HPLC
High Performance Liquid Chromatography
(Is an advance type of Liquid Chromatography)
(Is an advance type of Liquid Chromatography)
INTRODUCTION
It is a form of column chromatography used frequently in all field of Life science and Analytical Chemistry. HPLC is used to separate components of a mixture by using a variety of chemical interactions between the substance being analyzed and the chromatography column. We can analyzed the Vitamins, API, Enzymes, Photochemical, Toxins and Microbial toxin content with the help of HPLC.
2. Types of HPLC;-
Normal phase HPLC (NP-HPLC) was the first kind of HPLC chemistry used, and separates analytes based on polarity. This method uses a polar stationary phase and a non-polar mobile phase, and is used when the analyte of interest is fairly polar in nature. The polar analyte associates with and is retained by the polar stationary phase. Adsorption strengths increase with increase in analyte polarity, and the interaction between the polar analyte and the polar stationary phase (relative to the mobile phase) increases the elution time. The interaction strength not only depends on the functional groups in the analyte molecule, but also on steric factors and structural isomers are often resolved from one another. Use of more polar solvents in the mobile phase will decrease the retention time of the analytes while more hydrophobic solvents tend to increase retention times. Particularly polar solvents in a mixture tend to deactivate the column by occupying the stationary phase surface. This is somewhat particular to normal phase because it is most purely an adsorptive mechanism (the interactions are with a hard surface rather than a soft layer on a surface).
NP-HPLC had fallen out of favor in the 1970's with the development of reversed-phase HPLC because of a lack of reproducibility of retention times as water or protic organic solvents changed the hydration state of the silica or alumina chromatographic media. Recently it has become useful again with the development of HPLC bonded phases which utilize a partition mechanism which provides reproducibility.
2.2. Reversed phase chromatography:-
Reversed phase HPLC (RP-HPLC or RPC) consists of a non-polar stationary phase and an aqueous, moderately polar mobile phase. One common stationary phase is a silica which has been treated with RMe2SiCl, where R is a straight chain alkyl group such as C18H37 or C8H17. The retention time is therefore longer for molecules which are more non-polar in nature, allowing polar molecules to elute more readily. Retention Time (RT) is increased by the addition of polar solvent to the mobile phase and decreased by the addition of more hydrophobic solvent.
3. PART OF HPLC
Generally all the HPLC have five modules.
Solvent reservoir is the upper part of HPLC that contains all the solvents which is used during analysis. For the buffer solution must be used amber colored glass bottle. All the solvent bottles filled with solvents connected to tubes and Outlet of tubes connected with degasser (some models of HPLC have same module for degasser and Pump). The inlet of tubes assembled with filtration kits and have a specific Marking (Like A, B, C and D).
Solvent Arrangement:
· Always kept water in ‘’A’’ tube connected bottle because the Solvent parameter “A” is always default. If you don’t given the solvent % value in any solvent then automatically solvent tube “A” considered.
· In Solvent Tube “D” connected with amber colored bottle is used always for Buffer solution because Buffer solution may be Photo sensitive in nature.
· Generally we are using Organic solvents as a mobile phase Methanol and Acetonitrile due to convenient for identification of bottles we have denoted “B” for Methanol and “C” for Acetonitrile.
· All the bottles should be labeled with solvent or reagent name, date of preparation and prepared by.
3.2. Vacuum Degasser:
Vacuum degasser is used for the Degassing of solvents (We can used a separate vacuum degasser for the proper degassing of solvents). The outlet of solvent tubes connected with Vacuum degasser and the outlet of Vacuum degasser connected with the pump.
Degassing: Removal of air bubbles from the solvents that is called Degassing and for that purpose which tools used that is called Degasser.
3.3. Pump:
Pump is used for the constant flow of the solvents through column. Pumps vary in pressure capacity, but their performance is measured on their ability to yield a consistent and reproducible flow rate. Modern HPLC systems have been improved to work at much higher pressures, and therefore be able to use much smaller particle sizes in the columns (< 2 micrometres).
There are three types of pumps according to the application.
3.3.1. Isocratic pump:
Have a single port for the solvent Pumping.
3.3.2. Binary Pump:
Two port for solvent Pumping.
3.3.3. Quaternary pump:
Have four ports for solvent Pumping.
Solvent Flow system: Two type of Solvent flow system
3.3.4. Isocratic Flow: continues flow in same Solvent composition and same flow rate.
3.3.5. Gradient Flow: Solvent composition and Flow rate has been changed according to given (in method) flow rate, solvent composition followed by time.
3.4. Sample injection port (Automatic or Manual):
For optimum column efficiency, the sample should not be too large, and should be introduced onto the column as a "plug" of vapour - slow injection of large samples causes band broadening and loss of resolution. The most common injection method is where a microsyringe is used to inject sample through a rubber septum into a flash vapouriser port at the head of the column. The temperature of the sample port is usually about 50C higher than the boiling point of the least volatile component of the sample. For packed columns, sample size ranges from tenths of a microliter up to 20 microliters.
3.4.1. Manual Injector:
Manual injector used as sample insertion port in the Mobile phase flow. We have filled the syringe manually and injected in the injection port after that load the injection with the help of Injection loader toward clock wise direction.
3.4.2. Auto sampler: Auto sampler is a robotic tools used for the automatically injection of sample in the injector port. They have followed all the given information in the Method (Software of HPLC) like- Injection volume and positions of vials in tray.
3.5. Column Compartment:
Column compartment is used for the holding of column and provided the required temperature for the proper analysis. The temperature of Column compartment is given in the method of Analysis.
That may be constant of programmed according to sample. Generally we are used only constant temperature.
There are two general types of column, packed and capillary (also known as open tubular). Packed columns contain a finely divided, inert, solid support material (commonly based on diatomaceous earth) coated with liquid stationary phase. Most packed columns are 1.5 - 10m in length and have an internal diameter of 2 - 4mm. Capillary columns have an internal diameter of a few tenths of a millimeter. They can be one of two types; wall-coated open tubular (WCOT) or support-coated open tubular (SCOT). Wall-coated columns consist of a capillary tube whose walls are coated with liquid stationary phase. In support-coated columns, the inner wall of the capillary is lined with a thin layer of support material such as diatomaceous earth, onto which the stationary phase has been adsorbed. SCOT columns are generally less efficient than WCOT columns. Both types of capillary column are more efficient than packed columns.
In 1979, a new type of WCOT column was devised - the Fused Silica Open Tubular (FSOT) column;
These have much thinner walls than the glass capillary columns, and are given strength by the polyimide coating. These columns are flexible and can be wound into coils. They have the advantages of physical strength, flexibility and low reactivity.
Parameters of Columns:
The internal diameter (ID) of an HPLC column is a critical aspect that determines quantity of analyte that can be loaded onto the column and also influences sensitivity. Larger columns are usually seen in industrial applications such as the purification of a drug product for later use. Low ID columns have improved sensitivity and lower solvent consumption at the expense of loading capacity.
Most traditional HPLC is performed with the stationary phase attached to the outside of small spherical silica particles (very small beads). These particles come in a variety of sizes with 5μm beads being the most common. Smaller particles generally provide more surface area and better separations, but the pressure required for optimum linear velocity increases by the inverse of the particle diameter squared.
This means that changing to particles that are half as big, keeping the size of the column the same, will double the performance, but increase the required pressure by a factor of four. Larger particles are more often used in non-HPLC applications such as solid-phase extraction.
Many stationary phases are porous to provide greater surface area. Small pores provide greater surface area while larger pore size has better kinetics especially for larger analytes. For example a protein which is only slightly smaller than a pore might enter the pore but not easily leave once inside.
Column temperature
For precise work, column temperature must be controlled to within tenths of a degree. The optimum column temperature is dependent upon the boiling point of the sample. As a rule of thumb, a temperature slightly above the average boiling point of the sample results in an elution time of 2 - 30 minutes. Minimal temperatures give good resolution, but increase elution times. If a sample has a wide boiling range, then temperature programming can be useful. The column temperature is increased (either continuously or in steps) as separation proceeds.
3.6. Detector:
Absorbance is the logarithm of the ratio of the intensities of the incident light (Io) and the transmitted light (I). It is related according to the Beer-Lambert Law to the molar absorptivity (molar extinction coefficient, ), the thickness of the substance (i.e., the path length of the cell, b) and the molar concentration of the substance (c):
The ordinate of the chromatogram represents the detector signal, which in general, is proportional to the analyte concentration in the cell. Since chromatographic systems permit the quantitative analysis of sample components representing many orders of magnitude - from ppm to percent concentrations - one may select, various amplification ranges so that the visual display of components (both small and large).
Operation:-
The sample to be analyzed is introduced in small volume to the stream of mobile phase and is retarded by specific chemical or physical interactions with the stationary phase as it traverses the length of the column. The amount of retardation depends on the nature of the analyte, stationary phase and mobile phase composition. The time at which a specific analyte elutes (comes out of the end of the column) is called the retention time and is considered a reasonably unique identifying characteristic of a given analyte. The use of pressure increases the linear velocity (speed) giving the components less time to diffuse within the column, leading to improved resolution in the resulting chromatogram. Common solvents used include any miscible combinations of water or various organic liquids (the most common are methanol and acetonitrile). Water may contain buffers or salts to assist in the separation of the analyte components, or compounds such as Trifluoroacetic acid which acts as an ion pairing agent.
A further refinement to HPLC has been to vary the mobile phase composition during the analysis; this is known as gradient elution. A normal gradient for reversed phase chromatography might start at 5 % methanol and. progress linearly to 50 % methanol over 25 minutes, depending on how hydrophobic the analyte is. The gradient separates the analyte mixtures as a function of the affinity of the analyte for the current mobile phase composition relative to the stationary phase. This partitioning process is similar to that which occurs during a liquid-liquid extraction but is continuous, not step-wise. In this example, using a water/methanol gradient, the more hydrophobic components will elute (come off the column) under conditions of relatively high methanol; whereas the more hydrophilic compounds will elute under conditions of relatively low methanol. The choice of solvents, additives and gradient depend on the nature of the stationary phase and the analyte. Often a series of tests are performed on the analyte and a number of generic runs may be processed in order to find the optimum HPLC method for the analyte - the method which gives the best separation of peaks.
