Are you planning to learn or buy an HPLC (High Performance Liquid Chromatography)? Buying an HPLC can be a tough job especially when you do not have expertise in it. The interesting thing for you is that we are with you. After helping 1000+ buyers buying the right HPLC, we have planned to make the most detailed guide on how to buy an HPLC without having technical knowledge.
The guide is easy to read and follow whether you are in your early 20’s or 50+ age. Anyhow, if you still need our help in selecting the right model of HPLC, our technical team is available 24/7 for your help free.
Which things you need to need to know before buying a HPLC?
A brief knowledge about HPLC and its applications
Your careful attention for 15 minutes to read this guide.
Yes, only 15 minutes you need to understand, how to buy HPLC and you will pass through the whole steps.
In this guide, you will cover.
HPLC Overview
Working Principle of HPLC
Types of HPLC based on Operational Method
Main Parts of HPLC
Quality Parameters Checking of HPLC
Trouble Shooting of Common Problems, which occurs during Operation.
HPLC Applications
Merits and demerits of HPLC
Famous brands of HPLC
High Performance Liquid Chromatography (HPLC)
In this chapter you are going to learn about chromatography and its types, its major concern is about High performance liquid chromatography (HPLC). Chromatography is a separating technique, in which separation is done on the basis of the structure of the molecule in two-phase (i.e. Mobile phase and Stationary phase)
The stationary phase is a solid or a liquid material packed in a column and a mobile phase is a liquid or a gas that flows through the stationary phase. The mobile phase flows through the stationary phase and carries the components of the mixture with it. The component of a mixture, which shows stronger interactions with the stationary phase, will retain in the stationary phase while the component of the mixture, which dissolves in the mobile phase, will move with it and elute first.
HPLC stands for High performance Liquid chromatography or high-pressure liquid chromatography as it uses a high-pressure liquid mobile phase than gases used in Gas chromatography.
HPLC is an analytical technique used for the separation of components of the organic mixture of compounds when such compounds are nonvolatile, thermally unstable, and have relatively high molecular weights.
Working principle of HPLC
In the previous chapter you learn about the introduction of HPLC, now in this chapter, you will learn about the working principle of HPLC. The working principle of HPLC is based on the distribution of the sample between a mobile phase (eluent) and a stationary phase (packing material of the column). Molecule retarded in stationary phase and separate on the basis of difference in retention time. The component of a mixture which retain in the stationary phase elute later, as compared to that which dissolves in the mobile phase.
Types of HPLC
This section will help you to learn different types of HPLC.
Types of HPLC, Based on mode of separation
1. Normal Phase HPLC
In this type of HPLC, components are separated on the basis of polarity. In the Normal phase HPLC, the polar stationary phase is used with the non-polar mobile phase. The stationary phase is usually silica and the mobile phase is Hexane, Methylene Chloride, Chloroform, Diethyl ether, and mixtures of these.
As the Stationary phase is polar in normal phase HPLC therefore, the Retention time of Polar samples on the polar surface of the column is longer and they elute later than less polar materials.
2. Reverse Phase HPLC
In this type of HPLC, the stationary phase is non-polar in nature, while the mobile phase is a polar liquid i.e. mixtures of water and Methanol or Acetonitrile. It works on the principle of hydrophobic interactions hence retention time of non-polar material is longer than polar material.
Note: Reverse phase chromatography is more commonly used as drugs are usually Hydrophilic.
3. Size-exclusion HPLC
In this type of HPLC, the Column used is filled with a material having control pore size and separate the components on the basis of size. Large size molecules pass through the column while small size molecules penetrate inside the pores of the packing material and due to having large retention time elute later.
4. Ion-Exchange HPLC
In this type of HPLC, The stationary phase has a charged surface of opposite charge to the sample ions.
The sample with a strong charge will be attracted to the ionic surface, due to this attraction its retention time will increase and elute later. The mobile phase is an aqueous buffer, where both pH and ionic strength are used to control elution time.
(Learn) Types of HPLC Based on Elution technique:
Isocratic Elution:
A separation in which the composition of the mobile phase remains the same throughout the procedure. In isocratic elution, peak width increases due to high retention time which directly relates to the number of theoretical plates. This leads to the disadvantage that late eluting peaks become broad and flat.
Gradient Elution:
A separation in which the composition of the mobile phase is changed during the procedure. Gradient elution decreases the retention of later-eluting components and gives narrow peaks and also improves the peak shape and height.
Main parts of HPLC
Now it is the time to learn about some main parts of HPLC. The components of HPLC are following,
1. Solvent Reservoir
The mobile phase contained in a glass reservoir. In HPLC, the mobile phase is normally a mixture of polar and non-polar liquid components whose compositions are changed by changing the composition of the sample.
2. Pump
A pump sucks the mobile phase from the solvent reservoir and forces it to pass through the system’s column and detector. The pressure of the pump depends on a number of factors including column dimensions, the particle size of the stationary phase, the flow rate, and the composition of the mobile phase. Depending on these factors the operating pressures can be generated up to 42000 kPa (about 6000 psi).
3. Sample Injector
There are two types of injectors used for HPLC i.e. a single injection or an automated injection system. An injector for the HPLC system should provide an injection of the liquid sample within the range of 0.1-100 mL of volume with high reproducibility and under high pressure.
4. Columns
The column is known as the heart of the HPLC. Columns are usually made up of polished stainless steel polished internally to a mirror finish. They are of different lengths and internal diameters. They are commonly filled with a stationary phase (a solid or a liquid) with a particle size of 3–10 µm.
Types of columns:
Following are the types of columns
Standard analytical column: Its length is 10-30 cm and inter diameter is 4-5 mm
Microbore columns: Its length is 10-25 cm and inter diameter is 1-2 mm
Semi preparative: Its length is 10-25 cm and inter diameter is 7-10 mm
Preparative columns: Its length is 10-25 cm and inter diameter is 20-40 mm
5. Detector
The HPLC detector is present at the end of the column to detect the analytes as they elute from the chromatographic column.
Following are different detector used in HPLC.
Diode array detectors (DAD):
These are microprocessor-controlled photodiode array spectrophotometers in which the light from an ultraviolet source passes through the flow cell into a polychromator which disperses the bean so that the full spectrum falls on the array of diodes.
UV/VIS Absorption Detectors:
Different compounds absorb different amounts of light in the UV and visible regions. A beam of UV light is shined through the analyte after it is eluted from the column. A detector is on the opposite side measure how much light is absorbed and transmitted.
The Fluorescence Detector
Measure the ability of a compound to absorb then re-emit light at given wavelengths, some compounds will absorb specific wavelengths of light which raising it to a higher energy state. When the compound returns to its ground state, it will release a specific wavelength of light which can be detected.
Electrochemical Detectors (ECDs):
Used for compounds that undergo oxidation and reduction reactions. The detector measures the current produced as the result of oxidation and reduction reaction of the analyte at a suitable electrode. The current level is directly proportional to the concentration of the analyte present.
Conductivity Detector:
This detector records the mobile phase conductivity changes as different sample components are eluted from the column.
6. Data Collection Devices
Signals from the detector collected on chart recorders vary in their ability to process, store and reprocess chromatographic data. The computer integrates the response of the detector to each component and places it into a chromatograph that is easy to read.
Quality parameters of HPLC
Here, in this chapter, you are going to learn about some quality parameters of HPLC. The Quality parameters of HPLC are as following
Wavelength
Temperature of column
Flow rate
Run time
Pressure
Injection volume
Type of elution( isocratic or gradient)
But mind it these parameters are varied from compound to compound. For pregabalin parameters of HPLC have their own fixed values which are listed below
| Wavelength | UV 210 nm |
| Temperature | 30°C |
| Flow Rate | 1mL/min |
| Run Time | 1.3 times the retention time of pregabalin |
| Injection Volume | 20µL |
Trouble shooting of common problems
The best way to learn HPLC is to learn some basic trouble shooting. Some normally occurring errors are as follow with there solutions
- Abnormal pressure
No pressure reading, no flow
| Possible Cause | Solution |
| Power off | turn on power |
| Controller setting | verifies proper setting |
| Air trapped in pump head | Degas pump before running mobile phase |
| Faulty check valve | replace check valve |
| Major leak | Tighten the valve or replace fitting |
High back pressure
| Possible cause | Solution |
| Improper mobile phase | use a correct mobile phase |
| Improper column | use proper column |
| Injector blockage | remove blockage |
| Low column temperature | raises column temperature |
Low back pressure
| Flow set too slow | set flow rate |
| Column temperature too high | maintain column temperature |
| Leak in system | locate and correct the temperature |
| column | use proper temperature |
2. Column leak
| Loose end fitting | tighten end fitting |
| Column packing in the ferrule | rinse ferrule |
| Improper frit thickness | use proper frit |
3. Problems with the chromatogram
Many issues in the liquid chromatography system appear due to changes in the chromatogram. Some of these can be solved by changes in the instrument; however, other problems require modification of the assay procedure. Setting the proper column type, pre-column or guard column, tubing’s, detector cell and mobile phase are keys to good chromatography.
HPLC applications
HPLC gives information about the resolution, identification, and quantification of a compound. It is also used in chemical separation and purification. The other applications of HPLC include:
Pharmaceutical Applications
To control drug stability.
Tablet dissolution study of pharmaceutical dosages form.
Pharmaceutical quality control.
Environmental Applications
Detection of phenolic compounds in drinking water.
Bio-monitoring of pollutants.
Applications in Forensics
Quantification of drugs in biological samples.
Identification of steroids in blood, urine etc.
Forensic analysis of textile dyes.
Determination of cocaine and other drugs of abuse in blood, urine etc.
Food and Flavour
Measurement of Quality of soft drinks and water.
Sugar analysis in fruit juices.
Analysis of polycyclic compounds in vegetables.
Preservative analysis.
Applications in Clinical Tests
Urine analysis, antibiotics analysis in blood.
Used to isolation metabolites from biological fluids.
It is used to remove impurities.
It is used to determine drug estimation from drug formulations.
Advantages and disadvantages of HPLC
Advantages
The advantages of HPLC are as follows:
It can test both raw materials and finished products.
It is helpful in solving product failure problems.
It can detect contaminants and other impurities.
It can determine product stability and shelf life.
The testing can be done even with just a small sample size.
It enables you to modify the testing depending on the needed quantification level.
The results it produced are reliable.
It is helpful in developing better products.
Disadvantages
The disadvantages of HPLC are as follows:
Coelution
Coelution separation of two compounds with similar structures. Coelution makes the elution of the mixture difficult.
Adsorbed Compounds
Some compounds bind strongly that they are essentially never released from the beads in the column and are never measured in the solution.
Cost
Although HPLC is much more efficient but is much more costly than developing other assays for separating compounds. Small companies cannot afford this.
Complexity
This requires a highly skilled technician to monitor the column at all times and make sure that the process is running exactly as planned.
Famous Brands of HPLC
You will learn about the latest models of HPLC of famous brands. Following are the Famous brands of HPLC.
Most recent series of Shimadzu is New Nexera Series
| Sr. No. | Model | Features |
| 1 | Nexra XS/X3 | It is compatible with wide range of analysis conditions from conventional HPLC analysis to ultra-high separation analysis. |
| 2 | Nexra XR | This is UHPLC like model is the new standard for the shimadzu LC series. |
| 3 | Nexra Lite | This is conventional HPLC model. |
Latest model of Agilent HPLC is 1260 Infinity II LC System
Specifications of Agilent model are as follow
| Column Capacity | 4 |
| Depth | 468mm |
| Flow range | 0.05 to 5 mL/min |
| Injection range | 0.1-100 µL 0.1-900 µL with Extended Injection Range Option |
| Line voltage | 100-240 VAC |
| Pump type | Isocratic, Gradient |
| System pressure operating range | up to 600 bar |
| Width | 435 mm |
Latest models of waters HPLC are as follow with their features
| Sr. No. | Model | Features |
| 1 | Alliance HPLC system | The industry-standard HPLC platform with versatile, dependable performance, flexible system configurations. |
| 2 | Arc HPLC system | Count on the Arc HPLC System for high-efficiency separations |
| 3 | Breeze QS HPLC | With a choice of multiple components to build your own liquid chromatography system, the Breeze QS HPLC System delivers out standing . |
| 4 | Acquity Arc system | The ACQUITY Arc System is a quaternary-based, modern LC system for scientists working with established methods |
| 7 | Specialized LC systems | Portfolio of purpose-built solutions for specific applications and workflows. |
| 6 | Supercritical Fluid chromatography | Supercritical fluid chromatography (SFC) systems provide an environmentally-friendly approach to analytical and purification applications. |
Which brand HPLC you are using? Please comment in the section below. Do not forget to share the guide with your colleagues and friends.
Very informative. Good work.