HIGH-PERFORMANCE

LIQUID CHROMATOGRAPHY

(HPLC)

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Separation techniques

▶ The analyzed samples are mostly in the form of a mixture of different substances. For

the analysis of mixed substances, these substances may need to be separated before

the analysis.

▶ Because the substances in a mixture usually interfere the signals of other substances.

(Interference)

▶ The chemical composition of Folia mentha includes menthol, limonene, pulegon, caryophilin,

pinen. Pharmaceutical preparations include excipients as well as the Active pharmacetical

ingredients. Also, some pharmaceutical preparations include more than one active ingredient.

▶ In order to quantify the drug concentration in blood, the drug molecule needs to be separated

from the blood constituents.

▶ The substance of interest (analyte) needs to be separated from the matrix to eliminate

the interference.

▶ If the analytes are separated from the mixture, their individual signals can be detected

and they can be quantified correctly. 2

Why do we use HPLC?

▶ HPLC is a method that is used for the separation, identification and

quantification of analytes in a mixture.

▶ For example;

▶ Active pharmaceutical ingredients in a drug preparation.

▶ Impurities and degredation products in a drug preparation.

▶ Active compunds in drogues

▶ Chemical compunds in foods and food supplements

▶ Enzymes, amino acids, proteins, polysaccharides etc. in the organism

▶ HPLC is the most important instrument of a quality control laboratory for

many industries.

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What is chromatography?

▶ Chromatgoraphy is a set of separation technique which allows qualitative and

quantitative analysis of various substances in a mixture.

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What is chromatography?

▶ All chromatographic methods have two immiscible phases (medium) called

«stationary phase» and «mobile phase».

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While the stationary phase remains constant, the mobile phase moves continuously through the stationary phase.

The separation of the substances are due to the affinity of them to either stationary and mobile phase.

The duty of the stationary phase is to keep and retain the substances. The duty of the mobile phase is to push and move the substances.

While mobile phase moves continuously through the stationary phase, the substances in the mixture are introduced into the mobile phase and carried along with it. Different substances move with different speed rates.

Since their speed rate is different, each substance forms a group of its molecules and move as a group. Hence, the substances in the mixture gets separated.

Column chromatography

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History of chromatgoraphy

▶ Chromatography was firstly used by Russian botanist Mikhail Tswett in 1906.

▶ He introduced a mixture of leaf pigments (chlorophyll, carotene, xanthophyll) along with petroleum ether into a column filled with calcium carbonate. The pigments got separated inside the column.

▶ Because these pigments have different colors, they formed groups with different colors in the column. This technique was named as chroma-tography which means colorful photography.

▶ Sample : The solution of pigment mixture

▶ Analyte : Pigments (chlorophyll, carotene, xanthophyll)

▶ Stationary phase : Calcium carbonate

▶ Mobile phase : Petroleum ether

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▶ Since the physicochemical properties of the substances in the mixture are

different, the substances are retained by the stationary phase in different

rate of forces. So they move with different speed rates.

▶ Each substance moves with a certain speed within a group of molecules.

Hence, each substance make a group and moves along with it.

• Movement

• Speed rate

• Retention

• Retention time

• Elusion time

• Time to leave the stationry phase

Chromatogram and peak

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▶ While moving, the molecule groups of the substance show a normal

distribution in stationary phase.

▶ The molecules leaving the stationary phase are measured with a proper

detection method and the signal is recorded electronically. The graph that

shows signal versustime is called «chromatogram».

▶ The concentration profile of each substance is called peak.

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▶ X axis shows time, Y axis shows the detector response (signal).

▶ tR1: Retention time of substance B (the time when B leaves the stationary phase)

▶ tR2: Retention time of substance A (the time when A leaves the stationary phase)

▶ t0: The time when solvent leaves the stationary phase

▶ W1, W2 : Width of the substance peaks. This value shows the band width in the column.

▶ Peak height: The distance between the top of the peak and the bseline. Increases with the concentration of the substance.

▶ Peak area: Area between the peak and baseline. Increases with the concentration of the substance. Mostly, this value is used for quantitation of the analytes.

Qualitative info

Quantitative info

Stationary and mobile phase

▶ Chromatographic systems always have two immiscible phases.

▶ The substances in the mixture moves through the stationary phase along with

the mobile phase. The mobile phase acts as a carrier.

▶ The substances in the mixture have different affinities to mobile and

stationary phases.

▶ The substances which have high affinity to mobile phase, have high a speed

rate so they move fast and leave the column in a short time. Their retention

time is short.

▶ The substances which have high affinity to stationary phase, have a low speed

rate so they move slowly and leave the column in a long time. Their retention

time is greater.

▶ The interaction between the substances and phases define the separation

mechanism (Adsorption or partition) 12

Separation principles of chromatography

1. Adsorption chromatography

2. Partition chromatography

• Adsorption is the adhesion of substances to a surface. This

surface needs to be a solid in chromatography.

• Partition is the distribution of a substance between (in) two

liquid phases.

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1. Adsorption chromatography

▶ If the stationary phase is a solid, then the stationary phase acts as an adsorbent.

▶ While the substances in the mixture move along with the mobile phase, some of them get adsorbed onto the stationary phase and some of them continue moving with the mobile phase.

▶ Because the mobile phase has a continuous flow, the adsorbed substances have to leave the stationary phase and moves with the mobile phase after a certain period.

▶ In this way, different substances with different adsorption interests leave the stationary phase in different times and get separated according to their speed rates.

▶ The principal of separation in the systems with a solid stationary phase is adsorption.

▶ The substances have different speed rates based on their adsorption rate.

▶ The adsorption rate determines the retention time of a substance.

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2. Partition chromatography

▶ In some chromatographic systems, the stationary phase consists of an inert solid coated with a liquid.

▶ This means that the stationary phase is a liquid in these systems. Mobile and stationary phases are immiscible. The substances in the sample dissolves in both phases. The dissolved substances fragment (distribute) between stationary and mobile phases in different rates.

▶ The substances highly distributed in mobile phase move quickly along with the mobile phase. The substances highly distributed in stationary phase tend to spend more time in the stationary phase hence move slowly. In this way, the substances have different speed rates and get separated.

▶ The principal of separation in the systems with a liquid stationary phase is partition.

▶ The substances have different speed rates based on their partition coefficients.

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Classification of chromatography

Based on chromatographic bed shape

1. Planar chromatography

1. Paper chromatography

2. Thin layer chromatography

2. Column chromatography

1. Gas chromatography

2. High-performance liquid chromatography

Based on the type of solid phase

1. Liquid chromatography

1. Liquid-solid chromatography

2. Liquid-liquid chromatography

2. Gas chromatography

1. Gas-solid chromatography

2. Gas-liquid chromatography

Based on separation mechanism (type of solid phase)

1. Adsorption chromatography (Solid stationary phase)

1. Liquid-solid chromatography (liquid mobile phase)

2. Gas-solid chromatography (gas mobile phase)

2. Partition chromatography (Liquid stationary phase)

1. Liquid-liquid chromatography (liquid mobile phase)

2. Gas-liquid chromatography (gas mobile phase)

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High-performance liquid

chromatography

▶ The columns used in HPLC are prepared by filling a stainless steel column with silicate particles which are coated with a liquid film.

▶ Because the stationary phase is liquid, HPLC is based on partition and classified as liquid-liquid chromatography.

▶ The size of the particles are very small (μm) and are filled in the column by compression.

▶ The mobile phase is pumped trough the column with high pressure.

▶ While the analytes in the sample pass through the column, they show different affinities to stationary and mobile phases. So, the analytes with a high affinity to stationary phase are retained in the column, move slower and leave the column late.

▶ The substances that leaves the column are monitored with a suitable detector. So the time of elution (retention time) their concentration are detected.

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High-performance liquid

chromatography▶ HPLC system consist of 5 main parts

▶ Pump

▶ Injector

▶ Column

▶ Dedector

▶ Recorder

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High performance liquid chromatography

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High-performance liquid

chromatography

▶ Pump system enables the movement of the mobile phase in the column with high presure.

▶ Enjector delivers the sample into the column by mixing it with mobile phase in small volumes.

▶ Column is the part where the separation occurs. It is manufactured by filling a stainless steel column with fine particles.

▶ The substances enter the detector after getting separated in the column. Detector determines their concentration and the time when they left the column.

▶ The detected signals are converted into numerical values and graphs by the recorder.

▶ The analysis results are recorded as “chromatogram” which is a graph of the detector response versus time.

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Detectors used in HPLC

▶ Ultraviolet / visible region (UV/VIS)

▶ Fluorescence

▶ Infrared

▶ Refractive index

▶ Electrochemical

▶ Mass spectrometer

▶ The concentration profile of each substance is called peak.

▶ The graph that shows the peaks of the substances are «chromatogram».

▶ Chromatograms are obtained by plotting detector response versus time.

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Quantitative determination by HPLC –

UV detection

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UV light can be used to quantify the substances in a

mixture after separationg them.

▶ The absorbed photon amount is correlated with the substance amount.

▶ If the concentration is doubled, light being absorbed gets twice as much.

▶ Detector measures the intensity of the light

▶ The decrease in the intensity of the light creates a signal and the signals are

recorded as peaks.

There are two techniques in HPLC

▶ Normal phase ▶ Stationary phase : Polar

▶ Mobile phase : Apolar (hexane, octanol etc.)

▶ Reversed phase Ters faz

▶ Stationary phase : Apolar

▶ Mobile phase : Polar (water, buffer, methanol, acetonitrile, etc.)

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Normal and reversed phase

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Normal and reversed phase

▶ In normal phase,

▶ The stationary phase is polar. The most polar substance in the sample is

retained more, moves slowly and leaves the column lastly. So its retention

time is long. Less polar substance moves faster. Least polar substance moves

the fastest and leaves the column first, hence its retention time is short.

▶ In reversed phase,

▶ The stationary phase is nonpolar. The most polar substance in the sample

moves quickly and leaves the column first.

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How to separate substances?

▶ Mostly, as a first step, stationary phase is chosen according to the

physicochemical properties of the analytes. Then, a mobile phase is prepared

by mixing diffrent solutions and solvents to achieve a suitable polarity.

▶ In reversed phase HPLC, mobile phase is prepared by mixing water, methanol,

acetonitrile, buffer solutions etc.

▶ The temperature of the column affects the retention times

▶ Flow rate of the mobile phase and pressure in the column also affects the

retention times of the analytes.

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References

▶ Principles of Instrumental Analysis, D.A. Skoog, D.M. West, II. Ed. 1981

▶ Analitik Kimya II, F. Onur, A.Ü. Eczacılık Fakültesi Yayınları No. 101, 2011

▶ Analitik Kimya Pratikleri Kantitatif Analiz, F. Onur (Ed.), A.Ü. Eczacılık

Fakültesi Yayınları No. 111, 2014

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