1. HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)

2. 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.

3. 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.

4. What is chromatography?
Chromatgoraphy is a set of separation technique which allows qualitative and quantitative analysis of various substances in a mixture.

5. What is chromatography?
All chromatographic methods have two immiscible phases (medium) called «stationary phase» and «mobile phase».
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.

6. Column chromatography

7. 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

8. 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

9. Chromatogram and peak
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.

11. 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

12. 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)

13. Separation principles of chromatography
Adsorption chromatography
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.

14. 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.

15. 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.

16. Classification of chromatography
Based on separation mechanism (type of solid phase)
Adsorption chromatography (Solid stationary phase)
Liquid-solid chromatography (liquid mobile phase)
Gas-solid chromatography (gas mobile phase)
Partition chromatography (Liquid stationary phase)
Liquid-liquid chromatography (liquid mobile phase)
Gas-liquid chromatography (gas mobile phase)
Based on the type of solid phase
Liquid chromatography
Liquid-solid chromatography
Liquid-liquid chromatography
Gas chromatography
Gas-solid chromatography
Gas-liquid chromatography
Based on chromatographic bed shape
Planar chromatography
Paper chromatography
Thin layer chromatography
Column chromatography
Gas chromatography
High-performance liquid chromatography

17. 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.

18. High-performance liquid chromatography
HPLC system consist of 5 main parts
Pump
Injector
Column
Dedector
Recorder

19. High performance liquid chromatography

20. 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.

21. 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.

22. Quantitative determination by HPLC – UV detection
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.

23. 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.)

24. Normal and reversed phase

25. 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.

26. 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.

27. 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