Laboratory Instrumentation Chromatography

8/3/2019 Laboratory Instrumentation Chromatography

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HISTORY

INTRODUCTION

Mikhail Semyonovich Tsvet (18721919)

was a Russian botanist who inventedadsorption chromatography technique in

1906 during his research on

plant pigments. Tsvet's work involved

the study of plant pigments, such as

chlorophylls and carotenoids. In 1901 he

developed the techniques of adsorption

chromatography. He passed solutions of

leaf colourants dissolved in a light petrol

mix through a column of powdered

chalk. The distinct colour bands formed

by the different pigments could then beand analysed.The method was described on 30 December 1901 at the XI

Congress of Naturalists and Physicians in St. Petersburg. The first printed

description was in 1905, in the Proceedings of the Warsaw Society of

Naturalists.

He first used the term "chromatography" in print in 1906 in his two papers

about chlorophyll in the German botanical journal, Berichte der Deutschen

botanischen Gesellschaft. In 1907 he demonstrated his chromatograph for

the German Botanical Society. However, scientists at the time could not

replicate his findings, believing that they were erroneous. However, 10

years after his death in 1919, others successfully repeated his results and
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The word chromatography is composed of two Greek roots, chroma

(colour) and graphein (to write), and its translation means colour

writing, which refers to visualizing the separated multicoloured rings onthe column. Another interpretation of this term links it to Tswetts

surname, which is colour in Russian. According to International Union of

Pure and Applied Chemistry (IUPAC) definition of chromatography in 1993,

chromatography is defined as the physical method of separation in which

the components to be separated are distributed between two phases, one

of which is stationary while the other moves in a definite direction.

Chromatography is the laboratory techniques for the separation of

mixtures. The mixture is dissolved in a fluid called the "mobile phase",

which carries it through a structure holding another material called the

"stationary phase". The various constituents of the mixture travel at

different speeds, causing them to separate. The separation is based on

differential partitioning between the mobile and stationary phases. Subtle

differences in a compound's partition coefficient result in differential

retention on the stationary phase and thus changing the separation. Any

Chromatography system is composed of three components, which are the

stationary phase, mobile phase and mixture to be separated. We can only

control stationary and mobile phase as mixtures are the problem we have

to deal with.

Stationary phase is a layer or coating on the supporting medium that

interacts with the analytes and is fixed in a place either in column or a

planar surface. It can be solid, liquid, gel or solid-liquid mixture. Its also

the part of the chromatographic system though which the mobile phase

flows where distribution of the solutes between the phases occurs. It

Mobile phase is the part of the chromatographic system which carries

the solutes through the stationary phase. It can be either liquids or
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Chromatography may be preparative or analytical. The purpose of

preparative chromatography is to separate the components of a mixture

for further use (and is thus a form of purification). Analyticalchromatography is done normally with smaller amounts of material and is

for measuring the relative proportions of analytes in a mixture.

There are many types of chromatography, types are as follows:

(a)Techniques by chromatographic bed shape

Column chromatography

Planar chromatography

Paper chromatography

Thin layer chromatography

(b)Techniques by physical state of mobile phase

Gas chromatography

Liquid chromatography

(c)Techniques by separation mechanism

Ion exchange chromatography

Size-exclusion chromatography


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COLUMN CHROMATOGRAPHY

Column chromatography is a method used to purify individual chemicalcompounds from mixtures of compounds. It is often used for preparative

applications on scales from micrograms up to kilograms. The main

advantage of column chromatography is the relatively low cost and

disposability of the stationary phase used in the process.

In column chromatography, the mobile phase is a solvent, and the

stationary phase is a finely divided solid, such as silica gel or alumina.

Chromatography columns vary in size and polarity. There is an element of

trial and error involved in selecting a suitable solvent and column for the

separation of the constituents of a particular mixture. A small volume of

the sample whose constituents are to be separated is placed on top of the

column. The choice of the eluting solvent should ensure that the sample is

soluble. However, if the sample was too soluble the mobile phase

(solvent) would move the solutes too quickly, resulting in the non-

separation of the different constituents.

There is an optimum flow rate for each particular separation. A faster flow

rate of the eluent minimizes the time required to run a column and

thereby minimizes diffusion, resulting in a better separation. However, the

maximum flow rate is limited because a finite time is required for analyte

to equilibrate between stationary phase and mobile phase, see Van

Deemter's equation. A simple laboratory column runs by gravity flow. The

flow rate of such a column can be increased by extending the fresh eluent

filled column above the top of the stationary phase or decreased by the

tap controls. Faster flow rates can be achieved by using a pump or by

using compressed gas (air, nitrogen, or argon) to push the solvent through

the column.
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The classical preparative chromatography column is a glass tube with a

diameter from 5 mm to 50 mm and a height of 5 cm to 1 m with a tap and

some kind of a filter (a glass frit or glass wool plug to prevent the loss of

the stationary phase) at the bottom. Two methods are generally used toprepare a column: the dry method, and the wet method.

The preparations of the two methods are further discussed as follows:

a) Prepare the column.

The column is packed using a simple dry-pack method.


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Plug a Pasteur pipette with a

small amount of cotton; use a

wood applicator stick to tamp

it down lightly. Take care thatyou do not use either too

much cotton or pack it too

tightly. You just need enough

to prevent the adsorbent from

leaking out.

Add dry silica gel adsorbent,

230-400 mesh -- usually the

jar is labelled "for flash

chromatography." One wayto fill the column is to invert

it into the jar of silica gel and

scoop it out.

Then tamp it down before

scooping more out.

Another way to fill the

column is to pour the gel into

the column using a 10 mL

beaker.


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When properly packed, the

silica gel fills the column to

just below the indent on the

pipette. This leaves a space

of 45 cm on top of the

adsorbent for the addition of

solvent. Clamp the filledcolumn securely to a ring

stand.

Whichever method you use to

fill the column, you must

tamp it down on the bench

top to pack the silica gel. You

can also use a pipette bulb to

force air into the column and

pack the silica gel.

b) Pre-elute the column.

The procedure for the experiment that you are doing will probably

specify which solvent to use to pre-elute the column. A non-polar

solvent such as hexanes is a common choice.


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Add hexanes (or other specified

solvent) to the top of the silica

gel. The solvent flows slowly

down the column; on the

column above, it has flowed

down to the point marked by

the arrow.

Monitor the solvent level, both

as it flows through the silica gel

and the level at the top. If you

are not in a hurry (or busy

doing something else), you can

let the top level drop by gravity,

but make sure it does not gobelow the top of the silica.

Again, the arrow marks how far

the solvent has flowed down

the column.


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Speed up the process by using a

pipette bulb to force the solvent

through the silica gel. Place the

pipette bulb on top of thecolumn, squeeze the bulb, and

then remove the bulb while it is

still squeezed. You must be

careful not to allow the pipette

bulb to expand before you

remove it from the column, or

you will draw solvent and silica

gel into the bulb.

When the bottom solvent level

is at the bottom of the column,

the pre-elution process is

completed and the column isready to load.

If you are not ready to load your

sample onto the column, the

column can be left at this point.

Just make sure that it does not

go dry, therefore keep the topsolvent level above the top of

the silica (as shown in the

picture to the left) by adding

solvent as necessary.

c) Load the sample onto the silica gel column.

Two different methods are used to load the column: the wet method

and the dry method: wet and dry.

In the wet method, the sample to be purified (or separated into

components) is dissolved in a small amount of solvent, such as


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hexanes, acetone, or other solvent. This solution is loaded onto the

column.

Wet loading method

The column at the left is being

loaded by the wet method. Once

it's in the column, fresh eluting

solvent is added to the top and

you are ready to begin the

elution process.

Sometimes the solvent of choice to load the sample onto the column is

more polar than the eluting solvents. In this case, if you use the wet

method of column loading, it is critical that you only use a few drops of

solvent to load the sample. If you use too much solvent, the loading

solvent will interfere with the elution and hence the purification or

separation of the mixture. In such cases, the dry method of column

loading is recommended.


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Dry loading method

First dissolve the sample to be

analyzed in the minimum amount

of solvent and add about 100 mgof silica gel. Swirl the mixture

until the solvent evaporates and

only a dry powder remains. Place

the dry powder on a folded piece

of weighing paper and transfer it

to the top of the prepared

column. Add fresh eluting solvent

to the top, the elution process

are ready.


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d) Elute the column.

Force the solvent through the column by pressing on the top of the

Pasteur pipette with a pipette bulb. Only force the solvent to the very

top of the silica: do not let the silica go dry. Add fresh solvent as

necessary.

The solvent being forced

through the column with a

pipette bulb. The series of 5

photos below show the colored

compound as it moves through

the column after successive

applications of the pipette bulb

process. The last two photos

illustrate collection of the

colored sample. Note that the

collection beaker is changed as

soon as the colored compound

begins to elute. The process is

complicated if the compound is

not colored. In such

experiments, equal sized

fractions are collected

sequentially and carefully

labelled for later analysis.


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e) Elute the column with the second elution solvent.

If you are separating a mixture of one or more compounds, at this point

you would change the eluting solvent to a more polar system.

f) Analyze the fractions.

If the fractions are colored, you can simply combine like-colored

fractions, although TLC before combination is usually advisable. If the

fractions are not colored, they are analyzed by TLC (usually). Once the

composition of each fraction is known, the fractions containing the

desired compound(s) are combined.


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PLANAR CHROMATOGRAPHY

Planar chromatography is a separation technique in which the stationary

phase is present as or on a plane. The plane can be a paper, serving as

such or impregnated by a substance as the stationary bed (paper

chromatography) or a layer of solid particles spread on a support such as

a glass plate (thin layer chromatography).

Different compounds in the sample mixture travel different distances

according to how strongly they interact with the stationary phase as

compared to the mobile phase. The specific Retention factor (Rf) of each

chemical can be used to aid in the identification of an unknown substance.

The retention factor(R) may be defined as the ratio of the distance

travelled by the substance to the distance travelled by the solvent.

Rvalues are usually expressed as a fraction of two decimal places but it

was suggested by Smith that a percentage figure should be used instead.

If Rvalue of a solution is zero, the solute remains in the stationary phase

and thus it is immobile. If Rvalue = 1 then the solute has no affinity for

the stationary phase and travels with the solvent front. To calculate the

Rvalue, take the distance travelled by the substance divided by the

distance travelled by the solvent (as mentioned earlier in terms of ratios).
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PLANAR

CHROMATOGRAPHY

PAPER

CHROMATOGRAPHY

THIN LAYER

CHROMATOGRAPHY

For example, if a compound travels 2.1 cm and the solvent front travels

2.8 cm, (2.1/2.8) the Rvalue = 0.75

Example of retention factor (R) calculation

Planar chromatography is divided into:

PAPER CHROMATOGRAPHY

Paper chromatography is an analytical chemistry technique for separating

and identifying mixtures that are or can be colored, especially pigments. It

is also used for testing the purity of compounds, identifying substances or

used in secondary or primary colors in ink experiments. The stationary

phase is usually a piece of high quality filter paper. The mobile phase is a

developing solution that travels up the stationary phase, carrying the

samples with it. Components of the sample will separate readily according
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to how strongly they adsorb on the stationary phase versus how readily

they dissolve in the mobile phase.

When a colored chemical sample is placed on a filter paper, the colorsseparate from the sample by placing one end of the paper in a solvent.

The solvent diffuses up the paper, dissolving the various molecules in the

sample according to the polarities of the molecules and the solvent. If the

sample contains more than one color, that means it must have more than

one kind of molecule. Because of the different chemical structures of each

kind of molecule, the chances are very high that each molecule will have

at least a slightly different polarity, giving each molecule a different

solubility in the solvent. The unequal solubilities cause the various color

molecules to leave solution at different places as the solvent continues to

move up the paper. The more soluble a molecule is, the higher it will

migrate up the paper. If a chemical is very nonpolar it will not dissolve at

all in a very polar solvent. This is the same for a very polar chemical and a

very nonpolar solvent.

Two-way paper chromatography, also calledtwo-dimensional

chromatography, involves using two solvents and rotating the paper 90

in between. This is useful for separating complex mixtures of similar

compounds, for example, amino acids. Paper chromatography is a useful

technique because it is relatively quick and requires small quantities of

material. This method has been largely replaced by thin layer

chromatography, however it is still a powerful teaching tool.
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Example of paper chromatography

Example of two-way paper chromatography

THIN LAYER CHROMATOGRAPHY


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Thin layer chromatography is a chromatography technique used to

separate mixture performed on a sheet of glass, plastic, or aluminium foil,

which is coated with a thin layer ofadsorbent material, usually silica

gel, aluminium oxide, or cellulose (blotter paper). This layer of adsorbentis known as the stationary phase. After the sample has been applied on

the plate, a solvent or solvent mixture (known as the mobile phase) is

drawn up the plate via capillary action because different analytes ascend

the TLC plate at different rates, separation is achieved

Compared to paper, it has the advantage of faster runs, better

separations, and the choice between different adsorbents. For even better

resolution and to allow for quantification, high-performance TLC can be

used.

Thin layer chromatography can be used to monitor the progress of a

reaction identify compounds present in a given mixture and determine the

purity of a substance. Examples of thin layer chromatography are

analyzing ceramides and fatty acids and detection of pesticides or

insecticides in food and water, analyzing the dye composition of fibers in

forensics and assaying the radiochemical purity ofradiopharmaceuticals,

or identification ofmedicinal plants and their constituents.

Example separation of black ink on a TLC plate
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Example of thin layer chromatography of an extract of green

leaves (spinach) in 7 stages of development. Carotene elutes

quickly and is only visible until step 2. Chlorophyll A and B are
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halfway in the final step and lutein the first compound staining

yellow.

G AS CHROMATOGRAPHY

Gas chromatography al known specifically as gas-liquid chromatography, it is

an analytical technique for separating compounds based primarily on their

volatilities. Gas chromatography provides both qualitative and

quantitative information for individual compounds present in a sample.

Compounds move through a GC column as gases, either because the

compounds are normally gases or they can be heated and vaporized into

a gaseous state. The compounds partition between a stationary phase,

which can be either solid or liquid, and a mobile phase (gas). The

differential partitioning into the stationary phase allows the compounds to

be separated in time and space.
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Gas Chromatographic System

The schematic diagram of a gas chromatograph:

Instrumental components

Carrier gas

The carrier gas must be chemically inert. Commonly used gases include

nitrogen, helium, argon, and carbon dioxide. The choice of carrier gas is

often dependent upon the type of detector which is used. The carrier gas


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system also contains a molecular sieve to remove water and other

impurities.

Sample injection port

For optimum column efficiency, the sample should not be too large, and

should be introduced onto the column as a "plug" of vapour - slow

injection of large samples causes band broadening and loss of resolution.

The most common injection method is where a microsyringe is used to

inject sample through a rubber septum into a flash vapouriser port at the

head of the column. The temperature of the sample port is usually about

50C higher than the boiling point of the least volatile component of the

sample. For packed columns, sample size ranges from tenths of a

microliter up to 20 microliters. Capillary columns, on the other hand, need

much less sample, typically around 10-3 mL. For capillary GC, split/splitless

injection is used. Have a look at this diagram of a split/splitless injector;


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The injector can be used in one of two modes; split or splitless. The

injector contains a heated chamber containing a glass liner into which the

sample is injected through the septum. The carrier gas enters the

chamber and can leave by three routes (when the injector is in split

mode). The sample vapourises to form a mixture of carrier gas,

vapourised solvent and vapourised solutes. A proportion of this mixture

passes onto the column, but most exits through the split outlet. The

septum purge outlet prevents septum bleed components from entering

the column.

Columns


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There are two general types of column,packedand capillary(also known

as open tubular). Packed columns contain a finely divided, inert, solid

support material (commonly based on diatomaceous earth) coated with

liquid stationary phase. Most packed columns are 1.5 - 10m in length and

have an internal diameter of 2 - 4mm.

Capillary columns have an internal diameter of a few tenths of a

millimeter. They can be one of two types; wall-coated open tubular

(WCOT) or support-coated open tubular (SCOT). Wall-coated columns

consist of a capillary tube whose walls are coated with liquid stationary

phase. In support-coated columns, the inner wall of the capillary is lined

with a thin layer of support material such as diatomaceous earth, onto

which the stationary phase has been adsorbed. SCOT columns are

generally less efficient than WCOT columns. Both types of capillary

column are more efficient than packed columns.

In 1979, a new type of WCOT column was devised - the Fused Silica Open

Tubular(FSOT) column;

These have much thinner walls than the glass capillary columns, and are

given strength by the polyimide coating. These columns are flexible and

can be wound into coils. They have the advantages of physical strength,

flexibility and low reactivity.


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Oven

The column is placed in an oven where the temperature can be controlled

very accurately over a wide range of temperatures. For precise work,

column temperature must be controlled to within tenths of a degree. The

optimum column temperature is dependent upon the boiling point of the

sample. As a rule of thumb, a temperature slightly above the average

boiling point of the sample results in an elution time of 2 - 30 minutes.

Minimal temperatures give good resolution, but increase elution times. If a

sample has a wide boiling range, then temperature programming can be

useful. The column temperature is increased (either continuously or in

steps) as separation proceeds.


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Detectors

There are many detectors which can be used in gas chromatography.

Different detectors will give different types of selectivity. A non-selective

detector responds to all compounds except the carrier gas, a selective

detectorresponds to a range of compounds with a common physical or

chemical property and a specific detectorresponds to a single chemical

compound. Detectors can also be grouped into concentration dependant

detectors and mass flow dependant detectors. The signal from a

concentration dependant detector is related to the concentration of solute

in the detector, and does not usually destroy the sample Dilution of with

make-up gas will lower the detectors response. Mass flow dependant

detectors usually destroy the sample, and the signal is related to the rate

at which solute molecules enter the detector. The response of a mass flow

dependant detector is unaffected by make-up gas. Have a look at this

tabular summary of common GC detectors:


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Detector TypeSupport

gasesSelectivity

Detectabi

lity

Dynam

ic

rangeFlame

ionization

(FID)

Mass flowHydrogen

and airMost organic cpds. 100 pg 107

Thermal

conductivity

(TCD)

Concentrati

onReference Universal 1 ng 107

Electron

capture

(ECD)

Concentrati

onMake-up

Halides, nitrates,

nitriles, peroxides,

anhydrides,

organometallics

50 fg 105

Nitrogen-

phosphorusMass flow

Hydrogen

and air

Nitrogen,

phosphorus10 pg 106

Flame

photometric

(FPD)

Mass flow

Hydrogen

and air

possiblyoxygen

Sulphur,

phosphorus, tin,

boron, arsenic,

germanium,

selenium, chromium

100 pg 103

Photo-

ionization

(PID)

Concentrati

onMake-up

Aliphatics,

aromatics, ketones,

esters, aldehydes,

amines,

heterocyclics,

organosulphurs,

some

organometallics

2 pg 107

Hall

electrolytic

conductivity

Mass flowHydrogen,

oxygen

Halide, nitrogen,

nitrosamine, sulphur


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The effluent from the column is mixed with hydrogen and air, and ignited.

Organic compounds burning in the flame produce ions and electrons

which can conduct electricity through the flame. A large electrical

potential is applied at the burner tip, and a collector electrode is located

above the flame. The current resulting from the pyrolysis of any organic

compounds is measured. FIDs are mass sensitive rather than

concentration sensitive; this gives the advantage that changes in mobile

phase flow rate do not affect the detector's response. The FID is a useful

general detector for the analysis of organic compounds; it has high

sensitivity, a large linear response range, and low noise. It is also robust

and easy to use, but unfortunately, it destroys the sample.

Top of Form

Bottom of Form


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Data Recorder System

The data recorder plots the signal from

the detector over time. This plot is

called a chromatogram. The

retention time, which is when the

component elutes from the GC system,

is qualitatively indicative of the type

of compound. The data recorder also

has an integrator component to calculate the area under the peaks or the

height of the peak. The area or height is indicative of the amount of each

component.


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LIQUID CHROMATOGRAPHY

Liquid chromatography is an analytical chromatographic technique that isuseful for separating ions or molecules that are dissolved in a solvent. If

the sample solution is in contact with a second solid or liquid phase, the

different solutes will interact with the other phase to differing degrees due

to differences in adsorption, ion-exchange, partitioning, or size. These

differences allow the mixture components to be separated from each

other by using these differences to determine the transit time of the

solutes through a column.

Simple liquid chromatography consists of a column with a fritted bottom

that holds a stationary phase in equilibrium with a solvent. Typical

stationary phases (and their interactions with the solutes) are: solids

(adsorption), ionic groups on a resin (ion-exchange), liquids on an inert

solid support (partitioning), and porous inert particles (size-exclusion). The

mixture to be separated is loaded onto the top of the column followed by

more solvent. The different components in the sample mixture pass

through the column at different rates due to differences in their partioning

behavior between the mobile liquid phase and the stationary phase. The

compounds are separated by collecting aliquots of the column effuent as

a function of time.

Schematic of a simple liquid chromatographic separation


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Conventional Liquid chromatography is most commonly used in

preparative scale work to purify and isolate some components of a

mixture. It is also used in ultratrace separations where small disposable

columns are used once and then discarded. Analytical separations of

solutions for detection or quantification typically use more sophisticated

high-performance liquid chromatography instruments. HPLC instruments

use a pump to force the mobile phase through and provide higher

resolution and faster analysis time.

High-Performance Liquid Chromatography (HPLC)


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High-Performance Liquid Chromatographic System

High-performance liquid chromatography (HPLC) is a form of liquid

chromatography to separate compounds that are dissolved in solution.

HPLC instruments consist of a reservoir of mobile phase, a pump, an

injector, a separation column, and a detector. HPLC is used in drug

analysis, toxicology, explosives analysis, ink analysis, fibers, and plastics

to name a few forensic applications.

Schematic of an HPLC instrument:


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Like all chromatography, HPLC is based on selective partitioning of the

molecules of interest between two different phases. Here, the mobile

phase is a solvent or solvent mix that flows under high pressure over

beads coated with the solid stationary phase. While travelling through the

column, molecules in the sample partition selectively between the mobile

phase and the stationary phase. Those that interact more with the

stationary phase will lag behind those molecules that partition


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preferentially with the mobile phase. As a result, the sample introduced at

the front of the column will emerge in separate bands (called peaks), with

the bands emerging first being the components that interacted least with

the stationary phase and as a result moved quicker through the column.The components that emerge last will be the ones that interacted most

with the stationary phase and thus moved the slowest through the

column. A detector is placed at the end of the column to identify the

components that elute. Occasionally, the eluting solvent is collected at

specific times correlating to specific components. This provides a pure or

nearly pure sample of the component of interest. This technique is

sometimes referred to as preparative chromatography.

Many different types of detectors are available for HPLC. The simplest and

least expensive is the refractive index detector (RI). Although this detector

is a universal detector, meaning it will respond to any compound that

elutes, it does not respond well to very low concentrations and as a result

is not widely used. On the other hand, detectors based on the absorption

of light in the ultraviolet and visible ranges (UV/VIS detectors and UV/VIS

spectrophotometers) are the most commonly used, responding to a wide

variety of compounds of forensic interest with good to excellent

sensitivity. The photodiode array detector (PDA) is especially useful since

it can produce not only a peak-based output (a chromatogram) but also a

UV/VIS scan of every component. In many ways, the ideal detector for

HPLC is a mass spectrometer (MS), which provides both quantitative

information and in most cases a definitive identification of each

component (qualitative information). However, HPLC-MS systems are

relatively complex and expensive and are not readily available in all labs.

Other detectors that are sometimes used include fluorescence detectors

(which are very sensitive) and electrochemical detectors.

Unlike in gas chromatography (GC) in which the mobile phase is an inert

gas, the mobile phase in HPLC can be one of many different solvents or

combinations of solvents. This imparts to HPLC a greater flexibility and


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range of application than has GC. Because the sample does not have to be

converted to the gas phase, compounds such as explosives that break

down at high temperatures are much more amenable to HPLC than GC.

For HPLC, all that is required is that the sample be soluble in the solventsselected for the analysis. In addition, there are several types of HPLC

defined by the type of mobile phase and stationary phase that is used. For

forensic applications, one of the most commonly used types of HPLC is

referred to as reversed phase. In this type of HPLC, the mobile phase is

a solvent or mix of solvents that are polar, meaning that different parts

of the individual solvent molecules carry a partial positive or negative

charge. Water, methanol (methyl alcohol), ethanol (ethyl alcohol), and

acetone are examples of polar solvents. The stationary phase in reverse

phase HPLC is a non-polar material such as a long chain hydrocarbon

molecule. In this type of HPLC, components in the sample will partition

and separate based on their degree of interaction with the stationary

phase relative to the mobile phase. In other words, the separation is

based primarily on the relative polarity of the sample molecules. Reverse

phase HPLC is used in drug analysis (LSD for example), analysis of cutting

agents such as sugars, explosives, and gunshot residue (GSR), and

forensic toxicology.

Normal phase HPLC uses a polar stationary phase and a non-polar mobile

phase, but this is not widely used in forensic applications. Size exclusion

chromatography (SEC) is more common and separates compounds based

on relative sizes. The stationary phase in SEC is composed of a gel with

different sizes of microscopic pores through it. The larger the molecule,

the longer it takes for it to navigate through the pores and reach the

detector. SEC is useful for the analysis of large molecules that come in a

range of sizes such as plastic polymers, proteins, and nitrocellulose, a

component of GSR. Chiral chromatography, a relatively recent

development, is making inroads into forensic science since it is capable of

separating enantiomers, molecules that are mirror images of each other.

This capability is particularly valuable in forensic toxicology and drug


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analysis. Finally, ion exchange chromatography is available for detection

species such as nitrate (NO,3-) and other ions.

ION - EXCHANGE CHROMATOGRAPHY

The most popular method for the purification of proteins and other

charged molecules is ion exchange chromatography. It uses an ion

exchange mechanism to separate analytes based on their respective

charges. It is usually performed in columns but can also be useful in

planar mode. Ion exchange chromatography uses a charged stationary

phase to separate charged compounds including anions, cations, amino

acids, peptides, and proteins.

In cation exchange chromatography, positively charged molecules are

attracted to a negatively charged solid support. Conversely, in anion

exchange chromatography, negatively charged molecules are attracted to

a positively charged solid support. In conventional methods the

stationary phase is an ion exchange resin that carries charged functional
http://en.wikipedia.org/wiki/Anionhttp://en.wikipedia.org/wiki/Cationhttp://en.wikipedia.org/wiki/Amino_acidhttp://en.wikipedia.org/wiki/Amino_acidhttp://en.wikipedia.org/wiki/Peptidehttp://en.wikipedia.org/wiki/Proteinhttp://en.wikipedia.org/wiki/Ion_exchange_resinhttp://en.wikipedia.org/wiki/Functional_grouphttp://en.wikipedia.org/wiki/Anionhttp://en.wikipedia.org/wiki/Cationhttp://en.wikipedia.org/wiki/Amino_acidhttp://en.wikipedia.org/wiki/Amino_acidhttp://en.wikipedia.org/wiki/Peptidehttp://en.wikipedia.org/wiki/Proteinhttp://en.wikipedia.org/wiki/Ion_exchange_resinhttp://en.wikipedia.org/wiki/Functional_group


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groups which interact with oppositely charged groups of the compound to

be retained.

A sample is introduced, either manually or with an autosampler, into asample loop of known volume. A buffered aqueous solution known as the

mobile phase carries the sample from the loop onto a column that

contains some form of stationary phase material. This is typically a resin

or gel matrix consisting of agarose or cellulose beads with covalently

bonded charged functional groups. The target analytes (anions or cations)

are retained on the stationary phase but can be eluted by increasing the

concentration of a similarly charged species that will displace the analyte

ions from the stationary phase.
http://en.wikipedia.org/wiki/Functional_grouphttp://en.wikipedia.org/w/index.php?title=Autosampler&action=edit&redlink=1http://en.wikipedia.org/wiki/Buffer_solutionhttp://en.wikipedia.org/wiki/Agarosehttp://en.wikipedia.org/wiki/Cellulosehttp://en.wikipedia.org/wiki/Covalent_bondhttp://en.wikipedia.org/wiki/Functional_grouphttp://en.wikipedia.org/w/index.php?title=Autosampler&action=edit&redlink=1http://en.wikipedia.org/wiki/Buffer_solutionhttp://en.wikipedia.org/wiki/Agarosehttp://en.wikipedia.org/wiki/Cellulosehttp://en.wikipedia.org/wiki/Covalent_bond


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S IZE-EXCLUSION CHROMATOGRAPHY

Size-exclusion chromatography (SEC) is also known as gel permeation

chromatography (GPC) or gel filtration chromatography. It separates

molecules according to their size or more accurately according to their

hydrodynamic diameter or hydrodynamic volume. Smaller molecules are

able to enter the pores of the media and, therefore, molecules are

trapped and removed from the flow of the mobile phase. The average

residence time in the pores depends upon the effective size of the analyte

molecules. However, molecules that are larger than the average pore size

of the packing are excluded and thus suffer essentially no retention; such

species are the first to be eluted. It is generally a low-resolution

chromatography technique and thus it is often reserved for the final,

"polishing" step of purification. It is also useful for determining the tertiary

structure and quaternary structure of purified proteins, especially since it

can be carried out under native solution conditions.
http://en.wikipedia.org/wiki/Tertiary_structurehttp://en.wikipedia.org/wiki/Tertiary_structurehttp://en.wikipedia.org/wiki/Quaternary_structurehttp://en.wikipedia.org/wiki/Solutionhttp://en.wikipedia.org/wiki/Tertiary_structurehttp://en.wikipedia.org/wiki/Tertiary_structurehttp://en.wikipedia.org/wiki/Quaternary_structurehttp://en.wikipedia.org/wiki/Solution


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Size-exclusion chromatography is a widely used technique for the

purification and analysis of synthetic and biological polymers, such as

proteins, polysaccharides and nucleic acids.

The advantages of this method include good separation of large molecules

from the small molecules with a minimal volume of elute, and that various

solutions can be applied without interfering with the filtration process, all

while preserving the biological activity of the particles to be separated.

With size exclusion chromatography, there are short and well-defined

separation times and narrow bands, which lead to good sensitivity. There

is also no sample loss because solutes do not interact with the stationary

phase. Disadvantages are that only a limited number of bands can be

accommodated because the time scale of the chromatogram is short, and,

in general, there has to be a 10% difference in molecular mass to have a

good resolution
http://en.wikipedia.org/wiki/Proteinshttp://en.wikipedia.org/wiki/Polysaccharideshttp://en.wikipedia.org/wiki/Nucleic_acidshttp://en.wikipedia.org/wiki/Proteinshttp://en.wikipedia.org/wiki/Polysaccharideshttp://en.wikipedia.org/wiki/Nucleic_acids


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Exclusion chromatography separates molecules on the basis of size. A

column is filled with semi-solid beads of a polymeric gel that will admit

ions and small molecules (blue) into their interior but not large ones

(shown in red). When a mixture of molecules and ions dissolved in a

solvent is applied to the top of the column, the smaller molecules (and

ions) are distributed through a larger volume of solvent than is available

to the large molecules. Consequently, the large molecules move more

rapidly through the column, and in this way the mixture can be separated

(fractionated) into its components.


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Laboratory Instrumentation Chromatography

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