HPLC Made Easy: Understanding Principle, Instrumentation, and System Suitability

HPLC stands for High-Performance Liquid Chromatography or High-Pressure Liquid Chromatography. It is a technique in analytical chemistry used to separate, identify, and quantify each component in a mixture. Previously, HPLC analysis was carried out using low-pressure liquid chromatography, which took more time. Currently, Ultra High Performance Liquid Chromatography (UHPLC) is becoming more popular. HPLC analyzes only the compound that is dissolved in solvent. It identifies the compound that is dissolved and how much of the compound is dissolved. 

Principle:

The analysis takes place in a separation column between a stationary and a mobile phase. The stationary phase is a granular material with very small porous particles in a separation column. The mobile phase is a solvent or solvent mixture that is forced at high pressure through the separation column. The delivery pump controls the constant flow rate of the mobile phase. The sample is injected by using a syringe into the mobile phase, which flows from the pump to the column. The components are separated from one another according to varying degrees of interaction between the molecule and the stationary phase. After leaving the column, the separated components are detected by the detector. The detector transfers an electric signal as a chromatogram, which is displayed in HPLC software. According to the data, we can identify and quantify the components dissolved in the liquid sample. 

Basic Overview of Instrumentation & Working Procedure:

Here in the flow diagram, we can see different kinds of HPLC components like 

I. Solvent reservoir
II. Degassing unit

III. Solvent Delivery Pump
IV. Sample injector
V. Column Oven

VI. Column (Stationary Phase)

VII. Detector

VIII. Recorder

The solvent reservoir contains solvents like Mobile phase (a), 10% IPA Solution(b), 1:1 volume of Methanol & Water(c), and 1:1 volume of  Methanol & Acetonitrile (d). 

The degassing unit helps remove air bubbles dissolved in the mobile phase. Due to the standard atmospheric pressure, air is easily dissolved in the mobile phase. If air bubbles are not removed from the mobile phase, then, after passing through the delivery pump, it can create different kinds of troubleshooting, such as fluctuations of the mobile phase, baseline noise, etc.

The solvent delivery pump delivers the mobile phase at a constant flow rate to the column we have set in the method.

The sample injector injects the standard or test sample into the mobile phase.

A column oven is used to keep the column at a constant temperature set in the method. Unstable temperatures affect the separation of components in the column.

The column is also known as the stationary phase. Each compound in the column moves according to its difference in speed. Components move faster through the column if there is a strong affinity between the mobile phase and the component. The component moves more slowly through the column if it has more affinity for the column. So, according to speed, the component with high affinity for the mobile phase gets eluted first from the column.

The detector is used to detect the eluted compound. The workstation processes the data from the detector and gives it as a chromatogram.

Types of HPLC Techniques:

Based on the polarity of the stationary and mobile phases:

A. Normal phase HPLC: In this type of HPLC, the stationary phase is polar, and the mobile phase is nonpolar. Nonpolar compounds move faster and elute first than polar compounds due to less affinity between solute and stationary phase. Polar compounds spend more time in the column due to high affinity with the stationary phase and elute slowly. Usually, silica is the stationary phase, and the mobile phase is hexane, methylene chloride, chloroform, and diethyl ether.

B. Reverse Phase HPLC: In this mode, the stationary phase is nonpolar. N is nonpolar and hydrophobic in nature. The mobile phase is polar in nature; it may be a mixture of water and methanol or acetonitrile. In this phase, polar compounds are eluted first, then nonpolar compounds. Most pharmaceutical ingredients are polar in nature. Reverse-phase HPLC is more popular than normal-phase.

Based on the principle of separation:

A. Adsorption chromatography: Separation of compounds based on the difference in affinity of compounds towards the stationary phase. The most widely used stationary phase is unmodified silica, which allows high efficiency and high permeability. The functional group responsible for adsorption is the silanol group, which reacts with sample solutes by hydrogen bonding.

B. Ion Exchange Chromatography: Separation of compounds based on ion exchange of functional groups. In this ion exchange, resins are used to separate a mixture of similarly charged ions. The retention of the ions on the column depends on the ionic strength and pH of the mobile phase. The stationary phase has an ionically charged surface that is opposite the charge of the sample. This technique is used almost exclusively with ionic or ionizable samples.

C. Size exclusion chromatography (gel permeation chromatography): Separation is based on the different molecular sizes of the compounds separated by using different gels.

D. Chiral Phase Chromatography: Separation of optical isomers using chiral stationary phases.

Based on the elution technique:

A. Isocratic Separation: The same mobile phase combination is used throughout the process of separation.

B. Gradient Separation: The mobile phase combination is changed at a specific period during the analysis. The mobile phase combination of lower polarity is followed by gradually increasing the polarity.

INSTRUMENTATION:

I. SOLVENT RESERVOIR:

It stores the different types of solvents, like mobile phase, methanol, etc., in a glass reservoir. 

II. Degassing Unit: 

The mobile phase, or eluent, used for liquid chromatography may contain different kinds of invisible gases. When the mobile phase containing gases passes through the pump, it can cause a noisy baseline. The degasser has special polymer membrane tubing that removes gas. The numerous very small pores on the surface of the polymer tube allow only air to pass through it. Under a low-pressure container, it creates pressure differences inside and outside the tubing. Higher pressure inside the tubing allows the dissolved gas to pass through the pores.

III. SOLVENT DELIVERY PUMP:

It is located in the upper part of the HPLC. It allows the flow of the mobile phase from the reservoir to the system. It generates flow by the back-and-forth motion of a motor-driven piston. Piston motion produces the "pulse." There are mainly 2 types of pumps.

i. Constant Pressure pump: e.g., displacement-type pump

ii. Constant volume pump: e.g., reciprocating pump and syringe pump

IV. SAMPLE INJECTOR:

It is placed after the pump. The sample is injected by using a syringe in the flow of the mobile phase. Nowadays, an autosampler is the most widely used, which automatically injects at a scheduled time.

V. Column Oven:

The separation of components is influenced by the column temperature. Small temperature differences cause variation in results. So the column oven keeps the column temperature constant.

VI. Column:

The separation is carried out in a column, so it is also known as the heart of the column. Columns are generally made of cleaned stainless steel. They are about 50-300 mm long and have an inside diameter of 2-5 mm. They are normally loaded with a stationary stage with a molecular size of 3 to 5 micrometers. Columns are of three types: analytical column, preparative column, and guard column. The life of the column can be increased by using a guard column. It is placed between the injector and the column. 

VII. Detector:

The detector is used to observe the obtained separation. It is located in the last part of the column. The detectors used in HPLC are 

i. UV detectors, 

ii. Visible detectors

iii. PDA detector

iv. Refractive index detector

v. Mass spectrometer

vi. Fluorescence Detector

vii. Chemiluminescence detector

viii. Electrochemical detector

VIII. Recorder:

A change in eluent detected by a detector is in the form of an electronic signal; thus, it is not visible to our eyes. Nowadays, computer-based data processing is mostly used.

Application of HPLC:

• It is used in the resolution, identification, and quantification of compounds.
• It is used in chemical separation and purification.
• It is used in pharmaceutical quality control (assay, identification, dissolution test)
• It detects phenolic compounds in drinking water.
• Identification of steroids in blood, urine, etc.
• Forensic analysis of textile dyes.
• Determination of cocaine and other drugs of abuse in blood and urine.
• Sugar analysis in fruit juices.
• Measurement of quality of soft drinks and water.
• Quantification of drugs in biological samples.
• Analysis of bilirubin and biliverdin in hepatic disorders.
• Bio-monitoring of pollutants.

HPLC SYSTEM SUITABILITY:

System suitability is defined by ICH as "the checking of a system, before or during analysis of unknown, to ensure system performance. System suitability is to prove that the system is working properly before HPLC analysis. It is required to be done before every sample analysis.

I. Theoretical Plate or Column Efficiency:

The column contains a large number of separate layers called theoretical plates. The number of theoretical plates is used to determine the performance & effectiveness of the column. The number of theoretical plates depends upon the nature of the eluting substance and operating conditions like flow rate, mobile phase temperature, column packing, packing uniformity within the column, internal diameter, and length of the column. Its value should be 2000 or more.

The formula for electronic calculation is 

N = 5.54 (T_r/W_h)²

^{Where Tr =} Retention time of the peak corresponding to the component.

Wh = Peak width at half height of the peak

For manual calculation, the formula given below is used

N = 16 (T_r/W_b)²

Where Tr = Retention time of the peak corresponding to the component.
Wb = Peak width at the base of the peak

II. Resolution:

It is the ability to distinguish between the two peaks. It is a quantitative measure of how well two elution peaks can be differentiated in a chromatographic separation.

The formula for electronic calculation

R=1.18 (TR2-TR1)/(Wh1+Wh2)

Where TR1 = Retention time of the peak eluting first
TR2 = Retention time of the second peak eluting
W_{h1 =} Peak width at half height of the first peak
W_{h2 =} Peak width at half height of the second peak

The formula for manual calculation

R=2.0 (TR2-TR1)/(Wb1+Wb2)

Where TR1 = Retention time of the peak eluting first
TR2 = Retention time of the second peak eluting

Wb_{1 =} Peak width at the base of the first peak
Wb_{2 =} Peak width at the base of the second peak

III. Symmetry factor and tailing factor:

The tailing factor is a measure of peak asymmetry in HPLC, calculated at 5% of the peak height.

It is the distance from the front slope of the peak to the back slope divided by twice the distance from the center line of the peak to the front slope. The general limit is from 0.8 to 1.8. It should not be more than 2.

Formula:

TF == W0.05/2f

Where W0.05 = peak width at 5% height

f = maximum to leading at 5% height from the baseline

👍️ The symmetry factor (also called the asymmetry factor) measures peak symmetry but is calculated at 10% of the peak height.

Formula:

As=b/a

a = Distance from the peak maximum to the front half-width at 10% height

b=Distance from the peak maximum to the back half-width at 10% height

Acceptance Criteria:
The tailing factor for a perfectly symmetrical peak will be 1.0.

A tailing factor of less than 1.0 means fronting in peak shape.
A tailing factor of more than 1.0 means tailing in peak shape.

IV. Retention Time

Retention time is a measure of the time taken for a solute to pass through a chromatography column. It is calculated as the time from injection to detection.

V. % Relative Standard Deviation (%RSD):

It is defined as the standard deviation expressed as the % of the mean. In HPLC, 5 or 6 replicate injections of standard solution are performed. Its value should be less than 2. The % RSD is calculated by the following formula:

%RSD= SD/X1 * 100

Where SSD = Standard deviation

X1 = Mean 

VI. Capacity Factor (K):

It is the measure of the retention of a peak that is independent of column geometry or mobile phase flow rate. Generally, it should be NLT 2.0.

K = time spent by a substance in the stationary phase

Time in the mobile phase.

or

K = Amount of a K = Amount of substance in the stationary phase

Amount of substance in the mobile phase

VII. Peak-to-valley Ratio:

It is employed when baseline separation between two peaks in related substance analysis is not achieved.

Peak-to-valley ratio (P/V) = H_p/H_v

Hp = height above the extrapolated baseline of the minor peak
Hv = height above the extrapolated baseline at the lowest point of the curve separating the minor peak and major peak