Chromatogtaphy

Introduction to Chromatography (HPLC)

2

Chromatography began at the turn of 20th century when

Ramsey {Ramsey., Proc.Roy. soc.A76,111 (1905)}

separated mixtures of gases and vapours on adsorbents

like charcoal and Michael Tswett [Tswett, M.,

Ber.deut.botan. Ges., 24, 316 & 386(1906)] separated

plant pigments by liquid chromatography.

William Ramsey Michael Tswett

3

Tswett is credited as being the "Father of

Chromatography" principally because he coined the term

chromatography (literally colour writing) and

scientifically described the process.

4

MICHAEL TSWETT

He was a Russian botanist.

His main interest was to find out the composition of the green plant pigment, chlorophyll.

There were two schools of thought on this subject.

One school thought that chlorophyll was a single solute.

The other school thought that chlorophyll was a mixture of different solutes.

5

A glass column 1-5 cm in diameter and 20-100 cm long was taken.

The column was packed with the slurry of Calcium Carbonate made in petroleum ether(60-80).

MICHAEL TSWETT’S EXPERIMENT

6

The solution of chlorophyll was allowed to move down slowly and then the column was washed with ethyl alcohol for few hours.

A concentrated solution of chlorophyll was prepared in petroleum ether(60-80) and a small volume (~10cm3) was put on the top of the calcium carbonate column in the glass column as a thin band.

MICHAEL TSWETT’S EXPERIMENT

7

COTTON PLUG

SLURRY OF CaCO3 POWDER IN PETROLEUM ETHER

COTTON PLUG

CONCENTRATED SOLUTION OF CHLOROPHYLL IN PETROLEUM ETHER

WASHING/ ELUTION WITH ETHANOL

8

β CHLOROPHYLL

α CHLOROPHYLL XANTHOPHYLL VIOLA XANTHINE CAROTENE

COTTON PLUG

SLURRY OF CaCO3 POWDER IN PETROLEUM ETHER

COTTON PLUG

CONCENTRATED SOLUTION OF CHLOROPHYLL IN PETROLEUM ETHER

WASHING/ ELUTION WITH ETHANOL

9

• Each band was removed from the column and identified.

• It was concluded that chlorophyll is a mixture

of five different solutes. α –chlorophyll, β - chlorophyll,

xanthophyll, viola xanthene, and carotene .

10

α CHLOROPHYLL and β CHLOROPHYLL

11

XANTHOPHYLL

12

VIOLA XANTHENE

13

CAROTENE

14

• Michael Tswett’s main problem was resolved and

in the process he discovered a simple, time saving

and relatively less laborious method of separating

complex solutes from a mixture.

• For example α – chlorophyll (MW 893) differs

from β – chlorophyll by only one methyl group

yet these two solutes are easily separated in

the simplest apparatus.

15

• Today the term chromatography is used for

separations involving colourless solutes also. This

is done to honour the discoverer of the

technique.

• Michael Tswett coined the term “Chromatography for this

new technique of separation.

(Chromatus = colour, graphein = to write)

• He visualized the coloured bands from

chlorophyll as coloured writing.

16

As a chemist, a researcher or simply as an

inquisitive person, two questions come to our

mind on the results of Tswett’s experiment.

1. Why do the five solutes separate on the column?

2. How do the five solutes separate on the column?

17

Migration velocities increase from β- chlorophyll to carotene.

“The five solutes are separated on the column

because of the differences in their migration

velocities”

18

“The five solutes have different migration velocities

across the column of CaCO3, because they have

different degrees of adsorption on the surface of

CaCO3”

19

Carotene has the least degree of adsorption on

CaCO3 surface and hence the highest

migration velocity while β- chlorophyll has the

highest degree of adsorption on CaCO3

surface and hence the lowest migration velocity of

the five solutes.

20

This is the basis of “Adsorption column

chromatography”.

21

Adsorbents

The most important agent in chromatography is the adsorbent.

Therefore, utmost care is to be bestowed in the selection of

proper adsorption material and also the process of building

up a column with it and the maintenance of the column.

22

It must be insoluble in the solution under analysis and the

solvent that is used for elution

Essential requirements to be satisfied by every adsorbent

It should not chemically react with the mixture under analysis.

It should generally be colourless, but this is not a rigid

rule as there are instances where coloured adsorbents

have been used successfully.

Particle-size of the adsorbent material is of primary importance

for successful separation.

23

Too loose , large paricles.

Too tight , small paricles.

24

Slaked lime (120 mesh) and magnesia are two cheap

adsorbents.

Fuller's earths are also good adsorbents but unlike the

previous ones they are acidic in reaction. On moistening

they darken considerably.

Calcium carbonate is a cheap adsorbent of uniform size.

• Sodium sulphate, talc, magnesium sulphate, calcium

phosphate, calcium hydroxide; barium carbonate,

magnesium carbonate, magnesium trisilicate, calcium

sulphate, soda ash, titania, charcoal, sucrose, starch are some

of the other materials.

25

• Adsorbents may be classified as follows according to the

force with which they hold the ions.

Type Examples

Weak Sucrose and starch

Intermediate Calcium carbonate, calcium phosphate, calcium hydroxide and

magnesium hydroxide

Strong Alumina, charcoal and Fuller's earth.

26

From Tswett’s experiment if we change slurry

of CaCO3 and replace it with slurry of Al2O3,

will the five solutes form chlorophyll separate

and will the sequence of separation remain the

same?

In Tswett’s experiment if we replace chlorophyll

extract with a mixture of A+B+C+D+E solutes,

will the solutes separate and what will be the

sequence of their separation?

27

In Tswett’s experiment if we replace chlorophyll

extract with a mixture of A+B+C+D+E solutes,

will the solutes separate and what will be the

sequence of their separation?

If we have understood the concept of

adsorption, then it should not be difficult to

answer the above two questions.

28

In the first instance, we have changed the adsorbent, other

things remaining unchanged.

The five adsorbates have a new surface of Al2O3.

If they exhibit differences in the degree of their

adsorption on the surface of Al2O3, they will get

separated.

29

In the second instance, we have changed the five

solutes, other things remaining unchanged.

Although the surface of CaCO3 is the same as

that in Tswett’s experiment, the five solutes are

different.

They will get separated, if they exhibit differences in the degree of

their adsorption on the surface of CaCO3.

30

Let us consider a situation where in the glass

column we put a liquid which remains stationary

even if the tap is opened.

If we allow three solutes A, B and C to pass

through the chromatographic column and realize

their separation after some time, we can ask

again the same two questions which we did in

adsorption chromatography.

Another liquid which is immiscible with the stationary

liquid moves through it.

31

Why do the three solutes separate?

How do the three solutes separate?

The three solutes separate because they have

different migration velocities.

Solute ‘A’ has the highest migration velocity of the

three and reaches the end of the column first.

Solute ‘C’ has the lowest migration velocity of the

three and reaches the end of the column last.

32

The migration velocity increases from solute ‘C’

to solute ‘A’.

Solute ‘A’ travels the fastest because it is more

soluble in the liquid which moves across the

stationary liquid.

Solute ‘C’ on the other hand is more

soluble in the stationary phase and

hence lags behind.

33

The principle of separation here is differences in solubility

or partitioning of solutes between the stationary and mobile

liquid phases.

It is called as “Partition Column Chromatography”.

34

If they do not show differences with

reference to any property they cannot be

separated.

For solutes to get separated from a

mixture, they must exhibit some

differences with reference to some property

like solubility, boiling point, adsorption,

partitioning etc.,

35

Stationary phase: Phase which remains

stationary. It could be a solid or a liquid bonded to an inert

solid support.

Mobile phase: A phase which moves across the

stationary phase. It could be a gas or a liquid.

Elution: The process of washing a chromatographic

column with a liquid or a gas.

Solutes: This term is used in place of compounds or

substances.

36

Normal phase mode of separation: Here the stationary phase

is relatively more polar than the mobile phase.

Reverse phase mode of separation: Here the stationary phase

is relatively less polar than the mobile phase.

37

MOBILEPHASE

STATIONARY PHASE

PRINCIPLE OF SEPARATION

CONTAINER NAME OF THE CHROMATOGRAPHIC

TECHNIQUE

LIQUID SOLID DIFFERENCES IN ADSORPTION

COLUMN LIQUID-SOLID ADSORPTION COLUMN

CHROMATOGRAPHY

LIQUID SOLID DIFFERENCES IN ADSORPTION

THIN LAYER LIQUID-SOLID ADSORPTION THIN LAYER

CHROMATOGRAPHY

LIQUID LIQUID DIFFERENCES INPARTITIONING

COLUMN LIQUID-LIQUID PARTITION COLUMN

CHROMATOGRAPHY

LIQUID LIQUID DIFFERENCES INPARTITIONING

THIN LAYER LIQUID-LIQUID PARTITION THIN LAYER

CHROMATOGRAPHY

GAS LIQUID DIFFERENCES INPARTITIONING

COLUMN GAS-LIQUID-PARTITIONCOLUMN CHROMATIGRAPHY

GAS SOLID DIFFERENCES IN ADSORPTION

COLUMN GAS-SOLID ADSORPTION COLUMN

CHROMATOGRAPHY

38

GAS LIQUID/ SOLUTION

CLASSIFICATION OF DIFFERENT CHROMATOGRAPHIC TECHNIQUES

Stationary phase is solid Stationary phase is a liquid coated on the surface of an inert solid support

gas - solid - adsorption chromatography

gas - liquid - partition chromatography

Mobile phase is Gas and all the solutes being separated are in gaseous phase under experimental conditions

Mobile phase is liquid and all the solutes being separated are in liquid/solution state

39

Liquid / Solution Chromatography

ADSORPTION : Separation of solutes on the basis of their degree of adsorption on the surface of an adsorbent.

PARTITION : Separation of solutes on the basis of their differences in partition coefficient of solutes in two mutually immiscible solvents. ION EXCHANGE : Separation of similarly charged ions based on the differences in the degree of their affinity towards the ion exchange resin. SIZE EXCLUSION : Separation of solutes based on the differences in their molecular weights or sizes. ELECTROPHORETIC : Separation based on the movement of charged solutes to opposite poles under the applied electrical field.

AFFINITY : Separation of solutes based on specific affinity of solutes on the specific sites of the stationary phase.

40

• Let us consider a chromatographic column on which

three solutes A, B and C are separated in that order.

• Let there be sensor or a detector fixed at the other

end of the column.

• The detector can sense and count the number of

molecules of solute entering into it.

• The detector does not sense and count the

number of molecules of mobile phase entering

into it.

41

42

• As we can see from the figure, first the mobile phase

will enter into the detector and no response will be

generated.

DETECTOR

A

B

C

COTTON PLUG

COTTON PLUG

43

Molecules of solute ‘B’ enter the detector next.

They are sensed and counted.

Mobile phase enters into the detector again and is not sensed and

counted. Molecules of solute ‘C’ enter the detector next.


Mobile phase enters into the detector again and is not

sensed and counted.

Molecules of solute ‘A’ enter the detector next.


Mobile phase enters into the detector again and

is not sensed and counted.

44

45

• If we plot a graph of graph of detector response as a

function of time, the above description can be converted

into the following picture.

•

• This is called a chromatogram.

A

B

C

DETECTORRESPONSE

TIME

46

A

B

C

DETECTORRESPONSE

TIME

tRA

tRB

tRC

DETECTORRESPONSE

TIME

X

Y

DETECTORRESPONSE

TIME

AUCA

AUCB

AUCC

TIME

DETECTORRESPONSE

t wA t wB t wC

47

• A normal chromatogram has certain characteristic features.

All the peaks are bell shaped symmetrical peaks.

Neighbouring peaks are separated by some distance.

Each peak has a characteristic retention time

(distance between center of the peak and the

reference point) under given experimental conditions.

This is useful in qualitative Chromatographic analysis.

48

Each peak has certain area under curve (AUC) and

AUC α Concentration of Solute. This is useful in

quantitative chromatographic analysis. A separate

calibration graph is needed for each peak in a

chromatogram.

Each peak has certain width at base which

increases with increase in retention time.

49

Both qualitative and quantitative chromatographic

analysis are relative methods of analysis in which

the standard solutes and the sample solutes are

chromatographed under the same experimental

conditions.

Volumetric and Gravimetric methods, on the other

hand, are the examples of absolute methods of

quantitative analysis.

50

• It is observed in practical Chromatographic

separations that the spots or bands or peaks are

always broad. They are never very sharp.

What could be the reason for this?

• If we observe our earlier Chromatogram

carefully, we see that for each solute, solute

molecules start eluting at a particular time and the

elution is complete after a few seconds.

51

• It means that all the molecules of the same solute do

not travel across the stationary phase with the same

speed. Some travel faster than others. Therefore

some reach the detector first and others latter.

• In other words molecules of the same solute spread across

the stationary phase which results in band broadening.

52

• We can visualize four important causes leading to molecular

spreading and band broadening.

• We must find out at least some important causes for molecular


53

• We should know in the beginning that the particles of

stationary phases are approximately spherical in nature and

the particles are not exactly of the same size.

• When we say that the particle size is 100µ, it really

means that it is the average particle size and that every

particle is not of exact 100µ diameter.

• Secondly, the spherical particles are always

porous in nature. That is, the surface is not

smooth.

54

Let us now imagine a magnified picture of a chromatographic column

• We can see that when the solute molecules travel across the stationary

phase, they have different paths to flow down the column. Each path has

different path length.

COTTON PLUG

DETECTOR

SAMPLE SOLUTION

WASHING WITH LIQUID MOBILE PHASE

55

• The molecular spreading results in band broadening.

• This cause of molecular spreading leading to band broadening

is known as, “Eddy Diffusion” or “Multiple path flows Effect”.

• Solute molecules travelling in shorter paths reach the

detector early.

• Solute molecules travelling in longer paths reach the

detector late.

• As a result of multiple path flows, molecules of the same

solute spread across the stationary phase .

56

1. Eddy diffusion. (difference in the mobile phase stream flow due to non homogeneity of stationary phase) in packed columns.

57

1. No or minimum Eddy diffusion. (difference in the mobile phase stream flow due to non homogeneity of stationary phase) in packed columns.

58

Can Eddy diffusion be avoided?

• Even if we bring the stationary phase particles closer

together than what they are in the above picture, there

will always be some distance between the

neighbouring particles.

• What is achieved by compact packing of stationary

phase particles in the column probably is that the

numbers of multiple path flows are minimized and

the extent of molecular spreading leading to band

broadening is reduced.

• Eddy diffusion, therefore, cannot be avoided, it can be optimized.

59

• Let us now have a magnified picture of two neighbouring

stationary phase particles.

60

• In the above picture we have three streams in which solute

molecules are moving between two stationary phase

particles.

• Two streams flow closer the surface of the stationary

phase particles.

DETECTOR

61

• Solute molecules in these streams experience resistance

to flow from the surface of the stationary phase particles.

• Their migration velocity will be relatively smaller than those

solute molecules traveling in the middle.

• This obviously results in molecular spreading adding

to band broadening.

• This cause is known as mobile phase mass transfer

and cannot be eliminated.

62

• In closely packed column the contribution of

this cause called “Mobile phase mass transfer” will

be more than in a loosely packed column.

• In a loosely packed column the contribution of

Eddy diffusion will be more.

• In practice we have to strike a balance.

63

Let us now consider two pictures of two magnified porous particles.

• In both the above pictures A and B, while solute molecules travel across the stationary phase , some of them enter inside the pores and remain stagnant for sometime before they are flushed out by the mobile phase which is continuously flowing across the chromatographic column.

•There is a small difference however.

DETECTOR

A

DETECTOR

B

64

• In picture A the pore is shallow and the solute molecules remain

stagnant along with the mobile phase for short time. This cause is

known as “Stagnant Mobile Phase Mass Transfer”.

• In picture B, solute molecules enter into a deep pore. Inside the deep pore

they have enough time to interact with the liquid stationary phase coated on

the surface of the stationary phase particles.They remain in the deep pore for a

longer period of time as that compared to solute molecules in a shallow pore.

• In either case, the solute molecules which enter into the pores

have relatively smaller migration velocity as when compared to

those which do not enter into the pores.

• This cause is known as “Stationary Phase Mass Transfer”.

• This results in molecular spreading and band broadening.

65

• Eddy diffusion, mobile phase mass transfer, stagnant

mobile phase mass transfer, and stationary phase

mass transfer are the four main causes for molecular


66

• The causes for molecular spreading leading to

band broadening cannot be eliminated.

• They can be optimized by choosing stationary phase

particles with uniform size having shallow pores and

packing compactly in a chromatographic column.

67

• Let us now consider in quantitative terms what we

have discussed qualitatively about molecular

spreading and band broadening in chromatographic

separations.

68

• In order to do that let us make the following assumptions:

tR - retention time of a solute

L cm - Length of chromatographic column

tO - retention time of the mobile phase

u - migration velocity of the mobile phase

uX - migration velocity of the solute

ns - number of moles of solute in the stationary phase

nm - number of moles of solute in the mobile phase

R - fraction of moles of solute in the mobile phase

69

• Let us imagine that a chromatographic column of

Lcm length is divided into N small sections of equal

length or height.L cm

• Each small section is called a “Theoretical Plate”

because it is a theoretical concept.

• No such sections or plates exist in reality.

70

• The solute molecules constantly get exchanged

between the stationary phase and the mobile phase

when they travel across the column.

• The solute molecules travel in the forward direction along

with the mobile phase only when they are in the mobile

phase . They remain stagnant when they are in the

stationary phase.

• The length or height of each theoretical plate is such that

when solute molecules travel that distance a new

equilibrium is established.

• There will be, therefore ‘N’ number of such equilibria

across the length of the column.

71

• It is then easy to imagine that more the number of

theoretical plates associated with the given length of

chromatographic column, more will be such equilibria

and solute molecules will have less time and chances

to spread across the stationary phase.

• It means that more the N (number of theoretical

plates), lesser is the molecular spreading and

sharper will be the resulting bands or peaks.

72

• In other words we can say that higher the N associated

with a given length of chromatographic column higher is

“ column separation efficiency.”

• We also know that L / N = H, height equivalent to a

theoretiacal plate. N and H are inversely proportional.

• It can, therefore, be concluded that higher the N or

lower the H associated with a given length of

chromatographic column, higher is the column separation

efficiency.

73

• The following three factors govern the column separation

efficiency.

1. Particle size of stationary phase.

2. Length of chromatographic column.

3. Mobile phase velocity.

74

Particle size of stationary phase

• We normally assume stationary phase particles to be

spherical in shape.

• If we reduce the particle size diameter from 100µ

to 50µ, 25µ, 10µ, 05µ, the surface area will increase as

follows.

75

Particle size in diameter (microns)

Surface area (m2/g)

100 0.01538

50 0.0307

25 0.0615

10 0.153

5 0.307

76

0

20

40

60

80

100

120

0 0.1 0.2 0.3 0.4

Series1

Parti

cle

size

in d

iam

eter

(mic

rons

)

Surface area (m2/g)

77

• We can see that as the particle size of spherical particles is

reduced, the surface area increases exponentially.

• When the surface area increases, the amount of the liquid

phase coated also increases proportionately. More the liquid

phase loading more will be the interactions of solute

molecules with the liquid stationery phase, less will be

molecular spreading and band broadening.

• Then we can say that lower the particle size higher will be N

and higher will be the column separation efficiency.

78

• However we have also to remember that as the

stationary phase particles become smaller there is more

resistant to mobile phase flow.

•We have therefore to optimize the particle size without

sacrificing analytical times. Because to achieve good

separation with reasonable analytical time is practically

important and cannot be overlooked.

79

• If with a given length of say 100cm long column,

N = 10,000 then with L = 200cm, N= 20,000, keeping

particle size of stationary phase the same.

• Thus, longer the chromatographic column, higher will be N

and column separation efficiency.

• With longer columns, however, retention time of solutes

will increase and peaks will become broader and analytical time

will increase.

80

• We have to, therefore, optimize the column length just

as we optimized the particle size. Our main aim being

good separation with reasonable analytical time.

81

For example for 10µ diameter porous stationary phase particles

we get a graph as shown below.

• It can be seen that initially as the

mobile phase velocity increases, H

decreases i.e. separation efficiency

increases.

• It reaches at the maximum with

mobile phase velocity ‘x’.

Mobile phase velocity

L/N

X

82

• We may, therefore, be tempted to work at the mobile phase

velocity ‘x’ because at this point we get the maximum

separation efficiency.

• From the above discussion it becomes clear that for getting

good separation efficiency with reasonable separation time

we have to optimize particle size, column length and

mobile phase velocity.

• However at this mobile phase velocity if the separation

times are long, then we will have to work at higher mobile

phase velocity at the cost of lower separation efficiency to

achieve reasonable analytical time.

83

A TYPICAL CHROMATOGRAM

It is a plot of the detector response in millivolts as a function of time (usually time is the abscissa and millivolts the ordinate)

Retention Time

Solvent

A

B

CInject

Detector Response

100

0

Time in minutes 0

84

• The above Fig. illustrates how resolution can be enhanced by

exploiting the selectivity effect of the mobile phase (i.e, by

switching from 60% ACN to 60% MeOH). It is important to

note that small change of selectivity can have a major effect on

resolution as resolution is proportional to (α -1). Columns of

different bonded phases (i.e,C8, phenyl, CN ,polar- embedded

etc) can also provide different selectivity effects.

85

• Finally, the plate number of the column (N) should be maximized by using a

longer column or a more efficient column. Increasing N is not an efficient way to

achieve resolution since Rs is proportional to √N. Doubling N by doubling the

column length increases analysis time by 2 but only increases resolution by √2 or

by 41%. In contrast, increasing α from 1.05 to 1.1 will almost double the

resolution. For complex samples with many peaks, increasing N is a viable and

the most direct approach.

86

Concept of tailing and fronting

Individual solute molecules act independently of one

another during the chromatographic process.

As, a result they produce a randomized aggregation of retention

times after repeated sorptions and desorptions.

The result of a given solute is a distribution, or peak, whose shape

can be approximated as being normal or Gaussian.

Nonsymmetrical peaks usually indicate that some undesirable

interaction has taken place during the chromatographic process.

87

•Ideal Guassian Broad Fronting Tailing Doublet

Rate theory

Faulty injection.Degraded column.

88

The figure below shows some shapes that can occur in actual samples.

89

Broad peaks like (b) are more common in packed columns and

usually indicate that the kinetics of mass transfer is too slow.

Asymmetric peaks can be classified as fronting (c) or tailing (d)

depending on the location of asymmetry.

The extent of asymmetry is defined as the tailing factor (TF).

90

TF = b/a

Both a and b are measured at 10% of the peak height shown.

As can be seen from the equation, a tailing peak will have a TF

greater than one.

The opposite symmetry, fronting, will yield a TF less than one.

91

The doublet peak like (e) can represent a pair

of solutes that are not adequately separated.

• Repeatability of a doublet peak should be

verified because such peak shape can also

result from faulty injection technique, too

much sample, or degraded columns.

Flow path for method developmentDefine Goal

Detection

Chromatography selection (Stationary

phase & Mobile phase)

Sample preparation

Measurement

Matrix Substitution

System suitability

Stability

Validation

Reporting

DETECTION• Prepare a solution of 10 µg/mL in MeOH:water mixture in acidic, basic or neutral conditions •Assess against their corresponding blanks for UV absorbance and Fluorescence•Tune the ionisation parameter on LC-MS to assess fragmentation and potential suitability for LC-MS-MS

Results may indicate likely detection mechanism and detectorThe most common HPLC detectors are

– UV/VIS– Fluorescence– Refractive index– Electro chemical– LCMS

DETECTIONDetection method chosen depending on :Requirements of assayUV detection – easiest and most widely available

But, has moderate selectivity and sensitivityFluorescence detection- greater sensitivity( 10-1000 times

higher than that of UV) and selective,But greater care is needed for IS selection

E.C.D detection-comparable sensitivity to fluorescence and good selectivity (due to response to specific functional group undergoing oxidation or reduction,

But has serious problem with stability at high sensitivity work

LCMS highest sensitivity and specificity,But involves large cost

Separation

Most difficult and time consuming part

-SAMPLE PREPARATION -Selectively enrich biological sample for quantification at appropriate level are possible

-CHROMATOGRAPHIC SEPARATIONResolve and quantify property of analyte/ potential metabolite and IS in presence of endogenous material in short time as possible

Chromatographic separationFundamental Resolution Equation Rs = 1 √n k α-1 4 1+ k αHow can resolution be changed ?Efficiency : √n - Column length, particle size, Column Quality, TemperatureRetention : k - Eluent Strength, Column stationary phase,

1+ k Temperature Selectivity : α-1 - Stationary Phase (column), Mobile phase, α Mobile phase additives, Temperature

In bioanalysis capacity factor more than 4 to 5 - Matrix effect

Sample preparation • OBJECTIVE • To carry through as high percentage as possible of

the analyte(s) from original sample to the determination steps (good recovery)

• To ensure that the analyte remain in their original state

• To carry through as few of the matrix components from the original sample to the determination step

• Preparation of sample in solution form, compatible for introduction in specific instrument (HPLC, LCMS, GCMS, RIA etc)

Sample preparation

In general there are three different methods of sample preparation prior to assay: -– Protein Precipitation– Liquid - Liquid Extraction– Solid Phase Extraction

Protein Precipitation• Denaturation and precipitation of proteins is done by

an addition of an acid, solution containing a heavy metal ion or water miscible organic solvents (Methanol, ACN)

• Following admixture of precipitant the sample is centrifuged to produce a clear supernatant

• Precipitation by addition of organic solvent• Water miscible organic solvents ACN(x1 vol),

MeOH(x 2 vol), ethanol or acetone• Use solvent in which analyte is highly soluble• Mixture of two solvents (MeOH and DMSO)

Protein Precipitation• Precipitation by addition of inorganic compounds• Acidic or anionic precipitants trichloroacetic acid

and/or perchloric acid (10-20 % w/v)• Mobile phase should contain a high molar

concentration of buffer to protect column• Should not be used for compound which are prone to

acid hydrolysis • Cationic precipitants such as copper and zinc in

alkaline solution e.g Zinc sulphate in NaOH or barium hydroxide or copper sulphate in potassium hydroxide

Protein Precipitation• Disadvantage Cannot be used for quantifying very low concentrations

due to dilution of sample Late eluting peak may co-elute with drug peak

distorting peak measurementsFurther precipitation may occur if supernatant is

allowed to stand which could clog the chromatographic column(can be avoided)

Liquid -Liquid Extraction

• Based on extraction of analyte depending on its partition between an aqueous and an immiscible organic phase

• The degree of extraction depends on partition coefficient (KD) which is dependent on

• Organic solvent• pH • Ionic strength• Optimised for maximum recovery and minimum co-

extract


• Analyte possessing sufficient lipophilic character gets extracted in organic layer

• The organic layer is collected, dried and reconstituted in solvent, mobile phase or a solution of a particular pH.

• Properties of ideal solvent • Environmentally acceptable, non toxic and not highly

inflammable, immiscible with water, convenient specific gravity,suitable volatility, high chemical stability, not prone to form emulsion

• Desirable compromise is required


• Most popular solvents are heptane, hexane, toluene, diethyl ether , ethyl acetate, dichloromethane and chloroform

• It is best to use least polar solvent that will effectively extract drug.

• No advantage in selecting solvent with too much extracting power it may reduce selectivity of assay

• Acid drugs extracted under acidic condition (pH 2 units below pKa)

• Basic drugs extracted under basic conditions (pH 2 units above pKa)


• Wherever possible high pH is desirable to ensure cleaner extract

• To limit the carry-over• Back extraction into aqueous phase with pH adjusted so

that analytes ionize again• Highly polar ionic drug are extracted by formation of

neutral ion pair complex • Addition of ion pairing agents tetralkyammonium salts

for acids and alkyl sulphonates (heptane sulphonic acid for base)


Disadvantages- Emulsion Formation- Time consuming- adsorption of analytes on glassware - Require large volume of organic solvent - Ionizable compounds require specific pH

adjustment for extraction

Solid Phase ExtractionSolid Phase ExtractionSPE utilizes the principle of selective retention of an analyte on a chemically modified silica surface/sorbent.There are four steps involved in SPE

– Conditioning– Application– Rinsing– Elution

Solid Phase ExtractionSolid Phase ExtractionAdvantages

– Faster Sample preparation – Average time cut by 2/3

– Less solvent and reagent consumption– Less hazardous waste– Greater Recoveries– Greater Accuracy– Greater Specificity– Less sample volume handling– Useful for both hydro and lipophilic drug– Easy automation

Disadvantages– Expensive

Concentration of sampleEVAPORATION OF SAMPLESimple but its practical application problematic-time and attention required-Loss of sample-In three ways : degradation, volatilization and incomplete transfer-Overcome by use Low Volume Evaporator-Sample is swept with a inert gas nitrogen.

Standardisation• External standardization•Internal standardizationDetector response is recorded as a ratio of the signal of the analyte to that of internal standard.

Calibration Check

Literature search for methods •Internet •Pubmed•Analytical Abstracts (Royal Society of chemist) www.rsc.org•Analytical Profiles

Rules of one and two

• Two basic rules applied while modifying the method

• Rule of one states• “In altering any chromatographic parameter and

this include the extraction step only one parameter is changed at any one time

• Rule of two states• “A chromatographic observation is not valid until

it has been repeated”

Method ValidationMethod validation is a procedure used to ascertain and prove that a test method consistently yield what it is expected to do with adequate accuracy and precision.The method validation parameters are

SpecificityAbility of an assay to distinguish a drug from its metabolite, other drugs and endogenous constituents of biological matrix.

CriteriaResponse of interfering peaks (if any) at the retention time of an analyte should be ≤ 20% of the LLOQ (Low limit of quantitation) response and should be ≤ 5% of the internal standard response.

Blank Plasma on LCMS Blank Plasma on HPLC

Sensitivity • The lowest level of concentration of an analyte that

can be measured in biological matrixCriteria• The response of LLOQ should be at least 3 times

greater than any interference peak.• The between batch accuracy must be ≤ 20%• The between batch precision must be ≤ 20%

Low Limit of Quantitation on LCMS

Low Limit of Quantitation on HPLC

LinearityA minimum of five non-zero calibration curve standards including LLOQ and highest concentration should yield:

• r2≥ 0.98∀ ± 20% deviation of LLOQ from nominal

concentration∀ ± 15% deviation of standards other than LLOQPrecision & Accuracy (-PA)• A PA batch is five non-zero calibration curve

standards including LLOQ and highest concentration with five sets of: -

• LLOQ Quality Control Sample• LQW Quality Control Sample• Middle Quality Control Sample• High Quality Control Sample

Acceptance criteria• A minimum of five sets of PA batch should run on

different days• Precision and accuracy must be ≤15% for:• within PA batch• Intra day PA batch• Inter day / Between PA batchesStability• The following stability exercise should be conducted

during method validation:• Standard stock solution• Bench top• Autosampler• Freeze them (I, II& III cycle)• Long term stability (Storage condition)

Recovery• This is carried out to ascertain maximum and

consistent extraction of drug from biological fluid.Ruggedness• It is carried out by analysing one PA batch processed

using different sets of reagents, different columns, different analyst and run on different instrument.

Method life cycleValidation

Development Optimization

As a part of GLP all the exercise carried out during method validation are well documented by competing specific pre-designed form, log books and note books.

The complete method validation should be audited by an independent quality assurance person for• Conduction of activities as per related SOPs• Pre recording and documentation of data• Data Analysis• Accurate reporting

GLP Requirements

References•Hill, H.M. Analysis of drugs in biological fluid, in :

Townshed, A (ed.) Encyclopedia of Analytical sciences, Volume 6, London, Academic Press, 1995

•Smyth, M.R.(ed.0 Chemical Analysis in Complex Matrices, New York, Ellis Harwood Limited, 1992

•Snyder, L.R. Kirkland, J.J. and Glajch, Practical HPLC Method Development, 2nd edition, New York, John Wiley and Sons, Inc., 1997

•Venn, R.F.(ed.) Principle and Practice of bioanalysis, London, Taylor and Francis

123

References

1.Heftmann, Chromatography Part A: Fundamentals and

Techniques, 6th edition, published by Elsevier, 2004.

2.Skoog, Holler, Nieman, Principles of Instrumental Analysis,

5th edition, published by Thomson Brook/Cole, 2005.

3.Mendham, Denny, Barnes, Thomas, Vogel’s text book of

Quantitative Chemical Analysis, 6th edition, published by

Person Education, 2007.