What is HPLC?

• High Performance Liquid Chromatography

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• High Pressure Liquid Chromatography (usually true]

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• Hewlett Packard Liquid Chromatography

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• High Priced Liquid Chromatography

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• HPLC is really the automation of traditional liquid chromatography under conditions which provide for enhanced separations during shorter periods of time!

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• Probably the most widely practiced form of quantitative, analytical chromatography today due to the wide range of molecule types and sizes which can be separated using HPLC or variants of HPLC!!

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This highly efficient technique uses solvent under pressure to elute the column. Although generally used as an analytical system, larger capacity columns having good resolving power can be used in preparative systems.

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The general arrangement and operation bears more resemblance to gas chromatography, but HPLC is the more powerful analytical technique as it permits analysis of non-volatile and thermally labile compounds. However, development of the technique had to await the technology for preparing the specialized stationary phases which is so crucial for success.

Liquid Column Chromatography

A sample mixture is passed through a column packed with solid particles which may or may not be coated with another liquid.

With the proper solvents, packing conditions, some components in the sample will travel the column more slowly than others resulting in the desired separation.

Types of HPLC:

• Normal–phase chromatography.
• Reversed-phase chromatography.

There are a wide variety of stationary phases available for HPLC :

• Normal Phase.�- Polar stationary phase and non-polar solvent. e.g. silica gel

• Reverse Phase.

- Non-polar stationary phase and a polar solvent.

e.g. silica gel - C18

Stationary Phases:

• Polar (Normal Phase): Silica, alumina

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• Non-Polar (Reversed Phase): ODS OctaDecylSilyl (ODS groups or C18 groups) Silica gel, C₁₈, C₈

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Chromatography Stationary Phases

relatively polar surface

relatively nonpolar surface

Silica Gel

“normal phase”

“reversed phase”

Basic liquid chromatography modes are named according to the mechanism involved:

 1. Liquid/Solid Chromatography (adsorption chromatography)

A. Normal Phase LSC

B. Reverse Phase LSC

 2. Liquid/Liquid Chromatography (partition chromatography)

A. Normal Phase LLC

B. Reverse Phase LLC

 3. Ion Exchange Chromatography

 4. Gel Permeation Chromatography (exclusion chromatography)

Four Basic Liquid Chromatography

• Based on distribution of solute between a liquid mobile phase and a stationary phase.
• The small diameter particles are used as stationary phase support.

• The table shows relation between various

parameters of HPLC.

• Trendline:

• Stationary phase have small particulate size and

high surface areas.

• Columns: 20 cm or less
• Mobile phase pumped at high pressures of

400Bar, 6000 psi.

• Flow rates: 1-3 cm3 per min

Separation:

Separation:

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Separation:

Composition of the System of HPLC

• Solvent reservoirs
• Solvent Delivery System (Pump)
• Injector for introducing sample
• Column
• Detectors
• Waste Collector
• Recorder (Data Collection)

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HPLC

Solvent Reservoirs

Pump

Injector

Column

Detectors

The solvents used for HPLC must be highly pure, contain no solid matter and will require degassing immediately before use by sonification or by displacement of dissolved gases with helium.

Any formation of air bubbles within the system totally upsets the pressure maintenance.

Criteria of solvents for HPLC use

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DEGASSING: The practice of removing air from the mobile phase is called degassing which can be achieved by bubbling He (Helium) gas into the mobile phase and this degassing removes dissolved oxygen from the Mobile Phase.

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The presence of oxygen in mobile phase causes bubble formation resulting in air in the flow system and pump pressure will change causing spike in the chromatogram (due to air bubble formation in the detector cell).

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The Equipment of HPLC

HPLC Column: The column is a stainless steel tube packed with a special stationary phase consisting of extremely fine, regularly sized, spherical particles.

Owing to the small size of the particles, typically 5-10µm in diameter, any solvent contained in the matrix of each particle is close to the external solvent and equilibration can take place readily. This particular feature makes HPLC much more efficient than column chromatography using supports, in which the much larger particles have deep interstices (small opening between things) predominantly filled with stagnant, non-equilibrating solvent.

Guard (or pre-) Column :

They are placed between injector and

analytical columnIt

-The job of the guard column is to to remove or retain sample and solvent impurities that could be irreversibly adsorbed onto the analytical column ,thus prevent contamination of the analytical column which decrease its performance.

-A precolumn is used to protect the column against high-pH mobile phases

-They are very short columns about 3 cm , packed with material similar to that contained in the analytical column, for example C18.

Analytical column are typically 10-25 cm long and 5-10 mm internal diameter (id).

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Columns are made of stainless steel lined with glass to prevent metal interaction with solvent or solutes.

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Particle size from 3-20 μm. Particles ≤ 5 μm yield >10,000 theoretical plates/ meter.

Analytical Columns:

Recently high speed and high performance microcolumns with 1-4mm(id) and length

of 3 to 7.5 cm having the advantages of speed and minimal solvent consumption.

Picture of an HPLC column

Several column types based on the stationary phases�

• Normal phase column

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• Reverse phase column

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• Size exclusion column

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• Ion exchange column

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• Chiral column

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Normal phase

• In this column type, the retention is governed by the interaction of the polar parts of the stationary phase and solute.
• For retention to occur in normal phase, the packing material (stationary phase) must be more polar than the mobile phase with respect to the sample. Example of stationary phase: silica and Alumina

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Reverse phase

• In this column the packing material is relatively nonpolar and the solvent is polar with respect to the sample. Retention is the result of the interaction of the nonpolar components of the solutes and the nonpolar stationary phase.

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• Typical stationary phases are nonpolar hydrocarbons, waxy liquids, or bonded hydrocarbons (such as C18, C8, etc.) and the solvents are polar aqueous-organic mixtures such as methanol-water or acetonitrile-water.

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In reverse phase chromatography, a highly polar solvent system is used for elution. Under such conditions polar compounds prefer to stay in the mobile phase and are eluted before non-polar materials which have a greater affinity for the stationary phase-the reverse of usual adsorption systems (normal phase chromatography).

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Size exclusion

• In size exclusion the HPLC column is consisted of substances which have controlled pore sizes and is able to be filtered in an ordinary phase according to its molecular size.

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• Small molecules penetrate into the pores within the packing while larger molecules only partially penetrate the pores. The large molecules elute before the smaller molecules.

Ion exchange

• In this column type the sample components are separated based upon attractive ionic forces between molecules carrying charged groups of opposite charge to those charges on the stationary phase.

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• Separations are made between a polar mobile liquid, usually water containing salts or small amounts of alcohols, and a stationary phase containing either acidic or basic fixed sites.

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Chiral column

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Another very exciting development of the column uses stationary phases in which the surface has been covalently linked to a chiral material. Such chiral bonded phase supports permit the analytical separation of enantiomers, and the potential for such chiral columns seems boundless. These stationary phases are known as customized stationary phases which are really expensive.

HPLC Pumps

As a consequence of the highly packed nature of the stationary phase in HPLC columns, pressures of anything up to 7000 psi are necessary to force the eluting solvent along the column which is effectively performed in HPLC by using pump.

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The pump must be capable of maintaining a constant flow of solvent with no pressure surges and it must also be constructed of material which can withstand a wide range of organic solvents. The pump is also designed to permit the precise choice of a particular rate from a wide range of flow rates.

Constant Pressure Pumps

• They provide constant flow rate in the column thanks to a constant pressure. They use the low pressure of a gas contained in a cylinder to create the desired high pressure of the mobile phase.

Syringe Type Pumps

• They use a motorized syringe to get a constant solvent delivery. Changing the voltage of the motor, different flow rates can be obtained.

Reciprocating Piston Pumps

• They consist of a hydraulic chamber were a reciprocating piston is placed. This way, solvent gets in the pump when the piston moves back and is pushed into the column when the piston moves forward.

Elution may be with a single solvent or a mixture of solvents mixed at a certain ratio, when the process is described as isochratic elution, or the composition of the eluting system can be varied with time to permit gradient elution.

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The mixing of solvents for gradient elution may be carried out either at the low pressure side of the system, before the pump, or in the high pressure part. Both arrangements require microprocessor control.

Elution Process

Introduction of sample to the column

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Samples for analysis are introduced onto the column by injection through a multiport valve which permits precise, repeatable sample loading with minimal disturbance of solvent flow.

Basically, a solution of the sample is injected into an isolated, fixed volume loop of tubing which, on turning the valve, becomes part of the solvent delivery system.

Types of Injectors

• Rheodyne injector
• Septum injector
• Stop flow injector
• Auto-sampler

Valve-Type Injection in HPLC

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Widely used in HPLC. Allows reproducible introduction of sample into the pressurized mobile phase without interruption of flow even at high temperature.

Inject valve has two position: (a) Load (Fill) (b) Inject

Valve in the load position. Before injecting the sample the valve is turned to the load position.

Automated Injection

Sample is introduced from a vial held in a sample carousel using a syringe assembly. Valves are automatically actuated used to wash syringe needle and syringe assembly.

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Benefits of Auto injector

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Wide variability in injection volume (0.1 mL-2 mL). Precision is better or equal to manual injection. High sample throughput and less labor intensive.

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Chromatograms

If a detector that responds to solute concentration is placed at the end of the column and its signal (Response of the detector) is plotted as function of time (or of volume of the added mobile phase), a series of peaks is obtained. Such a plot, called a chromatogram, is useful for both qualitative and quantitative analysis. The positions of peaks on the time axis may serve to identify the components of the sample; the areas under the peaks provide a quantitative measure of the amount of each component.

APPLICATIONS OF CHROMATOGRAPHY

Chromatography has grown to be the premier method for separating closely related chemical species. In addition, it can be employed for qualitative identification and quantitative determination of separated species.

Qualitative Analysis

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A chromatogram provides only a single piece of qualitative information about each species in a sample, namely, its retention time or its position on the stationary phase after a certain elution period. It is a widely used tool for recognizing the presence or absence of components of mixtures containing a limited number of possible species whose identities are known. Positive spectroscopic identification would be impossible without a preliminary chromatographic separation on a complex sample.

Quantitative Analysis

Chromatography can provide useful quantitative information about the separated species. Quantitative column chromatography is based upon a comparison of either the height or the area of the analyte peak with that of one or more standards. For planar chromatography, the area covered by the separated species serves as the analytical parameter. If conditions are properly controlled, these parameters vary linearly with concentration.

Analyses Based on Peak Height

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The height of a chromatographic peak is obtained by connecting the base lines on either side of the peak by a straight line and measuring the perpendicular distance from this line to the peak. This measurement can ordinarily be made with reasonably high precision. Accurate results are obtained with peak heights only if variations in column conditions do not alter the peak widths during the period required to obtain chromatograms for sample and standards. The variables that must be controlled closely are column temperature, eluent flow rate, and rate of sample injection.

Analyses Based on Peak Areas

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Peak areas are a more satisfactory analytical variable than peak heights. On the other hand, peak heights are more easily measured and, for narrow peaks, more accurately determined. Most modern chromatographic instruments are equipped with digital electronic integrators that permit precise estimation of peak areas.

If such equipment is not available, manual estimate must be made. A simple method, which works well for symmetric peaks of reasonable widths, is to multiply the height of the peak by its width at one half the peak height.

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Peak area = Height of the peak x width at one-half of the peak height

Calibration and Standards

The most straightforward method for quantitative chromatographic analyses involves the preparation of a series of standard solutions that approximate the composition of the unknown. Chromatograms for the standards are then obtained and peak heights or areas are plotted as a function of concentration. A plot of the data should yield a straight line passing through the origin.

Methods for Describing Column Efficiency

A chromatographic column is made up of numerous discrete but contiguous narrow layers called theoretical plates. At each plate, equilibration of the solute between the mobile and stationary phase was assumed to take place. Movement of the solute down the column was then treated as a stepwise transfer of equilibrated mobile phase from one plate to the next.

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PARAMETERS USED IN HPLC

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• 1.Retention time
• 2.Retention volume
• 3.Seperation factor
• 4. Resolution
• 5. Height Equivalent to a Theoretical Plate (HETP)
• 6. Efficiency
• 7. Asymmetry factor

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• 1.Retention time:

Retention time is the difference in time between the point of injection and appearance of peak maxima. It is also defined as time required for 50% of a component to be eluted from a column. It is measured in minutes and seconds.

• 2.Retention volume:

Retention volume is the volume of carrier gas or liquid required to elute 50% of the component from the column. It is the product of retention time and flow rate.

Retention volume = Retention time × flow rate

• 3.Seperation factor:

Separation factor is the ratio of partition coefficient of the 2 components to be separated.

S=Ka/Kb

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Ka, Kb = Partition coefficients of a, b

If there is a more difference in partition coefficient between 2 compounds, the peaks are far apart and the separation factor is more. If the partition coefficient of 2 compounds are similar, then the peaks are closer and the separation factor is less.

• 4. Resolution:

Resolution is the measure of extent of separation of 2 components and the base line separation achieved.

Rs = 2 (Rt₁-Rt₂) / w₁ + w₂

• 5. Height Equivalent to a Theoretical Plate (HETP): Relationship between plate height and column variables

A theoretical plate is an imaginary or hypothetical unit of a column where distribution of solute between stationary phase and mobile phase has attained equilibrium. It can also be called as a functional unit of the column.

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A theoretical plate can be of any height, which describes the efficiency of separation. If HETP is less, the column is more efficient. If HETP is more, the column is less efficient.

HETP = length of the column / no. of theoretical plates

HETP is given by Van deemter equation:

HETP = A + B/u + Cu

Where A = Eddy diffusion term or multiple path diffusion which arises due to the packing of the column. This can be minimized by uniformity of packing.

B = Longitudinal diffusion term or molecular diffusion.

C = Effect of mass transfer.

u = flow rate or velocity of the mobile phase.

• 6. Efficiency:

Efficiency of a column is expressed by the theoretical plates.

n = 16 Rt²/ w²

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Where n = no of theoretical plates

Rt= retention time

w = peak width at base

Rt and w are measured in common units (cm or mm , min or sec ). No of theoretical plates is high, the column is said to be highly efficient. For GLC, a value of 600/meter is sufficient. But in HPLC, high values like 40,000 to 70,000/ meter are recommended.

7. Asymmetry factor:

• A chromatographic peak should be symmetrical about its centre and said to follow Gaussian distribution. But in practice due to some factors, the peak is not symmetrical and shows tailing or fronting.
• Fronting is due to saturation of stationary phase and can be avoided by using less quantity of sample. Tailing is due to more active adsorption sites and can be eliminated by support pretreatment.
• Asymmetry factor (0.95 to 1.05) can be calculated by AF = b/a (b, a calculated by 5% or 10% of the peak height).

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Causes of abnormal peaks in HPLC chromatogram

• Broad peaks occur due to the more conc. of sample, large injection volume, column deterioration.
• Ghost peaks occur due to the contamination of the column, compound from earlier injections.
• Negative peaks occur if mobile phase absorbance is larger than sample absorbance.
• Peak doubling occurs due to the co- elution of interfering compound, column over load, channeling in column.
• Base line spikes occur due to the air bubbles in the mobile phase and/or detector, column deterioration.

Identifying problem using a chromatogram

1.The above pattern occurs when the mobile phase is not suitable

I. Blank base(normal)

II. Noisy :

• occurs when there is contamination of mobile phase
• improper cleaning

• Improper mixing of mobile phase(90% of the time)

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• Problems with column(30% of the time)

After adjusting PH with buffer

Good resolution

• Low temperature
• Overloading of sample
• Flow rate
• Overfilling of column
• Insufficient mixing of mobile phase

6.High concentration of sample

6.High concentration of sample

RT=4.0

Occurs due to problems with mobile phase

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RT=3.

0

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RT=3.5

Due to problem in communication between detector and the software

Occurs when column lifetime is diminished

• Problem with injector
• Fluctuation in flow rate
• Error in detection

A=3010

A=3215

A=3516

ADVANTAGES OF HPLC

1. Separations are fast and efficient (high resolution power)
2. Continuous monitoring of the column effluent
3. It can be applied to the separation and analysis of very complex mixtures
4. Accurate quantitative measurements.
5. Repetitive and reproducible analysis using the same column.
6. Adsorption, partition, ion exchange and exclusion column separations are excellently made.

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7. HPLC is more versatile than GLC in some respects, because it has the advantage of not being restricted to volatile and thermally stable solute and the choice of mobile and stationary phases is much wider in HPLC.

8. Both aqueous and non aqueous samples can be analyzed with little or no sample pre treatment.

9. A variety of solvents and column packings are available, providing a high degree of selectivity for specific analyses.

10. It provides a means for determination of multiple components in a single analysis.

APPLICATIONS:

HPLC is one of the most widely applied analytical separation techniques.

Pharmaceutical:

Tablet dissolution of pharmaceutical dosages.

Shelf life determinations of pharmaceutical products.

Identification of counterfeit drug products.

Pharmaceutical quality control.

Environmental

Forensics

Clinical

Food and Flavor

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The Role of HPLC in Drug Analysis