HPLC Instrumentation and Applications. Other names for HPLC 1- High Speed Liquid Chromatography - As the separation is completed within few minutes. 2-

HPLC Instrumentation and

Applications

Other names for HPLC1- High Speed Liquid Chromatography

- As the separation is completed within few minutes.2- High Performance Liquid Chromatography

(HPLC)3- High Resolution Liquid Chromatography (HRLC)High performance is the result of many factors:

- Smaller particles of the stationary phase, uniform pore size, high pressure column slurry packing technique, accurate low volume of the sample injected, sensitive detector, and good pump system

IntroductionHigh pressure liquid chromatography (HPLC) is

an advanced form of liquid chromatography used to separate the components of a mixture (Analytes).

In HPLC chromatography: the mixture is dissolved in a solvent (mobile phase) and then forced to flow through a chromatographic column under a high pressure. In the column, the mixture is resolved into its components.

The separation occurs because each component in the mixture interacts differently with the stationary phase. Molecules that interact strongly with the stationary phase (yellow component) will move slowly through the column, while the molecules that interact less strongly (blue component) will move rapidly through the column.

The start to the detector

-Why high pressure?- In HPLC the stationary phase has two characters :- - Has small particles size (5- 10 µm). - - And packed under high pressure.- Reduction of the particle size of the stationary phase

leads to: - Leaving less space for the mobile phase to pass

through. - Decrease the flow rate of the liquid mobile phase.- Thus pressure from 1000 to 5000 psi, pound per

square inch (68 to 340 atm.) is applied to overcome the obstructive effect of the fine particles.

Classification of HPLCI- Types of HPLC according to the mechanism of

separation1- Adsorption chromatographyThe stationary phase is an adsorbent and

the separation is based on adsorption-desorption steps.

A- Normal phase chromatographyThe stationary phase is strongly polar (e.g.

silica gel) and the mobile phase is non polar such as (hexane or tetrahydrofuran).

Polar sample retained longer on the column.

B- Reversed Phase chromatography

The stationary phase is strongly non polar ( e.g. C-18 silica , hydrpophobic) while the mobile phase is polar (as a mixture of water and methanol or acetonitrile).

2- Size exclusion chromatography.

The column is packed with material having controlled pore sizes and the sample is screened or filtered according to its molecular size, there is no interaction between solute and stationary phase. The large molecules rapidly washed through the column, the smaller molecules penetrate inside the pores and elute later.

Large molecules

Small molecules

3- Ion exchange chromatographyThe stationary phase has an ionically charged

surface of opposite charge to the sample ions

- This technique is used only for ionic or ionisable samples.

Types of St. Ph

1- Anion exchange resin2- Cation exchange resinMatrix: is polymer of styrene with

divinyl benzene

Anaion exchange as:- strong anion as quaternary ammonium

form Matrix- (NR3)+ -Cl-

- weak anion as Matrix-NH2(CH3)-Cl-

Cation exchange as: sulfonic acid Matrix-(SO3)– H+

(strong). And Matrix-COO- H+ (weak)The stronger the charge on the sample, the stronger it

will be attached to the ionic surface and thus the longer it will take to elute.

The mobile phase is an aqueous buffer, where the PH is adjusted to control elution time.

Choice of separation technique1- Sample molecular weight less than 2000

Water soluble Ionic

Non- ionic

Reversed phase Chrom. (RPC)Ion exchnge Chrom.(IEC)

Reversed phase Chrom. (RPC)Exclusion Chrm. (EC) if soluble in tetrahydrofuran

Organic solvent soluble non polar solvent----Adsorption chromatography

Tetrahydrofuran------ Exclusion Chrm. (EC)

Polar solvent------Normal phase chromatography ------Reversed phase chromatography

2- Sample molecular weight greater than 2000

Water soluble

Organic solvent soluble

Reversed phase chromatography (RPC)

Ion exchnge Chrom. (IEC)

Exclusion Chrm. (EC)

Exclusion Chrm. (EC)

II- HPLC can be divided into two main types according to the uses:

1- Analytical type: which is useda- In identification and assay of the

components in a mixture .b- To know the number of components

in a mixture (screening).2- Preparative or semipreparative

type: used in isolation and purification.

The difference between analytical and preparative HPLC

1- Dimensions of the column.- Analytical, 1-6 mm i. d. - Preparative up to 3 cm i. d.2- Flow rate of mobile phase (pump).For analytical HPLC pumps should

has flow rates that range from 1 to 10 ml/min.

but for preparative HPLC, flow rates in excess of 100 ml/min.

3- Injected volume of the sample- in analytical HPLC range from 20 uL

to 1 mL, - but in preparative or semi

preparative from 1 ml to 5 ml or more.

- 4- Size of the loop of injection port.

Chromatographic processThe process begins by:- Injecting the solute

onto the column (zero time).

- The separation occurs as the analyte and mobile phase are pumped through the column

- Detection of components by detector is displayed on a chart or computer screen (chromatogram).

The advantages of HPLC1- High speed2- High resolution3- High sensitivity4- Re-usable column5- No destruction of the components6- The instrumentation are automatic,

computerized7- Sample is recovered completely8- Quantitative work is more easily and most

sensitive

Instrumentation of HPLCHPLC instrument includes:A- Reservoir for solvents (mobile phase)B- High pressure pumpC- Sample inlet device D- ColumnE- DetectorF- Recorder

A- Reservoir for solvents (mobile phase)

-Mobile phase is usually organic or aqueous or mixture of both.

- Mobile phase is placed in bottles of glass.

Mobile phase

Miscible with water, such as acetonitrile, methanol, or isopropanol.

Characters of mobile phase:

1- Pure 2 - Low viscosity

3-Chemically inert 4- Low price

5- Compatible with detector

6- Solubility of the sample

Mobile phase

Solvent A Solvent B

Water Organic solvent

Elution Techniques (Programinig)1- Isocratic elution:The mobile phase composition remains constant

throughout the separation procedure.

2- Gradient elution:The mobile phase composition is changed during

the separation process.

Gradient elution is divided into two types:

A- Continuous (linear)

B- Discontinuous (stepwise)

Isocratic and gradient elution curves

Stepwise (discontinuous)

Linear (continuous)

Time

% of B

10%

20%

30%

40%

50%

60%

70%

0 5` 10` 15` 20` 25` 30`

Isocratic elution

Gradient elution

Advantages of gradient elution technique

1- Shortening the time of analysis.2- Reduces tailing, gives sharp peak.3- Increases the sensitivity of analysis.4- Decreases the retention of the later-eluting components so that they elute faster.

PH of mobile phaseThe pH of the solvent (water) may be adjusted

using phosphate or perchlorate or trifloroacetate acid or sulphate buffer.

The selectivity of HPLC is affected by :1- Type of mobile phase, organic or aqueous.

2- The composition of the mobile phase, whether one solvent or more.

3- The pH of the mobile phase.

Effect of buffer used in separationof xanthene alkaloids

TFAIncomplete

separation

HClO4No separation for 2

and 3

H2SO4Best separation

30% MeCN

70% Water

45% MeOH

55% Water

Mobile Phase Composition Effect on Selectivity

Fast Slow and better separation

Methanol and water give slow and better separation while use of actonitrile and water give fast and bad separation

% of Mobile phase B (MeOH) and separation selectivity

High % of B givesfast and bad separation

Low % of B givesslow and slightly better separation

Some solvents used in HPLC and their polarity

N.B. Chlorinated solvents do not used in HPLC to prevent rusting of stainless parts of the instruments

Solvents PolarityWater

Dimethyl sulfoxideEthylene glycol

AcetonitrileMethanolAcetoneDioxaneEthanol

TetrahydrofuranI-propanol

10.27.26.95.85.15.14.84.34.03.9

Treatment of mobile phaseA- Filtration before entering the column.B- Degassing using degasser.- 1- Heating with stirring - 2- Applying vacuum, - 3- Passing nitrogen or helium- 4- UltrasoundC- Pre-saturation with the stationary phase in

case of liquid liquid chromatography.

B- PumpFunction of the pump:Pump is used for forcing the mobile phase through the columnThere are two types of pump:1- Constant pressure pumpIt is free from pulsation resulting in smooth baseline2- Constant flow pump It is able to give constant flow rate of

mobile phase

To column

Mobile phase

The main criteria for the pump

1- The pump should be capable of

delivering accurate and pulse free flow rate (e.g. 5 ml/min).

2- The pump should be capable of delivering high volume of solvent.

3- The pump should be capable of delivering high pressure up to 5000 psi.

C- Sample inlet device(Injection port)

1- Manual injection 2-Automated injection

The injection port consists of A- The injection valve. B- The sample loop.

Manual injection

1- The sample is typically dissolved in the mobile phase. 2- It is drawn into a syringe and injected into the loop via injection valve.

D- ColumnColumn in HPLC is either

Different shapes for columns used in HPLC

1- Analytical, 1-6 mm i. d.

2- Preparative up to 3 cm i. d.

Made from: Stainless

Shape: StraightLength: Variable

Other types of columns used in HPLC

Guard column:1- Protect the

analytical column2- Organization

of separation in HPLC

E- Detectors (Brain of HPLC)Characters of detector

1- High sensitivity

2- Low noise (straight base line)

3-Wide range of response to different compounds

4- Unaffected by temperature or mobile phase

5- Non destructive to the compounds

6- Provides qualitative and quantitative information about the detected sample

Types of Detectors1 -UV absorption detector - It is the most sensitive, sensitive

to ng of compound.- The most widely used, it measure

the UV absorption of the solute.

2 - Refractive index detector

-Not used in case of gradient elution -- Less sensitive-It measures the difference in RI between pure mobile phase and the column eluate (mobile phase + solute).

3- Mass spectrometer detector It is used with capillary column in analytical

HPLC to give information about nature of the material by giving the mass spectrum of the material.

4- Fluorescence detector- More sensitive than UV detector (1000 fold as UV)

- It is used with compounds which are naturally fluorescent. Or compound which

- can be converted to fluorescent- derivative.

5- Photodiode array detector- It is series of detectors each is responsible

for receiving a different wavelength.

6-Flame ionization detector- Used for substances whose boiling point is

higher than that of the mobile phase- - It is more sensitive than refractive index

detector

Applications of HPLC1- Isolation and purification of

biologically active natural products2- Control of synthetic reactions Identification of intermediates and

target compound.3- Biosynthesis study Detection of biogenetic

intermediates and enzymes involved.4-Control the microbiological processUsed for separation of antibiotic from

broth mixture

5- Pharmacokinetics study Pharmacokinetic study comprises the

measurement of drug metabolites concentration in body fluids, absorption, bioavailability and elimination of drugs

HPLC determines the drug and its metabolites in one step.

6- Stability test Rapid method of analysis in stability test.7- Quality control HPLC is used to know the identity, purity

and content of the ingredients (drugs, raw and pharmaceutical products,

8- Drugs metabolisms

Applications of HPLC in isolation and purification of natural products

I. Purification refers to the process of separation or extraction the target compound from other compounds or contaminants.

1- Separation of quinine and quinidine

N

H

HO

N

MeO

H

H

5`6`

7`

8` 1`

2`3`

4`

891

2

3

4

5

6

7

R

S

N

HO

H

N

R

H

5`6`

7`

8` 1`

2`3`

4`

91

2

3

4

5

6

7

H

8

R

S

1- Quinidine2- Qinine

12

2- Separation of Xanthines alkaloids

N

NH

N

N

O

1

23 4

98

756

O

2- 1,7 Dimethylxanthine

N

NN

HN

O

1

23 4

98

756

O

3- Theophylline1,3 Dimethylxanthine

HN

NN

N

O

1

23 4

98

756

O

1- Theobromine3,7 Dimethylxanthine

3- Separation of vitamin B-1, 2, 6Column: Primesep 150x4.6

mmFlow rate: 1ml/minDetection: UV 280

nmMobile phase:

MeCN/H2O (10/90)With H3BO4 bufferPH 3.0

4- Separation of ascorbic acid and dehydro-ascorbic acid

Column: Primesep50x4.6 mmFlow rate: 1ml/minDetection: UVMobile phase:

MeCN/H2O (10/90)With HCOOH buffer0.1%.

5- Separation of chloramphenicol from mixture of antibiotics

6- Separation of mixture of alkaloids1- Codeine2- Strychnine3- Papaverine4- Quinine5- Quinidine

II- Quantitative (assay) and qualitative determination of natural products

Quantification of compounds by HPLC Is the process of determination of the unknown

concentration of a compound in a known solution.

Identification of compound by HPLC through :- Comparison of retention time with authentic- Comparison of UV spectrum of the compound

with that of the authentic.- Comparison of the Mass spectrum with that of

the authentic.-

Quantification of oroidin in Axinella damicornis sponge (assay)

Oroidin is known alkaloid isolated from sponge Axinella damicornis and it was identified by HPLC through the comparison of retention time and UV absorption with data base on HPLC.

NH

Br

Br

O

HN

NH

HN NH2

1

2

34

56

7

8

9

10

11

1213

1415

+

Steps of assay1- Quantification was done by injection of

different known conc. of oroidin (authentic).2- determination of the peak area for each

concentration3- Followed by drawing the standard curve

(area under the peak against conc.).4- Injection of known weight of the sponge

extract and find the area under the peak.5- From the standard curve find the

corresponding concentration of oroidin in the injected weight.

6- Calculate the weight of oroidin in the sponge.

Standard curve of oroidin

160 mg was found to be the conc. of oroidin in one gram of the sponge