What Is HPLC?
Known as HPLC, High-Performance Liquid Chromatography is a form of column chromatography that is a popular way of separating, identifying, and quantifying components dissolved in a liquid solvent with a high analytical resolution in the lab. HPLC means High-Performance Liquid Chromatography, which is also known as “High-pressure Liquid Chromatography”, “High-speed Liquid Chromatography”, “High separation Liquid Chromatography”, “modern column Chromatography”, etc.
HPLC is an important branch of chromatography, using the high-pressure infusion system to put the single solvents with different polarity or different portion of mixed solvents and mobile phases into a stationary phase chromatographic column, in which each component is separated and then entered the detector for testing, so as to realize the analysis of the sample.
When you use HPLC in applications, the sample retention time will be different due to the interaction between the stationary phase, the sample mixture which is being analyzed, and the solvents. Due to different polarities in the analytes, the sample will interact between the two phases at a different rate when it passes through the column. Analytes that have the least amount of interaction with the stationary phase or the most amount of interaction with the mobile phase will escape the column faster.
It can pump a sample mixture or analyte in a solvent, which is carried by a moving carrier gas stream of helium or nitrogen, going through a column with chromatographic packing material at high pressure. Because of its outstanding characteristics, HPLC is widely used in industrial and scientific applications.
This is normally shown as column length * inside diameter. For example: 150*4.6, 50 * 2.1, etc. Generally, 4.6 of diameter is used on the conventional detector and 2.1 is used with MS detector.
HPLC column characteristics
1. High pressure: The mobile phase is a liquid. When flowing through the column, the resistance is large. In order to pass the column quickly, high pressure must be applied to the carrier liquid.
2. High speed: fast analysis speed, fast liquid carrier flow rate, much faster than classical liquid chromatography. Usually, one sample is analyzed for 15 to 30 minutes, and some samples can be completed within 5 minutes, usually less than 1 hour.
3. High efficiency: high separation efficiency. The stationary phase and mobile phase can be chosen for excellent separation and many times higher separation efficiency than industrial rectification columns and gas chromatography.
4. High sensitivity: UV detector up to 0.01 ng, injection volume in the order of μL.
5. Wide range of applications: More than 70% of organic compounds can be analyzed by high performance liquid chromatography, especially for the separation and analysis of high boiling point, macromolecule, strong polarity, and poor thermal stability compounds, showing advantages.
6. Columns can be used repeatedly: separate columns can be used to separate different compounds
7. Samples are small and easy to recycle: the sample is not destroyed after passing through the column, and a single component can be collected or prepared.
8. High column quality recognition; imported chromatographic filler with high purity and goof uniformity
9. High column efficiency and high peak capacity.
10. Excellent batch stability.
11. Multiple types of chromatographic columns, there are many different types of C18 column and series.
Complete column specifications, particularly, X series column has various fillers, different varieties of column length and internal specifications, which can meet the requirements of the majority of analysis experiments.
In addition, high-performance liquid chromatography has the advantages that the column can be used repeatedly, the sample is not destroyed, and it is easy to recycle, but it also has disadvantages. Compared with gas chromatography, it has its own advantages and complements each other. A disadvantage of high performance liquid chromatography is the “extra-column effect”. Between the injection and the detector, in any dead space other than the column (injector, column fitting, connecting tube and detection cell, etc.). if the flow pattern of the mobile phase changes, any diffusion of the separated material and Retention will significantly lead to a broadening of the chromatographic peak and a decrease in column efficiency. High performance liquid chromatography detectors are less sensitive than gas chromatography.
Types of HPLC
- Adsorption Chromatography
- Partition Chromatography
- Ion Chromatography
- Size Exclusion Chromatography
- Bonded-phase chromatography
- Affinity Chromatography
There are 2 kinds of HAWACH HPLC columns:
Xcheoma series: with many types of fixed phase; with complete varieties and high quality. C18-Aqueous; ODS C18 HPLC Column;C18-low pH; C18 Alkaline HPLC Column; C8-universal; C8 Low pH HPLC Columns; C8-Fluorine; C4; C30; C1;Phenyl; PAH HPLC Column; etc
Echroma series: less column type, slightly cheaper. C18: 5um; C8: 5um.
Application of HPLC Column
(1)food nutrition analysis: proteins, amino acids, sugars, pigments, vitamins, spices, minerals, etc
(2)food additive analysis: sweeteners, preservatives, colorants, antioxidants, etc
(3)food contamination analysis: mycotoxin, trace elements, polycyclic aromatic diameter, etc
2.Environmental analysis: such as pesticide residue
3.Life Sciences: (1)essential for the study of life sciences, genetic engineering, clinical chemistry and molecular biology
(2) Low molecular weight substance: separation and determination of amino acids,organic acids, organic amines, steroids, porphyrins, carbohydrates, vitamins, etc
(3)High molecular weight substance: purification, isolation and determination of peptides, RNA, proteins and enzymes
4.(1)Medical examination: determination of metabolites in body fluids; Pharmacokinetic study; clinical drug testing.
(2)Synthetic drugs: antibiotics, anti-depressants, sulfonamides
(3)Natural medicine: alkaloids
5.Inorganic analysis: analysis of yang and anion
HPLC columns are prone to clogging and damage, so some laboratories would use a protective column in front of the column to extend the service life of the HPLC column.
The protection column generally includes: card sleeve+ column core + peek joint.
Regeneration of HPLC Column
It is recommended to flush in the direction of the arrow of the column, and try not to recoil as far as possible. The reference solvent sequence used for flushing is water, methanol, chloroform, isopropanol. And it is better to flush the column with a solvent equivalent to 20 times of the column volume in the following order.