(HPLC)

High Performance Liquid Chromatography (HPLC) was developed in the late 1960s and early 1970s. Today it is widely applied for separations and purifications in a variety of areas including pharmaceuticals, biotechnology, environmental, polymer and food industries. HPLC has over the past decade become the method of choice for the analysis of a wide variety of compounds. Its main advantage over GC is that the analytes do not have to be volatile, so macromolecules are suitable for HPLC analysis. HPLC is accomplished by injection of a small amount of liquid sample into a moving stream of liquid (called the mobile phase) that passes through a column packed with particles of stationary phase. Separation of a mixture into its components depends on different degrees of retention of each component in the column. The extent to which a component is retained in the column is determined by its partitioning between the liquid mobile phase and the stationary phase. In HPLC this partitioning is affected by the relative solute/stationary phase and solute/mobile phase interactions. Thus, unlike GC, changes in mobile phase composition can have an enormous impact on your separation. Since the compounds have different mobilities, they exit the column at different times; i.e., they have different retention times, tR. The retention time is the time between injection and detection. There are numerous detectors which can be used in liquid chromatography. It is a device that senses the presence of components different from the liquid mobile phase and converts that information to an electrical signal. For qualitative identification one must rely on matching retention times of known compounds with the retention times of components in the unknown mixture. It is important to remember that any changes in operating conditions will affect the retention time which will affect the accuracy of identification. Thus HPLC is most often used when one is performing a target compound analysis, where one has a good idea of the compounds present in a mixture so reference standards can be used for determining retention times. For a sample of largely unknown composition qualitative identification can be determined by liquid chromatography-mass spectrometry. A mass spectrum of any or all peaks in the chromatogram is compared with spectra contained in spectral libraries on the system's computer. It should be noted that LC-MS systems are very complex, expensive instruments which are not commonly found in an academic teaching environment. Quantitative analysis is often accomplished with HPLC. An automatic injector providing reproducible injection volumes is extremely beneficial and is standard on modern commercial systems. HPLCs are really rather simple. The apparatus consists of a mobile phase reservoir which is just a clean solvent jug, a solvent delivery system consisting of a pump for delivering precise, reproducible and constant amount of mobile phase, a sample inlet, the column, a detector with associated electronics, and some kind of interface to the outside world such as a computer. The pump which is used to deliver the mobile phase solvent at a uniform rate often operates at pressures ranging from 500 - 5000 p.s.i. These high pressures are needed because the stationary phase column packing consists of very small,, tightly packed particles. It takes a lot of pressure to push the mobile phase through this stationary phase at a reasonable flow rate. Why the small particles? The mobile phase mass transfer term in the van Deemter equation is dependent on both the square of the particle diameter of the stationary phase as well as the column diameter. Thus there is always a good reason to go to smaller stationary phase particles and smaller columns. Good separation of a given pair of compounds by HPLC depends on the choice of column (which has already been done for you) and on the efficiency of the overall system. The relative position of the various components in the sample on the chromatogram is affected by a solute-solvent type of interaction with the column substrate competing with a solute-solvent interaction with the mobile phase. Column efficiency is concerned with the broadening of an initially compact band of solutes as it passes through the column. The broadening is a result of column design and of column operating conditions. For samples with a broad range of retention times, it is often desirable to employ solvent programming, whereby the mobile phase composition is varied continuously or in steps as the separation proceeds. This is the answer, in liquid chromatography, to what is called The General Elution Problem. This is treated chromatographic separations in general. Basically, the analysis of mixtures of widely varying composition frequently leads to a very wide spread in retention times. The longer the retention time, the broader the peak, so for those components which take a long time to elute, detector sensitivity is diminished and analysis times can be very long. With solvent programming, successively eluted substances experience stronger solute-mobile phase interactions and so emerge from a column more rapidly than they would under conditions in which the solvent was not varied. So long as one does not experience peak overlap (i.e. resolution remains tolerable), solvent programming gives a superior separation. HPLC is just one type of liquid chromatography, meaning the mobile phase is a liquid. In this lab you will use what is called reversed phase HPLC.

Reversed phase HPLC is the most common type of HPLC. What reversed phase means is that the mobile phase is relatively polar, and the stationary phase is relatively non-polar. Thus non-polar compounds will be more retained (i.e. have longer retention times) than a polar compound. In normal phase HPLC, the mobile phase is relatively non-polar and the stationary phase is relatively polar. Other more general types of HPLC include partition, adsorption, ion-exchange, size-exclusion, and thin-layer chromatography.

Many HPLC detectors available for universal & selective detection

  1. Filter based UV-vis detector –Typically set at 254 nm usingthe most prominent band inHg spectrum – can also use 313,365, 334 nm and other lines as well.
  2. Variable wavelength detectors – use continuum source like (D2 or H2) & a monochromator, select any λ, less sensitive.
  3. PDA - D2 or H2 source, disperse & focus on diode array, get complete spectrum every 1 sec, powerful, expensive, less sensitive, lots of data generated.

Cell for UV-VISdetectorfor HPLC- Low vol

Diode Array Detector

Fluorescence detector – normallyfixed wavelength filter fluorometerexcitation filter & emission filtercan be changed for particular λ of interestgives selectivity based on:

- ability to exhibit fluorescence

- excitation wavelength

- emission wavelength

Variable λ monochromator basedfluorescence detectors also available. Filter based detectors usually more sensitive.

Refractive index detector (RI) -responds to nearly all solutes but has poor sensitivity – detectschanges in refractive index as samplepasses through as long as solute hasdifferent RI than solvent – analogous to TCD in GC.

Electrochemical Detection

• Amperometric – fix potential &measure current (i)

• Conductometric – measure conductivity

• Coulometric – fix potential & integrate i

• Voltammetric – vary potential & measure i

• Potentiometric – measure potential

Can use 2 or 3 electrode design with Pt orcarbon electrodes (glassy C or C paste)Electrochem. detector nearly universal.

Merits of the various detectors

Detector / Selectivity / Sensitivity / Merits
Optical detection / UV/UV-VIS detector / 2 / 3 / A wide variety of substances can be detected that absorb light from 190 to 900 nm. Sensitivity depends strongly on the component.
Diode array detector (DAD, PDA) / 2 / 3 / A wide variety of substances can be detected that absorb light from190 to 900 nm. Sensitivity depends strongly on the component. The spectrum can be confirmed for each component.
Fluorescence (FL) detector / 3 / 4 / Components emitting fluorescence can be detected selectively with high sensitivity. This is often used for pre-column and post-column derivatization.
Differential refractive index (RI) detector / 1 / 1 / Any component that differs in refractive index from the eluate can be detected, despite its low sensitivity. Cannot be used to perform gradient analysis.
Evaporative light scattering detector (ELSD) / 1 / 2 / This detector atomizes the column eluate, and detects the scattered light of the resulting particulate components. Non-UV-absorbing components are detected with high sensitivity.
Electrical detection / Conductivity detector (CD) / 2 / 3 / Ionized components are detected. This detector is used mainly for ion chromatography.
Electrochemical detector (ECD) / 3 / 4 / Electric currents are detected that are generated by electric oxidation-reduction reactions. Electrically active components are detected with high sensitivity.
Corona® Charged Aerosol Detector® (Corona® CAD®) / 1 / 3 / This detector atomizes the column eluate and electrically detects the resulting particulate components treated with corona discharge. UV-nonabsorbing components can be detected with sensitivity higher than that of ELSD.

General

  • Separation of organic, inorganic, biological compounds, polymers, and thermally labile compounds.
  • Qualitative and quantitative methods Common Specific Applications.
  • Quantitative/qualitiative analyses of amino acids, nucleic acids, proteins in physiological samples.
  • Measuring levels of active drugs, synthetic byproducts, degradation products in pharmaceuticals.
  • Measuring levels of hazardous compounds such as pesticides and insecticides.
  • Monitoring environmental samples.
  • Purifying compounds from mixtures Limitations.
  • Qualitative analysis may be limited unless HPLC is interfaced with mass spectrometry.
  • Resolution is limited with very complex samples Complementary or Related Techniques.
  • Gas chromatography provides analyses volatile analytes with superior resolution.
  • Supercritical fluid chromatography provides analyses of volatile, non-volatile and thermally labile compounds.
  • Capillary electrophoresis provides superior analyses in many biological/pharmaceutical applications.
  • Ion Chromatography provides analyses of ionic compounds, as does capillary electrophoresis.

APPLICATION

The information that can be obtained using HPLC includes identification, quantification, and resolution of a compound. Preparative HPLC refers to the process of isolation and purification of compounds. This differs from analytical HPLC, where the focus is to obtain information about the sample compound.

Chemical Separations It is based on the fact that certain compounds have different migration rates given a particular column and mobile phase, the extent or degree of separation is mostly determined by the choice of stationary phase and mobile phase.

Purification: Purification is defined as the process of separating or extracting the target compound from a mixture of compounds or contaminants. Each compound showed a characteristic peak under certain chromatographic conditions. The migration of the compounds and contaminants through the column need to differ enough so that the pure desired compound can be collected or extracted without incurring any other undesired compound.

Identification Generally assay of compounds are carried using HPLC. The parameters of this assay should be such that a clean peak of the known sample is observed from the chromatograph. The identifying peak should have a reasonable retention time and should be well separated from extraneous peaks at the detection levels which the assay will be performed.

Other applications of HPLC: Other applications of HPLC includes

Pharmaceutical applications:

• Tablet dissolution study of armaceutical dosages form.

• Shelf-life determinations of parmaceutical products

• Identification of active ingredients of dosage forms

• Pharmaceutical quality control

Environmental applications:

• Detection of phenolic compounds in Drinking Water

• Identification of diphenhydramine in sedimented samples

• Bio-monitoring of pollutant

Forensics:

• Quantification of the drug in biological samples.

• Identification of anabolic steroids in serum, urine, sweat, and hair

• Forensic analysis of textile dyes.

• Determination of cocaine and metabolites in blood

Clinical:

• Quantification of ions in human urine Analysis of antibiotics in blood plasma.

• Estimation of bilirubin and bilivirdin in blood plasma in case of hepatic disorders.

• Detection of endogenous neuropeptides in extracellular fluids of brain.

FoodandFlavor:

• Ensuring the quality of soft drink and drinking water.

• Analysis of beer.

• Sugar analysis in fruit juices.

• Analysis of polycyclic compounds in vegetables.

• Trace analysis of military high explosives in agricultural crops.

References:

  1. "Handbook of Instrumental Techniques for Analytical Chemistry" Frank Settle, Editor: "High Performance Liquid Chromatography", Phyllis Brown, Kathryn DeAntonois, Prentice Hall, 1997, pp. 147-164.
  2. "Principles of Instrumental Analysis", 5th Edition. Skoog, Holler, Nieman, Saunders College Publishing, 1998, pp. 673-697, 725-766.
  3. Bergh J. J., Breytenbach, J. C. Stability-indicating High-performance Liquid- chromatographic Analysis of Trimethoprim in Pharmaceuticals. J. Chromatogr. 1987; 387: 528-531.
  4. Stubbs C., Kanfer, I. Stability-indi- cating High-performance Liquid-chromato- graphic Assay of Erythromycin Estolate in Pharmaceutical Dosage Forms. Int. J. Pharm. 1990; 3(2): 113-119.
  5. MacNeil L., Rice J. J., Muhammad N. Lauback R. G. Stability-indicating Liquid-chromatographic Determination of Cefapirin, Desacetylcefapirin and Cefapirin Lactone in Sodium Cefapirin Bulk and Injectable Formulations. J. Chromatogr. 1986; 361: 285-290.
  6. Bounine J. P., Tardif B., Beltran P. Mazzo D. J. High-performance Liquid-© 2009, JGPT. All Rights Reserved. 25 Bansal V. et al., Journal of Global Pharma Technology. 2010; 2(5): 22-26.
  7. chromatographic Stability-indicating Determination of Zopiclone in Tablets. J. Chromatogr. 1994; 677(1): 87-93.
  8. Lauback R. G., Rice J. J., Bleiberg B., Muhammad N., Hanna, S. A. 1984. Specific High-performance Liquid-chromato- graphic Determination of Ampicillin in Bulks, Injectables, Capsules and Oral Suspensions by Reversed-phase Ion-pair Chromatography. J. Liq. Chromatogr. 1984; 7(6): 1243-1265.
  9. Wiklund A E., Dag B., Brita S. Toxicity evaluation by using intact sediments and sediment extracts. Marine Pollution Bulletin (2005); 50(6): 660-667.
  10. Kwok Y. C., Hsieh D. P. H., Wong P. K. Toxicity identification evaluation (TIE) of pore water of contaminated marine sediments collected from Hong Kong waters. Marine Pollution Bulletin. 2005; 51(8-12): 1085-1091.
  11. Hongxia Yu., Jing C., Cui Y., Shang H., Ding Z., Jin H. Application of toxicity identification evaluation procedures on wastewaters and sludge from a municipal sewage treatment works with industrial inputs. Ecotoxicology and Environmental Safety. 2004; 57(3): 426-430.
  12. Ayerton J. Assay of ceftazidime in biological fluids using high-pressure liquid chromatography. J. Antimicrob. Chemother. 1981; 8: 227-231.
  13. Bowden R.E., Madsen P.O. High- pressure liquid chromatographic assay of sulbactam in plasma, urine and tissue. Antimicrob. Agents Chemother. 1986; 30: 31-233.
  14. Haginaka J., Yasuda H., Uno T., Nkagawa T. Alkaline degradation and determination by high-performance by high-performance liquid chromatography. Chem. Pharm. Bull. 1984; 32: 2752-2758
  15. Fredj G., Paillet Aussel M. F., Brouard A., Barreteau H., Divine C., Micaud M. Determination of sulbactam in biological fluids by high-performance liquid chromatography. J. Chromatogr. 1986; 383: 218-222.
  16. Rodenas V., Garcia M.S., Sanchez-Pedreno C., Albero M.I. Flow-injection spectrophotometric determination of frusemide or sulphathiazole in pharmaceuticals. J. Pharm. Biomed. Anal. 1997; 15: 1687-1693.
  17. Shah A.J., Adlard M.W., Stride J.D. A sensitive assay for clavulanic acid and sulbactam in biological fluids by high-performance liquid chromatography and precolumn derivatization. J. Pharm. Biomed. Anal. 1990; 5: 437-443.
  18. Abidi S.L. High-performance liquid chromatography of phosphatidic acids and related polar lipids. J.Chromatogr. 1991; 587: 193-203.
  19. Christie W.W., Gill S., Nordbäck J., Itabashi Y., Sanda S., Slabas A.R. New procedures for rapid screening of leaf lipid components from Arabidopsis. Phytochemical Anal. 1998; 9: 53-57.
  20. "Introduction to Modern Liquid Chromatography" by L.R. Snyder and J.J. Kirkland, 2nd Edition. John Wiley & Sons, 1979. LC-GC Magazine. Advanstar Communications.

HPLC Links

A general treatment of chromatography including an overview of plate and rate theory.

Tutorials on molecular spectroscopy and chromatography.