds of
interest eluting from the column. It is important to note that not all detectors will respond to all
compounds. It is possible to have more than one detector in series to analyse the solutes in the
mobile phase as they elute from the column. Most detectors will not identify compounds that elute
except for the mass detector (mass spectrometer),

Question 26

Probably the most widely used detector is the UV–vis detector.

Answer 26

Very few detectors will work at a wavelength of less than 200 nm. Most UV–vis detectors typically contain two lamps which produce light in the wavelength band between 200 and 900 nm. The deuterium lamp produces wavelengths at the lower wavelength range and a tungsten lamp produces higher wavelengths of light. A diffraction grating, or filter, is then used to filter the light from the lamp to produce a specific
wavelength (monochromatic light), which can be set by the user. This will only provide the absorbance at a single wavelength. It is important to know at which wavelength the solute being
tested will absorb, so spectral scans are made on samples to determine this.

More recently, photodiode array detectors (PDA) or diode array detector (DAD) have been
developed which can be used in HPLC. In these devices, polychromatic light is passed through the
eluant from the column and the unabsorbed light lands on an array of diodes. Each diode acts like a
filter or slit. The different wavelengths of the polychromatic light that passes through the sample
have been absorbed by the sample to different extents. Thus, the absorbance at each of the
wavelengths can be measured because, when the light photons strike a photodiode, only photons of
a particular wavelength will have enough energy to induce a current. So, having an array of diodes,
light intensities (and thus absorbances) at all the wavelengths can be measured simultaneously. This
enables an almost continuous spectrum of UV–vis wavelengths for the eluant to be recorded very
rapidly. As all wavelengths are recorded simultaneously, a photodiode detector can analyse species
that absorb at different wavelengths in the same sample.

Question 27

Not all compounds will absorb light, or absorb only very poorly, which means that the sensitivity is
lower.

Answer 27

Several other types of detector are commonly used. These include the fluorescence detector,
differential refractive index detector, infrared detector, electrochemical amperometric detector and
conductivity detector. Fluorescence detectors have very high sensitivities but obviously can only be
used to analyse fluorescent compounds. Some compounds can be modified to be “tagged” to enable
detection.
The differential refractive index detector is also known as a universal detector as it can be used to
monitor the presence of almost any compound. It works by detecting changes in the refractive index
of the eluant caused by the presence of analytes in the mobile phase. As the mobile phase
containing analytes in the sample leaves the column, it passes through a flow cell. A light beam
passes through the flow cell and a reference cell containing pure mobile phase and the refractive
indices in the two cells are compared.

Question 28

Although refractive index is a universal property, this

detector has the following disadvantages.

Answer 28

It cannot be used where there is a mobile phase gradient (i.e. where more than two solvents
are used in different concentrations during elution) as the refractive index will always be
changing.
It is very sensitive to small changes in temperature, so very careful temperature control is
required.
It is much less sensitive than UV or fluorescence.

Question 29

mass spectrometer

Answer 29

. A mass spectrometer produces a mass spectrum for every eluted compound that
passes through it. This spectrum can be compared with a library of spectra and the compound may
be identified. It is very important to ensure that analytes are fully separated in the chromatographic
process as two compounds arriving in the mass spectrometer at the same time will be very difficult
to identify.
Mass detectors have traditionally been used with gas chromatography as it is better suited to
analysing gaseous or volatile compounds. The difficulty is that the mass detector chamber is a
vacuum, and adding large quantities of liquid mobile phase is not possible. There are ways around
this, such as letting only a small part of the mobile phase into the chamber, or evaporating the
solvent in a so-called thermospray interface

Question 30

HPLC can be used to

determine what is in a sample (qualitative analysis) and how much is there (quantitative analysis).

Answer 30

If the HPLC does not have a mass spectrometer, the only way to determine what is present is to
compare the retention time of particular analytes in your sample with known retention times for
compounds using your column and the same chromatography conditions.

However, without using a mass spectrometer, it is not possible to be certain about a peak’s identity; all that
can be said is that it elutes at the same time. In most cases, this is all that is required as the
approximate composition of the sample is known.
In the case of quantitative analysis, how much of each analyte is present can be determined using
standards.

Question 31

HPLC USING A STANDARD

Answer 31

Using a standard range of known concentrations of the compounds that are being
analysed. These are then put through the HPLC as if they were samples and the peak height or peak
area for each of the compounds is recorded at each known concentration. This enables the
generation of a calibration curve, which can then be used to determine the concentration of a
particular analyte in the sample. This calibration curve can be used for each set of samples in the
same series but should be repeated each time a new series of samples is run.