High-Performance Liquid Chromatography (HPLC) is a technique of analytical chemistry used to identify, separate, and quantify each component in a mixture.
HPLC relies on pumps to pass the pressurized liquid solvent containing the sample mixture through a column filled with a solid adsorbent material.

## Principle for HPLC

The principle of HPLC is based on the distribution of the analyte (sample) between a mobile phase (eluent) and a stationary phase (packing material of the column).

Materials

Chromatography - This is a technique used in the separation of mixtures.

It is done by passing the mixture in solution or suspension through a medium in which the components are at different rates.

Mobile Phase

It is the liquid or a gas that flows through the system by moving the materials to be separated over the stationary phase (materials are moved at different rates).

The most common eluents in for polymers that dissolve at room temperature

E.g., Tetrahydrofuran, chloroform, dimethylformamide.

Stationary Phase

It is a porous solid that is packed in a glass or metal tube or that constitutes the walls of an open-tube capillary.

E.g. Glass, silica, or alumina.

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Silica Gel
Silica is an amorphous solid form of silicon dioxide, consisting of a porous 3D framework of alternating Oxygen and Silicon atoms with nanometer-scale voids and pores. The voids may contain water or any other liquids and may be filled with gas or vacuum.

## Types of HPLC

Based on the mode of separation, HPLC is classified as follows:

Normal Phase

Separation of polar analytes by partitioning onto the polar, bonded stationary phase.

Reverse Phase

Separation of non-polar analytes by partitioning onto the non-polar, bonded stationary phase.

## Instrumentation of HPLC

Instrumentation of HPLC consists as follows:
Mobile Phase Reservoir.
Degasser.
Pump.
Sample Injector.
Column Heater.
Column (Stationary Phase).
Detector.
Data Analyser.
Waste Container.

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i. Mobile Phase Reservoir
The reservoir holding the mobile phase is often a glass bottle.
The reagent bottle that holds the HPLC solvent can be used as a reservoir.
The reservoir and its attachment to the pumps should be made of materials that do not contaminate the mobile phase.
E.g., Teflon, glass, or stainless steel.
Light sensitive or photosensitive solvents have to be kept in amber-colored bottles.

ii. Degasser

It is a device used in drilling to remove gases from drilling fluid, which could otherwise form bubbles.

Degassing, also known as degasification, is the removal of dissolved gases from liquids, especially water or aqueous solutions.

The problems produced by the bubble formation can largely be prevented by degassing the mobile phase.

The air bubbles can also modify the flow of the mobile phase through the column due to the creation of dead volumes.

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iii. Pump

A pump is the most important part of the HPLC system.

It is positioned in the most upper stream of the liquid chromatography system, and it generates a flow of eluent from the reservoir to the system.

The purpose of a pump is to force the liquid through the HPLC system while it maintains a constant flow of the mobile phase.

This occurs regardless of the backpressure caused by the flow resistance of the HPLC column.

HPLC pumps are of three main types:

Reciprocating Pump

The solvent is withdrawn into a small chamber (when the solvent check valve is open), and it is pumped out of it (when the column check valve is open) by the back and forth motion of a motor-driven piston.

The solvent chamber volume is small (35 to 400 mL).

Have high output pressure (up to 10,000 psi).

It has a constant flow rate, which is independent of column backpressure and solvent viscosity, but the disadvantage of a pulsed flow which must be smoothed out using a pulse damper.

Displacement (or Syringe) Pump

The solvent is pumped out from a large chamber by using a plunger. This pump produces a pulse-free flow, which is independent of column back pressure and viscosity of the solvent. But it has a limited solvent capacity of ~250 mL and requires refilling of the solvent chamber for continual use.

Pneumatic (or Constant Pressure) Pump

It uses gas to pressurize the mobile phase that is present in a collapsible solvent container and uses a compressed gas tank.

iv. Sample Injector

The simplest method in HPLC is to use a syringe. The sample is introduced into the flow of eluent using the syringe.

The mostly used injection method is based on the sampling loops. The use of an autosampler (auto-injector) system is also widely used that allows repeated injections in a set scheduled-timing.

An autosampler is merely an automated injection valve with a mechanism to hold multiple samples and to load these samples into the sample loop as dictated by the system controller.

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HPLC Sample Vial

Autosampler vials used in HPLC are generally grouped depending on the diameter of vials, the height of the vial, and the thread finish.

The vials are primarily utilized to inject samples from an autosampler.

The vials are sold in several sizes, although 2 mL vials are more generally used. The Autosampler needle pierces through the cap during injection and withdraws the required aliquots of the sample from the vials.

v. Column (Stationary Phase)

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Columns are the main components of HPLC because the column is responsible for the separation of the sample components.

The sample passes through the column along with the mobile phase and separates in its components when it comes out from the column.

Mostly the column housing is made of stainless steel since it is tolerant of a large variety of solvents.

But, for the analysis of some analytes such as ionic compounds and biomolecules, contact with metals is not desired, so a polyether ether ketone (PEEK) column housing is used instead.

The pressure limitations in the column are the length of 10 to 30cm, extra-column band broadening, inside diameters of 4 to 10mm, and are packed with particles of size 5 to 10mm in diameter.

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Types of Columns are as follows:

Guard Column

A short guard column is usually placed before the expensive analytical column to protect it from damage due to:

Particulate matter.

Irreversible binding of contaminant to the stationary phase.

Bleeding. But a loss in resolution may result.

This occurs in tubings connecting between the column and other parts, such as the injection system, the detector region, etc. The smaller is the radius of the connecting tube; the less is the extra-column band broadening.

Column Packing Pellicular Particles

A thin porous layer (e.g., silica) is deposited on the surface of non-porous beads (e.g., a glass of a larger diameter of 30 to 40 mm).

These are usually used in guard columns, with a loss in resolution.

Porous Particles

Small-sized porous particles (e.g., silica of diameter of 3-10 mm) are used in analytical columns.

vi. Column Heater

The column heater is important to keep consistent temperature conditions.

It is also used to analyze sugar and organic acid.

It is also important to keep a stabilized temperature because there are possibilities that a small difference in temperature can result in different separation results.

So the columns are generally kept inside the column oven (column heater).

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vii. Detectors

A chromatographic detector is a device used in HPLC to detect the separation of the analytes used in the column as the presence of analyte changes the composition of the eluent.

## Types of Detectors in HPLC

There are different types of detectors available :
Ultra Violet (UV).
Visible (VIS).
PhotoDiode Array (PDA).
Refractive Index (RI).
Evaporative  Light Scattering (ELS).
Multi-Angle Light Scattering (MALS).
Mass Spectrometer (MS).
Conductivity (CD).
Fluorescence (FL).
Chemiluminescence (CL).
Optical Rotation (OR).
ElectroChemical (EC).

UV, VIS, and PDA Detectors

These detectors are categorized as absorbance detectors as they provide good sensitivity for light-absorbing compounds at ~pg level.

These are easy to operate and provide good stability.

UV detector is a commonly used detector for HPLC analysis. During the analysis, the sample goes through a clear color-less glass cell called a flow cell.

When the UV light is irradiated on the flow cell, the sample absorbs a part of UV light. The most commonly used is 254nm.

Compared to a UV detector, the VIS detector uses a longer wavelength (400 to 700 nm). Some detectors provide wider wavelength selection, covering both UV and VIS ranges (195 to 700nm) is called UV/VIS detector.

PDA is a 3D detector; thus, it analyzes light intensity, time, and wavelength.

PDA is convenient to determine the most suitable wavelength without repeating analyses.

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Refractive Index Detectors

RI is used to measure the change in the reflex index.

In this, glass is divided into two cells, namely sample cell and reference cell.

These cells are filled with the mobile phase.

When the effluent going through the sample cells does not contain any analyte; the solvent inside both cells is the same.

It is suitable for detecting all components. For example, samples that do not have UV absorption (such as sugar, alcohol, or inorganic ions) cannot be measured by a UV detector.

It is applicable for use with a solvent that has no UV absorbance. A UV detector cannot be used with a solvent which has UV absorbance.

RI detector is often used for the detection of sugars and SEC analysis.

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Evaporative Light Scattering Detector

It provides good sensitivity for non-volatile analytes at the ng level. Here the column effluent is nebulized and then evaporated to make it forming fine particles.

It is used in a similar way to a RI detector but can provide more sensitive detection with a stable baseline.

An advantage is that ELSD can be used for the gradient method, whereas RI cannot be used.

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Multi-Angle Light Scattering Detector

Multiangle light scattering (MALS) is a technique for measuring the light scattered by a sample into a plurality of angles.

It is used for determining the absolute molar mass and the average size of molecules in solution, which is done by detecting how they scatter light.

Molecular weight can be determined directly without the need for a calibration curve. MALS can also provide an absolute Molecular weight of the analyte with a very low detection limit.

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Mass Spectrometer

The analytes are detected based on their Molecular weight. The obtained information is useful for compound structure identification.

It can be used to quantify very low detection limits of elemental and molecular components.

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Conductivity Detector
Solutions containing ionic components will conduct electricity. The conductivity detector measures electronic resistance, and it is directly proportional to the concentration of ions present in the solution. Hence it is generally used for ion chromatography.

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Fluorescence Detector

The advantage of the fluorescence method is that it is highly sensitive for selective groups of compounds at ~fg level.

When we use a specific wavelength, the analyte atoms are excited, and then they emit a light signal (fluorescence). The intensity of this light is monitored to quantify the analyte concentration.

For compounds that do not have fluorescence absorbance or low absorbance they can be treated with fluorescence derivatives such as dansyl chloride. It is easy to operate and relatively stable.

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Chemiluminescence Detector

It is similar to FL, but instead of using a light source to excite the analyte atoms, the excitation is initiated by a chemical reaction. Since it does not rely on the external excitation source, the noise is small, results in a high signal to noise ratio, i.e., it provides even higher sensitivity than FL.

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Optical Rotation Detector

Specific for the optical isomer measurement. The column can separate R and L type optical isomers, but the general detectors (e.g., UV) cannot distinguish R and L.

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Electro-Chemical Detector

There are several different types of electrochemical detectors. The detection is based on polarography, coulometry, amperometry, and conductometry. They offer high sensitivity, convenience, and wide-spread applicability. It is especially suitable for use with a semi-micro or capillary type system.

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## Various Chromatographic Parameters in HPLC

A chromatogram is a graphical representation of separated eluents, which can be used to identify compounds and to determine their relative concentrations.

It is a visual output of the chromatograph (instrument).

It can also be defined as a visible record (such as a graph) showing the result of separating the components of a mixture.

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The various parameters in a chromatogram are as follows:
System suitability.
Retention time.
Retention volume.
Tailing factor.
Asymmetry.
Theoretical plates.
HETP.
Resolution.
System suitability.

System Suitability

It is defined by ICH as "the checking of a system, before or during the analysis of unknowns, to ensure system performance.

This may include such factors as plate count, tailing, retention, and/or resolution.

It is a test to determine the suitability and effectiveness of the chromatographic system before use. The performance of any chromatographic system may continuously change during their regular use, which can affect the reliability of the analytical results.

Retention Time

Retention time (RT/tr) is a measure of the time taken by a solute to pass through the column.

It is calculated as the time from injection for detection.

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Retention Volume (VR)

Retention volume for a solute is the volume of the mobile phase required to carry the solute through the column to elution.

Theoretical Plates

A theoretical plate is a hypothetical zone in which two phases, such as the liquid and vapor phases of a substance, establishes an equilibrium with each other. Such equilibrium stages may also be referred to as an ideal stage, or a theoretical tray.

It gives information regarding column efficiency/performance.

n = 16 \left( \frac{R_t}{W_b}\right)^{2}

Where,
n\ = No of theoretical plates.
R_t = Retention time.
W_b = Width of the peak.

HETP (Height Equivalent to the Theoretical Plate)

It is the number of theoretical plates in a chromatography column.

It is used to relate the column height with the number of theoretical plates.

It is numerically equal to the column length divided by the Platter of theoretical plates in the column.

\text {HETP (H)} = \frac{\text{L (Lenght of the column)}}{\text{N (No of theoretical plates)}}

HETP is given by Van Deemter equation as:

\text {HETP (H)} = A\ +\ \frac {B}{C}\ +\ C.u

A = Eddy diffusion term or multiple path diffusion, which arises due to the packing of the column.

B = Molecular diffusion that depends on the flow rate.

C = Effect of mass transfer that depends on the flow rate.

U = Flow rate.

Resolution

The resolution of elution is a quantitative measure of how well two elution peaks can be differentiated in chromatographic separation.

Resolution is generally defined as the difference in retention time between the two peaks, divided by the combined widths of the elution peaks.

Asymmetry

The asymmetry is a measure of peak tailing, and it is defined as the distance from the centerline of the peak to the back slope divided by the distance from the centerline of the peak to the front slope (with all measurements made at 10% of the maximum peak height).

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Ideal chromatographic peaks are Gaussian and symmetrical.

Asymmetrical peaks are said to either front or tail. Peak fronting or tailing can be caused by poor quality or polluted columns or by the dead volume of the system. Asymmetry can degrade the quality of separation.

Peak Tailing and Fronting

Peak tailing is the most common chromatographic peak shape distortion.

It occurs when the peak asymmetry factor (As) is greater than 1.2 — although peaks with As greater than 1.5 are acceptable for many assays.

Tailing occurs when some sites on the stationary phase retain the solute more strongly than other sites.

Peak fronting is the result of overloading the column with a sample.

Injection of too much sample and overloading effect results from the poor sample solubility in the stationary phase as too much sample is injected.

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Relative Retention Time (RRT)

It is the ratio of the retention time of analyte peak relative to that of another used as a reference obtained under identical conditions.

It is the comparison of the RT of one compound to another.

Retardation Factor (RF)
In chromatography, the retardation factor is the fraction of an analyte in the mobile phase of a chromatographic system, and in planar chromatography, the retardation factor (RF) is defined as the ratio of the distance traveled by the center of a spot to the distance traveled by the solvent front.

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Retention/Capacity Factor (K)

A retention or capacity factor means measuring the retention of the analyte on the chromatographic column.

It is defined as the ratio of an analyte in the retention of the stationary phase to the time it is retained in the mobile phase.

This is inversely proportional to the retardation factor.

References
1. Principle of HPLC by www.researchgate.net.
2. Instrumental Methods of Analysis by Willard HH, Merritt LL, Dean JA, and Settle FA. (2001), CBS Publishers & Distributors, Delhi. (PDF by Shodhganga).
4. Lesson 6: Detectors for HPLC | Shodex/ HPLC Columns.
5. Lesson 1: Introduction to HPLC | Shodex/ HPLC Columns.