Pharmacognosy- 1 PHG 222 - PSAU · Elution Techniques (Programinig) 1- Isocratic elution: The mobile phase composition remains constant throughout the separation procedure. 2- Gradient

Prof. Dr. Amani S. Awaad

Professor of PharmacognosyPharmacognosy Department,

College of Pharmacy Salman Bin Abdulaziz

University,

Al-Kharj. KSA.

Email: [email protected]

Pharmacognosy- 1

PHG 222

Chromatography

High Performance Liquid Chromatography

After studying this topic student should be

able to :

Define HPLC

Describe HPLC principle

Explain major components of HPLC and their function

Explain application of HPLC

Explain factors affected function of HPLC

Describe advantages & disadvantages of HPLC

• Originally referred to as High-Pressure Liquid Chromatography

• Now more commonly called High

Performance Liquid Chromatography

• HPLC is really the automation of traditional liquid chromatography under conditions which provide for enhanced separations during shorter periods of time, utilizing very small particles, small column diameters, and very high fluid pressures.

What is HPLC?

Introduction

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.

(HPLC) is an advanced form of liquid

chromatography used to separate the

components of a mixture (Analytes).

The separation occurs because each component inthe mixture interacts differently with the stationaryphase. Molecules that interact strongly with thestationary phase (yellow component) will moveslowly through the column, while the moleculesthat interact less strongly (blue component) willmove rapidly through the column.

The start to the detect

- Why high pressure?

In HPLC the stationary phase has two

characters:

- Has small particles size (5- 10 µm).

-And packed under high pressure.

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.

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.

• HPLC instrument includes:

• A- Reservoir for solvents (mobile phase)

• B- High pressure pump

• C- Sample inlet device

• D- Column

• E- Detector

• F- Recorder

Instrumentation of HPLC

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

- Mobile phase is placed in bottles ofglass.



A- Reservoir for solvents (mobile phase)

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

Miscible with water, such as acetonitrile,

methanol, or isopropanol.




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)

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




Elution Techniques (Programing)

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 phase

The 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.

Incomplete

separation

No

separation

for 2 and 3

Best

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

High Performance Liquid ChromatographyInstrumentation of HPLCElution Techniques (Programing)



Instrumentation of HPLCElution Techniques (Programing)


Some solvents used in

HPLC and their polar PolaritySolvents

10.2

7.2

6.9

5.8

5.1

5.1

4.8

4.3

4.0

3.9

Water

Dimethyl

sulfoxide

Ethylene glycol

Acetonitrile

Methanol

Acetone

Dioxane

Ethanol

Tetrahydrofuran

I-propanol

N.B. Chlorinated solvents

do not used in HPLC to

prevent rusting of

stainless parts of the

instruments


Instrumentation of HPLCElution Techniques (Programing)A- Reservoir for solvents (mobile phase)Treatment of mobile phase

C- Pre-saturation with the stationary phase in case of

liquid liquid chromatography.

A- Filtration before entering the column.

B- Degassing using degasser.

To get red of dissolved gases and

bubbles which disturb the detector

1- Heating with stirring

2- Applying vacuum,

3- Passing nitrogen or helium

4- Ultrasound


B- High pressure pump

• Function of the pump:

• Pump is used for forcing the mobile phase through the column

There are two types of pump:

1- Constant pressure pump

It is free from pulsation resulting in smooth baseline

2- Constant flow pump

It is able to give constant flow rate of mobile phase.

1- The pump should be capable of delivering accurateand pulse free flow rate (e.g. 5 ml/min).

2- The pump should be capable of delivering highvolume of solvent.

3- The pump should be capable of delivering highpressure 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.


Type of injection

1- Manual injection

2-Automated injection


D- ColumnInstrumentation of HPLC

Column in HPLC is either

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

2- Preparative up to 3 cm i.d.

Made from: Stainless. Shape: Straight.Length: Variable

Other types of columns used in HPC

Guard column:

1- Protect the analytical column

2- Organization of separation in HPLC


E- Detectors (Brain of HPLC)Instrumentation of HPLC

1 -UV absorption detector

2 - Refractive index detector

3- Mass spectrometer detector

4- Fluorescence detector

5- Photodiode array detector

6-Infra Red detector

Characters of detectorsTypes of detectors

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

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Types of detectors

1 -UV absorption detector

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).

It is the most sensitive, sensitive to ng of

compound.

- The most widely used, it measure the UV

absorption of the 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.



Types of detectors

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-Infra Red detector

-Used for substances whose boiling point is

higher than that of the mobile phase

-- It is more sensitive than refractive index

detector


F- Data ProcessingInstrumentation of HPLC

Using specific software that is connected to

HPLC machine

Receive the information from HPLC

machine and present it as a graph

The graph describes about qualitative data

(Retention time) and quantitative data (area

under curve)

1- Analytical type: which is used

a- In identification and assay ofthe 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.

Analytical Preparative

Dimensions of the

column

Analytical, 1-6

mm i.d

Preparative up

to 3 cm i.d.

Flow rate of

mobile phase

(pump)

should has flow

rates that range

from 1 to 10

ml/min.

flow rates in

excess of 100

ml/min.

Injected volume of

the sample

flow rates in

excess of 100

ml/min.

from 1 ml to 5

ml or more.

Size of the loop of

injection

port

HPLC can be divided into two

main types according to the uses:

The difference between analytical and

preparative HPLC


Types of HPLC according to the uses


The factors which influence the

HPLC performance

1. Internal diameter of column

- the smaller in diameter, the higher in sensitivity

2. Pump pressure

- the higher in pressure, the higher in separation

3. Sample size

4. The polarity sample, solvent and column

5. Temperature

- the higher in temperature, the higher in separation


Advantages & Disadvantages of HLPC

1- High speed

2- High resolution

3- High sensitivity

4- Re-usable column

5- No destruction of the components

6- The instrumentation are automatic,

computerized

7- Sample is recovered completely

8- Quantitative work is more easily and most

sensitive

I-Advantages

II-Disadvantages

Need a skill to run the instruments

Solvents consuming


4. Clinical test- Monitoring of hepatic chirosis

patient through aquaporin 2 in the urine.

5. Food and essence manufacture- sweetener analysis in the fruit juice- preservative analysis in sausage

1.Pharmaceuticals industry

-To control the drug stability-Quantity of drug determination from

pharmaceutical dosage forms, ex. Paracetamol determination in panadol tablet

-Quantity of drug determination from biological fluids, ex: blood glucose level

2. Analysis of natural contamination- Phenol & Mercury from sea water

3. Forensic test- Determination of steroid in blood, urine & sweat.- Detection of psychotropic drug in plasma

Applications of HPLC

Chemical

Environmental

Pharmaceuticals

Consumer

Products

Clinical

polystyr

enes

dyes

phthalat

es

tetracyclines

corticosteroids

antidepressants

barbiturates

amino acids

vitamins

homocysteine

Bioscience

proteins

peptides

nucleotides

lipids

antioxidants

sugars

polyaromatic hydrocarbons

Inorganic ions

herbicides

29

I need a quantitative

separation of

carbohydrates in some

of our products

as soon as possible. I’ll get

on it!

I’ll need a separation

technique. in my lab,

HPLC & GC

Which should I use?

Carbohydrates

1. fructose

2. Glucose

3. Saccharose

4. Palatinose

5. Trehalulose

6. isomaltose

Zorbax NH2 (4.6 x 250 mm)

70/30 Acetonitrile/Water

1 mL/min

Detect=Refractive Index

30

Separations Separation in based upon differential

migration between the stationary and

mobile phases.

Stationary Phase - the phase which

remains fixed in the column, e.g. C18,

Silica

Mobile Phase - carries the sample

through the stationary phase as it

moves through the column.

Injector

Detector

Column

Solvents

Mixer

Pumps

High Performance Liquid Chromatograph

Waste

31

Separations Injector

Detector

Column

Solvents

Mixer

Pumps

Chromatogram

Start Injection

mAU

time

High Performance Liquid Chromatograph

32


Detector

Column

Solvents

Mixer

Pumps

Chromatogram

Start Injection

mAU

time

33


Detector

Column

Solvents

Pumps

Mixer

Chromatogram

Start Injection

mAU

time

34


Detector

Column

Solvents

Pumps

Mixer

Chromatogram

Start Injection

mAU

time

35


Detector

Column

Solvents

Pumps

Mixer

Chromatogram

Start Injection

mAU

time

36


Detector

Column

Solvents

Pumps

Mixer

Chromatogram

Start Injection

mAU

time

37


Detector

Column

Solvents

Pumps

Mixer

Chromatogram

Start Injection

mAU

time

38


Detector

Column

Solvents

Pumps

Mixer

Chromatogram

Start Injection

mAU

time

39


Detector

Column

Solvents

Pumps

Mixer

Chromatogram

Start Injection

mAU

time

40


Detector

Column

Solvents

Pumps

Mixer

Chromatogram

Start Injection

mAU

time

41


Detector

Column

Solvents

Pumps

Mixer

Chromatogram

Start Injection

mAU

time

42


Detector

Column

Solvents

Pumps

Mixer

Chromatogram

Start Injection

mAU

time

43


Detector

Column

Solvents

Pumps

Mixer

Chromatogram

Start Injection

mAU

time

44


Detector

Column

Solvents

Pumps

Mixer

Chromatogram

Start Injection

mAU

time

45


Detector

Column

Solvents

Pumps

Mixer

Chromatogram

Start Injection

mAU

time

46


Detector

Column

Solvents

Pumps

Mixer

Chromatogram

Start Injection

mAU

time

47

The Chromatogram

Injection

to

tR

mAU

time

tR

to - elution time of unretained peak

tR- retention time - determines sample identity

Area or height is proportional

to the quantity of analyte.

Documents

Pharmacognosy- 1 PHG 222 - PSAU · Elution Techniques (Programinig) 1- Isocratic elution: The mobile phase composition remains constant throughout the separation procedure. 2- Gradient