HPLC - High Performance Liquid Chromatography What Is HPLC (High Performance Liquid Chromatography)?

Brief History and Definition

Liquid chromatography was defined in the early 1900s by the work of the Russian botanist, Mikhail S. Tswett. His pioneering studies focused on separating compounds [leaf pigments], extracted from plants using a solvent, in a column packed with particles. Tswett filled an open glass column with particles. Two specific materials that he found useful were powdered chalk [calcium carbonate] and alumina. He poured his sample [solvent extract of homogenized plant leaves] into the column and allowed it to pass into the particle bed. This was followed by pure solvent. As the sample passed down through the column by gravity, different colored bands could be seen separating because some components were moving faster than others. He related these separated, different-colored bands to the different compounds that were originally contained in the sample. He had created an analytical separation of these compounds based on the differing strength of each compounds chemical attraction to the particles. The compounds that were more strongly attracted to the particles slowed down, while other compounds more strongly attracted to the solvent moved faster. This process can be described as follows: the compounds contained in the sample distribute, or partition differently between the moving solvent, called the mobile phase, and the particles, called the stationary phase. This causes each compound to move at a different speed, thus creating a separation of the compounds. Tswett coined the name chromatography [from the Greek words chroma, meaning color, and graph, meaning writingliterally, color writing] to describe his colorful experiment. [Curiously, the Russian name Tswett means color.] Today, liquid chromatography, in its various forms, has become one of the most powerful tools in analytical chemistry.

Figure A: Tswett's Experiment

Liquid Chromatography (LC) Techniques

Liquid chromatography can be performed using planar [Techniques 1 and 2] or column techniques [Technique 3]. Column liquid chromatography is the most powerful and has the highest capacity for sample. In all cases, the sample first must be dissolved in a liquid that is then transported either onto, or into, the chromatographic device. Technique 1. The sample is spotted onto, and then flows through, a thin layer of chromatographic particles [stationary phase] fixed onto the surface of a glass plate [Figure B]. The bottom edge of the plate is placed in a solvent. Flow is created by capillary action as the solvent [mobile phase] diffuses into the dry particle layer and moves up the glass plate. This technique is called thin-layer chromatography or TLC.

Figure B: Thin-layer Chromatography

Note that the black sample is a mixture of FD&C yellow, red and blue food dyes that has been chromatographically separated. Technique 2. In Figure C, samples are spotted onto paper [stationary phase]. Solvent [mobile phase] is then added to the center of the spot to create an outward radial flow. This is a form of paper chromatography. [Classic paper chromatography is performed in a manner similar to that of TLC with linear flow.] In the upper image, the same black FD&C dye sample is applied to the paper.

Figure C: Paper Chromatography

Notice the difference in separation power for this particular paper when compared to the TLC plate. The green ring indicates that the paper cannot separate the yellow and blue dyes from each other, but it could separate those dyes from the red dyes. In the bottom image, a green sample, made up of the same yellow and blue dyes, is applied to the paper. As you would predict, the paper cannot separate the two dyes. In the middle, a purple sample, made up of red and blue dyes, was applied to the paper. They are well separated. Technique 3. In this, the most powerful approach, the sample passes through a column or a cartridge device containing appropriate particles [stationary phase]. These particles are called the chromatographic packing material. Solvent [mobile phase] flows through the device. In solid-phase extraction [SPE], the sample is loaded onto the cartridge and the solvent stream carries the sample through the device. As in Tswetts experiment, the compounds in the sample are then separated by traveling at different individual speeds through the device. Here the black sample is loaded onto a cartridge. Different solvents are used in each step to create the separation.

Figure D-1: Column Chromatography Solid-Phase Extraction [SPE]

When the cartridge format is utilized, there are several ways to achieve flow. Gravity or vacuum can be used for columns that are not designed to withstand pressure. Typically, the particles in this case are larger in diameter [> 50 microns] so that there is less resistance to flow. Open glass columns [Tswetts experiment] are an example of this. In addition, small plastic columns, typically in the shape of syringe barrels, can be filled with packing-material particles and used to perform sample preparation. This is called solid-phase extraction [SPE]. Here, the chromatographic device, called a cartridge, is used, usually with vacuum-assisted flow, to clean up a very complex sample before it is analyzed further. Smaller particle sizes [<10 microns] are required to improve separation power. However, smaller particles have greater resistance to flow, so higher pressures are needed to create the desired solvent flow rate. Pumps and columns designed to withstand high pressure are necessary. When moderate to high pressure is used to flow the solvent through the chromatographic column, the technique is called HPLC.

What Is High Performance Liquid Chromatography (HPLC)?

The acronym HPLC, coined by the late Prof. Csaba Horvth for his 1970 Pittcon paper, originally indicated the fact that high pressure was used to generate the flow required for liquid chromatography in packed columns. In the beginning, pumps only had a pressure capability of 500 psi [35 bar]. This was called high pressure liquid chromatography, or HPLC. The early 1970s saw a tremendous leap in technology. These new HPLC instruments could develop up to 6,000 psi [400 bar] of pressure, and

incorporated improved injectors, detectors, and columns. HPLC really began to take hold in the mid-to late-1970s. With continued advances in performance during this time [smaller particles, even higher pressure], the acronym HPLC remained the same, but the name was changed to high performance liquid chromatography. High performance liquid chromatography is now one of the most powerful tools in analytical chemistry. It has the ability to separate, identify, and quantitate the compounds that are present in any sample that can be dissolved in a liquid. Today, compounds in trace concentrations as low as parts per trillion [ppt] may easily be identified. HPLC can be, and has been, applied to just about any sample, such as pharmaceuticals, food, nutraceuticals, cosmetics, environmental matrices, forensic samples, and industrial chemicals.

Figure D-2: HPLC Column

What Is UltraPerformance Liquid Chromatography (UPLC Technology)?

In 2004, further advances in instrumentation and column technology were made to achieve very significant increases in resolution, speed, and sensitivity in liquid chromatography. Columns with smaller particles [1.7 micron] and instrumentation with specialized capabilities designed to deliver mobile phase at 15,000 psi [1,000 bar] were needed to achieve a new level of performance. A new system had to be holistically created to perform ultra-performance liquid chromatography, now known as UPLC technology. Basic research is being conducted today by scientists working with columns containing even smaller 1micron-diameter particles and instrumentation capable of performing at 100,000 psi [6,800 bar]. This provides a glimpse of what we may expect in the future.

How Does High Performance Liquid Chromatography Work?

The components of a basic high-performance liquid chromatography [HPLC] system are shown in the simple diagram in Figure E. A reservoir holds the solvent [called the mobile phase, because it moves]. A high-pressure pump [solvent delivery system or solvent manager] is used to generate and meter a specified flow rate of mobile phase, typically milliliters per minute. An injector [sample manager or autosampler] is able to introduce [inject] the sample into the continuously flowing mobile phase stream that carries the sample into the HPLC column. The column contains the chromatographic packing material needed to effect the separation. This packing material is called the stationary phase because it is held in place by the column hardware. A detector is needed to see the separated compound bands as they elute from the HPLC column [most compounds have no color, so we cannot see them with our eyes]. The mobile

phase exits the detector and can be sent to waste, or collected, as desired. When the mobile phase contains a separated compound band, HPLC provides the ability to collect this fraction of the eluate containing that purified compound for further study. This is called preparative chromatography [discussed in the section on HPLC Scale]. Note that high-pressure tubing and fittings are used to interconnect the pump, injector, column, and detector components to form the conduit for the mobile phase, sample, and separated compound bands.

Figure E: High-Performance Liquid Chromatography [HPLC] System

The detector is wired to the computer data station, the HPLC system component that records the electrical signal needed to generate the chromatogram on its display and to identify and quantitate the concentration of the sample constituents (see Figure F). Since sample compound characteristics can be very different, several types of detectors have been developed. For example, if a compound can absorb ultraviolet light, a UV-absorbance detector is used. If the compound fluoresces, a fluorescence detector is used. If the compound does not have either of these characteristics, a more universal type of detector is used, such as an evaporative-light-scattering detector [ELSD]. The most powerful approach is the use multiple detectors in series. For example, a UV and/or ELSD detector may be used in combination with a mass spectrometer [MS] to analyze the results of the chromatographic separation. This provides, from a single injection, more comprehensive information about an analyte. The practice of coupling a mass spectrometer to an HPLC system is called LC/MS.

Figure F: A Typical HPLC [Waters Alliance] System

HPLC Operation A simple way to understand how we achieve the separation of the compounds contained in a sample is to view the diagram in Figure G. Mobile phase enters the column from the left, passes through the particle bed, and exits at the right. Flow direction is represented by green arrows. First, consider the top image; it represents the column at time zero [the moment of injection], when the sample enters the column and begins to form a band. The sample shown here, a mixture of yellow, red, and blue dyes, appears at the inlet of the column as a single black band. [In reality, this sample could be anything that can be dissolved in a solvent; typically the compounds would be colorless and the column wall opaque, so we would need a detector to see the separated compounds as they elute.] After a few minutes [lower image], during which mobile phase flows continuously and steadily past the packing material particles, we can see that the individual dyes have moved in separate bands at different speeds. This is because there is a competition between the mobile phase and the stationary phase for attracting each of the dyes or analytes. Notice that the yellow dye band moves the fastest and is about to exit the column. The yellow dye likes [is attracted to] the mobile phase more than the other dyes. Therefore, it moves at a faster speed, closer to that of the mobile phase. The blue dye band likes the packing material more than the mobile phase. Its stronger attraction to the particles causes it to move significantly slower. In other words, it is the most retained compound in this sample mixture. The red dye band has an intermediate attraction for the mobile phase and therefore moves at

an intermediate speed through the column. Since each dye band moves at different speed, we are able to separate it chromatographically.

Figure G: Understanding How a Chromatographic Column Works Bands

What Is a Detector? As the separated dye bands leave the column, they pass immediately into the detector. The detector contains a flow cell that sees [detects] each separated compound band against a background of mobile phase [see Figure H]. [In reality, solutions of many compounds at typical HPLC analytical concentrations are colorless.] An appropriate detector has the ability to sense the presence of a compound and send its corresponding electrical signal to a computer data station. A choice is made among many different types of detectors, depending upon the characteristics and concentrations of the compounds that need to be separated and analyzed, as discussed earlier. What Is a Chromatogram? A chromatogram is a representation of the separation that has chemically [chromatographically] occurred in the HPLC system. A series of peaks rising from a baseline is drawn on a time axis. Each peak represents the detector response for a different compound. The chromatogram is plotted by the computer data station [see Figure H].

Figure H: How Peaks Are Created

In Figure H, the yellow band has completely passed through the detector flow cell; the electrical signal generated has been sent to the computer data station. The resulting chromatogram has begun to appear on screen. Note that the chromatogram begins when the sample was first injected and starts as a straight line set near the bottom of the screen. This is called the baseline; it represents pure mobile phase passing through the flow cell over time. As the yellow analyte band passes through the flow cell, a stronger signal is sent to the computer. The line curves, first upward, and then downward, in proportion to the concentration of the yellow dye in the sample band. This creates a peak in the chromatogram. After the yellow band passes completely out of the detector cell, the signal level returns to the baseline; the flow cell now has, once again, only pure mobile phase in it. Since the yellow band moves fastest, eluting first from the column, it is the first peak drawn. A little while later, the red band reaches the flow cell. The signal rises up from the baseline as the red band first enters the cell, and the peak representing the red band begins to be drawn. In this diagram, the red band has not fully passed through the flow cell. The diagram shows what the red band and red peak would look like if we stopped the process at this moment. Since most of the red band has passed through the cell, most of the peak has been drawn, as shown by the solid line. If we could restart, the red band would completely pass through the flow cell and the red peak would be completed [dotted line]. The blue band, the most strongly retained, travels at the slowest rate and elutes after the red band. The dotted line shows you how the completed chromatogram would appear if we had let the run continue to its conclusion. It is interesting to note that the width of the blue peak will be the broadest because the width of the blue analyte band, while narrowest on the column, becomes the widest as it elutes from the column. This is because it moves more slowly through the chromatographic packing material bed and requires more time [and mobile phase volume] to be eluted completely. Since mobile phase is continuously flowing at a fixed rate, this means that the blue band widens and is more dilute. Since the detector responds in proportion to the concentration of the band, the blue peak is lower in height, but larger in width

Identifying and Quantitating Compounds

In Figure H, three dye compounds are represented by three peaks separated in time in the chromatogram. Each elutes at a specific location, measured by the elapsed time between the moment of injection [time zero] and the time when the peak maximum elutes. By comparing each peaks retention time [tR] with that of injected reference standards in the same chromatographic system [same mobile and stationary phase], a chromatographer may be able to identify each compound.

Figure I-1: Identification

In the chromatogram shown in Figure I-1, the chromatographer knew that, under these LC system conditions, the analyte, acrylamide, would be separated and elute from the column at 2.85 minutes [retention time]. Whenever a new sample, which happened to contain acrylamide, was injected into the LC system under the same conditions, a peak would be present at 2.85 minutes [see Sample B in Figure I-2]. [For a better understanding of why some compounds move more slowly [are better retained] than others, please review the HPLC Separation Modes section on page 28]. Once identity is established, the next piece of important information is how much of each compound was present in the sample. The chromatogram and the related data from the detector help us calculate the concentration of each compound. The detector basically responds to the concentration of the compound band as it passes through the flow cell. The more concentrated it is, the stronger the signal; this is seen as a greater peak height above the baseline.

Figure I-2: Identification and Quantitation

In Figure I-2, chromatograms for Samples A and B, on the same time scale, are stacked one above the other. The same volume of sample was injected in both runs. Both chromatograms display a peak at a retention time [tR] of 2.85 minutes, indicating that each sample contains acrylamide. However, Sample A displays a much bigger peak for acrylamide. The area under a peak [peak area count] is a measure of the concentration of the compound it represents. This area value is integrated and calculated automatically by the computer data station. In this example, the peak for acrylamide in Sample A has 10 times the area of that for Sample B. Using reference standards, it can be determined that Sample A contains 10 picograms of acrylamide, which is ten times the amount in Sample B [1 picogram]. Note there is another peak [not identified] that elutes at 1.8 minutes in both samples. Since the area counts for this peak in both samples are about the same, this unknown compound may have the same concentration in both samples. Isocratic and Gradient LC System Operation Two basic elution modes are used in HPLC. The first is called isocratic elution. In this mode, the mobile phase, either a pure solvent or a mixture, remains the same throughout the run. A typical system is outlined in Figure J-1.

Figure J-1: Isocratic LC System

The second type is called gradient elution, wherein, as its name implies, the mobile phase composition changes during the separation. This mode is useful for samples that contain compounds that span a wide range of chromatographic polarity [see section on HPLC Separation Modes]. As the separation proceeds, the elution strength of the mobile phase is increased to elute the more strongly retained sample components.

Figure J-2: High-Pressure-Gradient System

In the simplest case, shown in Figure J-2, there are two bottles of solvents and two pumps. The speed of each pump is managed by the gradient controller to deliver more or less of each solvent over the course of the separation. The two streams are combined in the mixer to create the actual mobile phase composition that is delivered to the column over time. At the beginning, the mobile phase contains a higher proportion of the weaker solvent [Solvent A]. Over time, the proportion of the stronger solvent [Solvent B] is increased, according to a predetermined timetable. Note that in Figure J-2, the mixer is downstream of the pumps; thus the gradient is created under high pressure. Other HPLC systems are designed to mix multiple streams of solvents under low pressure, ahead of a single pump. A gradient proportioning valve selects from the four solvent bottles, changing the strength of the mobile phase over time [see Figure J-3].

Figure J-3: Low-Pressure-Gradient System

HPLC Scale [Analytical, Preparative, and Process] We have discussed how HPLC provides analytical data that can be used both to identify and to quantify compounds present in a sample. However, HPLC can also be used to purify and collect desired amounts of each compound, using a fraction collector downstream of the detector flow cell. This process is called preparative chromatography [see Figure K]. In preparative chromatography, the scientist is able to collect the individual analytes as they elute from the column [e.g., in this example: yellow, then red, then blue].

Figure K: HPLC System for Purification: Preparative Chromatography

The fraction collector selectively collects the eluate that now contains a purified analyte, for a specified length of time. The vessels are moved so that each collects only a single analyte peak. A scientist determines goals for purity level and amount. Coupled with knowledge of the complexity of the sample and the nature and concentration of the desired analytes relative to that of the matrix constituents, these goals, in turn, determine the amount of sample that needs to be processed and the required capacity of the HPLC system. In general, as the sample size increases, the size of the HPLC column will become larger and the pump will need higher volume-flow-rate capacity. Determining the capacity of an HPLC system is called selecting the HPLC scale. Table A lists various HPLC scales and their chromatographic objectives.

Table A: Chromatography Scale

The ability to maximize selectivity with a specific combination of HPLC stationary and mobile phases achieving the largest possible separation between two sample components of interestis critical in determining the requirements for scaling up a separation [see discussion on HPLC Separation Modes]. Capacity then becomes a matter of scaling the column volume [Vc] to the amount of sample to be

injected and choosing an appropriate particle size [determines pressure and efficiency; see discussion of Separation Power]. Column volume, a function of bed length [L] and internal diameter [i.d.], determines the amount of packing material [particles] that can be contained (see Figure L).

Figure L: HPLC Column Dimensions

In general, HPLC columns range from 20 mm to 500 mm in length [L] and 1 mm to 100 mm in internal diameter [i.d.]. As the scale of chromatography increases, so do column dimensions, especially the cross-sectional area. To optimize throughput, mobile phase flow rates must increase in proportion to cross-sectional area. If a smaller particle size is desirable for more separation power, pumps must then be designed to sustain higher mobile-phase-volume flow rates at high backpressure. Table B presents some simple guidelines on selecting the column i.d. and particle size range recommended for each scale of chromatography. For example, a semi-preparative-scale application [red X] would use a column with an internal diameter of 1040 mm containing 515 micron particles. Column length could then be calculated based on how much purified compound needs to be processed during each run and on how much separation power is required.

Table B: Chromatography Scale vs. Column Diameter and Particle Size

HPLC Column Hardware

A column tube and fittings must contain the chromatographic packing material [stationary phase] that is used to effect a separation. It must withstand backpressure created both during manufacture and in use. Also, it must provide a well-controlled [leak-free, minimum-volume, and zero-dead-volume] flow path for the sample at its inlet, and analyte bands at its outlet, and be chemically inert relative to the separation system [sample, mobile, and stationary phases]. Most columns are constructed of stainless steel for highest pressure resistance. PEEK [an engineered plastic] and glass, while less pressure tolerant, may be used when inert surfaces are required for special chemical or biological applications. [Figure M-1].

Figure M-1: Column Hardware Examples

A glass column wall offers a visual advantage. In the photo in Figure M-2, flow has been stopped while the sample bands are still in the column. You can see that the three dyes in the injected sample mixture have already separated in the bed; the yellow analyte, traveling fastest, is just about to exit the column.

Figure M-2: A Look Inside a Column

Separation Performance Resolution The degree to which two compounds are separated is called chromatographic resolution [RS]. Two principal factors that determine the overall separation power or resolution that can be achieved by an HPLC column are: mechanical separation power, created by the column length, particle size, and packed-bed uniformity, and chemical separation power, created by the physicochemical competition for compounds between the packing material and the mobile phase. Efficiency is a measure of mechanical separation power, while selectivity is a measure of chemical separation power. Mechanical Separation Power Efficiency If a column bed is stable and uniformly packed, its mechanical separation power is determined by the column length and the particle size. Mechanical separation power, also called efficiency, is often measured and compared by a plate number [symbol = N]. Smaller-particle chromatographic beds have higher efficiency and higher backpressure. For a given particle size, more mechanical separation power is gained by increasing column length. However, the trade-offs are longer chromatographic run times, greater solvent consumption, and higher backpressure. Shorter column lengths minimize all these variables but also reduce mechanical separation power, as shown in Figure N.

Figure N: Column Length and Mechanical Separating Power [Same Particle Size]

Figure O: Particle Size and Mechanical Separating Power [Same Column Length]

For a given particle chemistry, mobile phase, and flow rate, as shown in Figure O, a column of the same length and i.d., but with a smaller particle size, will deliver more mechanical separation power in the same time. However, its backpressure will be much higher. Chemical Separation Power Selectivity The choice of a combination of particle chemistry [stationary phase] and mobile-phase composition the separation systemwill determine the degree of chemical separation power [how we change the speed of each analyte]. Optimizing selectivity is the most powerful means of creating a separation; this may obviate the need for the brute force of the highest possible mechanical efficiency. To create a separation of any two specified compounds, a scientist may choose among a multiplicity of phase combinations [stationary phase and mobile phase] and retention mechanisms [modes of chromatography]. These are discussed in the next section.

HPLC Separation Modes

In general, three primary characteristics of chemical compounds can be used to create HPLC separations. They are: Polarity Electrical Charge Molecular Size First, lets consider polarity and the two primary separation modes that exploit this characteristic: normal phase and reversed-phase chromatography. Separations Based on Polarity A molecules structure, activity, and physicochemical characteristics are determined by the arrangement of its constituent atoms and the bonds between them. Within a molecule, a specific arrangement of certain atoms that is responsible for special properties and predictable chemical

reactions is called a functional group. This structure often determines whether the molecule is polar or non-polar. Organic molecules are sorted into classes according to the principal functional group(s) each contains. Using a separation mode based on polarity, the relative chromatographic retention of different kinds of molecules is largely determined by the nature and location of these functional groups. As shown in Figure P, classes of molecules can be ordered by their relative retention into a range or spectrum of chromatographic polarity from highly polar to highly non-polar.

Figure P: Chromatographic Polarity Spectrum by Analyte Functional Group

Water [a small molecule with a high dipole moment] is a polar compound. Benzene [an aromatic hydrocarbon] is a non-polar compound. Molecules with similar chromatographic polarity tend to be attracted to each other; those with dissimilar polarity exhibit much weaker attraction, if any, and may even repel one another. This becomes the basis for chromatographic separation modes based on polarity. Another way to think of this is by the familiar analogy: oil [non-polar] and water [polar] dont mix. Unlike in magnetism where opposite poles attract each other, chromatographic separations based on polarity depend upon the stronger attraction between likes and the weaker attraction between opposites. Remember, Like attracts like in polarity-based chromatography.

Figure Q: Proper Combination of Mobile and Stationary Phases Effects Separation Based on Polarity

To design a chromatographic separation system [see Figure Q], we create competition for the various compounds contained in the sample by choosing a mobile phase and a stationary phase with different polarities. Then, compounds in the sample that are similar in polarity to the stationary phase [column packing material] will be delayed because they are more strongly attracted to the particles. Compounds whose polarity is similar to that of the mobile phase will be preferentially attracted to it and move faster.

In this way, based upon differences in the relative attraction of each compound for each phase, a separation is created by changing the speeds of the analytes. Figures R-1, R-2, and R-3 display typical chromatographic polarity ranges for mobile phases, stationary phases, and sample analytes, respectively. Lets consider each in turn to see how a chromatographer chooses the appropriate phases to develop the attraction competition needed to achieve a polarity-based HPLC separation.

Figure R-1: Mobile Phase Chromatographic Polarity Spectrum

A scale, such as that shown in Figure R-1, upon which some common solvents are placed in order of relative chromatographic polarity is called an eluotropic series. Mobile phase molecules that compete effectively with analyte molecules for the attractive stationary phase sites displace these analytes, causing them to move faster through the column [weakly retained]. Water is at the polar end of mobile-phase-solvent scale, while hexane, an aliphatic hydrocarbon, is at the non-polar end. In between, single solvents, as well as miscible-solvent mixtures [blended in proportions appropriate to meet specific separation requirements], can be placed in order of elution strength. Which end of the scale represents the strongest mobile phase depends upon the nature of the stationary phase surface where the competition for the analyte molecules occurs.

Figure R-2: Stationary Phase Particle Chromatographic Polarity Spectrum

Silica has an active, hydrophilic [water-loving] surface containing acidic silanol [silicon-containing analog of alcohol] functional groups. Consequently, it falls at the polar end of the stationary-phase scale shown in Figure R-2. The activity or polarity of the silica surface may be modified selectively by chemically bonding to it less polar functional groups [bonded phase]. Examples shown here include, in

order of decreasing polarity, cyanopropylsilyl- [CN], n-octylsilyl- [C8], and n-octadecylsilyl- [C18, ODS] moieties on silica. The latter is a hydrophobic [water-hating], very non-polar packing.

Figure R-3: Compound/Analyte Chromatographic Polarity Spectrum

Figure R-3 repeats the chromatographic polarity spectrum of our sample [shown in Figure P]. After considering the polarity of both phases, then, for a given stationary phase, a chromatographer must choose a mobile phase in which the analytes of interest are retained, but not so strongly that they cannot be eluted. Among solvents of similar strength, the chromatographer considers which phase combination may best exploit the more subtle differences in analyte polarity and solubility to maximize the selectivity of the chromatographic system. Like attracts like, but, as you probably can imagine from the discussion so far, creating a separation based upon polarity involves knowledge of the sample and experience with various kinds of analytes and retention modes. To summarize, the chromatographer will choose the best combination of a mobile phase and particle stationary phase with appropriately opposite polarities. Then, as the sample analytes move through the column, the rule like attracts like will determine which analytes slow down and which proceed at a faster speed. Normal-Phase HPLC In his separations of plant extracts, Tswett was successful using a polar stationary phase [chalk in a glass column; see Figure A] with a much less polar [non-polar] mobile phase. This classical mode of chromatography became known as normal phase.

Figure S-1: Normal-Phase Chromatography

Figure S-1 represents a normal-phase chromatographic separation of our three-dye test mixture. The stationary phase is polar and retains the polar yellow dye most strongly. The relatively non-polar blue dye is won in the retention competition by the mobile phase, a non-polar solvent, and elutes quickly. Since the blue dye is most like the mobile phase [both are non-polar], it moves faster. It is typical for normal-phase chromatography on silica that the mobile phase is 100% organic; no water is used. Reversed-Phase HPLC The term reversed-phase describes the chromatography mode that is just the opposite of normal phase, namely the use of a polar mobile phase and a non-polar [hydrophobic] stationary phase. Figure S-2 illustrates the black three-dye mixture being separated using such a protocol.

Figure S-2: Reversed-Phase Chromatography

Now the most strongly retained compound is the more non-polar blue dye, as its attraction to the nonpolar stationary phase is greatest. The polar yellow dye, being weakly retained, is won in competition by the polar, aqueous mobile phase, moves the fastest through the bed, and elutes earliest like attracts like. Today, because it is more reproducible and has broad applicability, reversed-phase chromatography is used for approximately 75% of all HPLC methods. Most of these protocols use as the mobile phase an aqueous blend of water with a miscible, polar organic solvent, such as acetonitrile or methanol. This typically ensures the proper interaction of analytes with the non-polar, hydrophobic particle surface. A C18bonded silica [sometimes called ODS] is the most popular type of reversed-phase HPLC packing. Table C presents a summary of the phase characteristics for the two principal HPLC separation modes based upon polarity. Remember, for these polarity-based modes, like attracts like.

Table C: Phase Characteristics for Separations Based on Polarity

Hydrophilic-Interaction Chromatography [HILIC] HILIC may be viewed as a variant of normal-phase chromatography. In normal-phase chromatography, the mobile phase is 100% organic. Only traces of water are present in the mobile phase and in the pores of the polar packing particles. Polar analytes bind strongly to the polar stationary phase and may not elute. Adding some water [< 20%] to the organic mobile phase [typically an aprotic solvent like acetonitrile] makes it possible to separate and elute polar compounds that are strongly retained in the normalphase mode [or weakly retained in the reversed-phase mode]. Water, a very polar solvent, competes effectively with polar analytes for the stationary phase. HILIC may be run in either isocratic or gradient elution modes. Polar compounds that are initially attracted to the polar packing material particles can be eluted as the polarity [strength] of the mobile phase is increased [by adding more water]. Analytes are eluted in order of increasing hydrophilicity [chromatographic polarity relative to water]. Buffers or salts may be added to the mobile phase to keep ionizable analytes in a single form. Hydrophobic-Interaction Chromatography [HIC] HIC is a type of reversed-phase chromatography that is used to separate large biomolecules, such as proteins. It is usually desirable to maintain these molecules intact in an aqueous solution, avoiding contact with organic solvents or surfaces that might denature them. HIC takes advantage of the hydrophobic interaction of large molecules with a moderately hydrophobic stationary phase, e.g., butyl-bonded [C4], rather than octadecyl-bonded [C18], silica. Initially, higher salt concentrations in water will encourage the proteins to be retained [salted out] on the packing. Gradient separations are typically run by decreasing salt concentration. In this way, biomolecules are eluted in order of increasing hydrophobicity. Separations Based on Charge: Ion-Exchange Chromatography [IEC] For separations based on polarity, like is attracted to like and opposites may be repelled. In ionexchange chromatography and other separations based upon electrical charge, the rule is reversed. Likes may repel, while opposites are attracted to each other. Stationary phases for ion-exchange separations are characterized by the nature and strength of the acidic or basic functions on their surfaces and the types of ions that they attract and retain. Cation exchange is used to retain and separate positively charged ions on a negative surface. Conversely, anion exchange is used to retain and separate negatively charged ions on a positive surface [see Figure T]. With each type of ion exchange, there are at least two general approaches for separation and elution.

Figure T: Ion-Exchange Chromatography

Strong ion exchangers bear functional groups [e.g., quaternary amines or sulfonic acids] that are always ionized. They are typically used to retain and separate weak ions. These weak ions may be eluted by displacement with a mobile phase containing ions that are more strongly attracted to the stationary phase sites. Alternately, weak ions may be retained on the column, then neutralized by in situ changing the pH of the mobile phase, causing them to lose their attraction and elute. Weak ion exchangers [e.g., with secondary-amine or carboxylic-acid functions] may be neutralized above or below a certain pH value and lose their ability to retain ions by charge. When charged, they are used to retain and separate strong ions. If these ions cannot be eluted by displacement, then the stationary phase exchange sites may be neutralized, shutting off the ionic attraction, and permitting elution of the charged analytes.

Table D: Ion-Exchange Guidelines

When weak ion exchangers are neutralized, they may retain and separate species by hydrophobic [reversed-phase] or hydrophilic [normal-phase] interactions; in these cases, elution strength is determined by the polarity of the mobile phase [Figure R-1]. Thus, weak ion exchangers may be used for mixed-mode separations [separations based on both polarity and charge]. Table D outlines guidelines for the principal categories of ion exchange. For example, to retain a strongly basic analyte [always positively charged], use a weak-cation-exchange stationary phase particle at pH > 7; this assures a negatively charged particle surface. To release or elute the strong base, lower the pH of the mobile phase below 3; this removes the surface charge and shuts off the ion-exchange retention mechanism. Note that a pKa is the pH value at which 50% of the functional group is ionized and 50% is neutral. To assure an essentially neutral, or a fully charged, analyte or particle surface, the pH must be adjusted to a value at least 2 units beyond the pKa, as appropriate [indicated in Table D]. Do not use a strong-cation exchanger to retain a strong base; both remain charged and strongly attracted to each other, making the base nearly impossible to elute. It can only be removed by swamping the strong cation exchanger with a competing base that exhibits even stronger retention

and displaces the compound of interest by winning the competition for the active exchange sites. This approach is rarely practical, or safe, in HPLC and SPE. [Very strong acids and bases are dangerous to work with, and they may be corrosive to materials of construction used in HPLC fluidics!] Separations Based on Size: Size-Exclusion Chromatography [SEC] Gel-Permeation Chromatography [GPC] In the 1950s, Porath and Flodin discovered that biomolecules could be separated based on their size, rather than on their charge or polarity, by passing, or filtering, them through a controlled-porosity, hydrophilic dextran polymer. This process was termed gel filtration. Later, an analogous scheme was used to separate synthetic oligomers and polymers using organic-polymer packings with specific poresize ranges. This process was called gel-permeation chromatography [GPC]. Similar separations done using controlled-porosity silica packings were called size-exclusion chromatography [SEC]. Introduced in 1963, the first commercial HPLC instruments were designed for GPC applications [see Reference 3]. All of these techniques are typically done on stationary phases that have been synthesized with a pore-size distribution over a range that permits the analytes of interest to enter, or to be excluded from, more or less of the pore volume of the packing. Smaller molecules penetrate more of the pores on their passage through the bed. Larger molecules may only penetrate pores above a certain size so they spend less time in the bed. The biggest molecules may be totally excluded from pores and pass only between the particles, eluting very quickly in a small volume. Mobile phases are chosen for two reasons: first, they are good solvents for the analytes; and, second, they may prevent any interactions [based on polarity or charge] between the analytes and the stationary phase surface. In this way, the larger molecules elute first, while the smaller molecules travel slower [because they move into and out of more of the pores] and elute later, in decreasing order of their size in solution. Hence the simple rule: Big ones come out first. Since it is possible to correlate the molecular weight of a polymer with its size in solution, GPC revolutionized measurement of the molecular-weight distribution of polymers that, in turn, determines the physical characteristics that may enhance, or detract from, polymer processing, quality, and performance [how to tell good from bad polymer]. Conclusion We hope you have enjoyed this brief introduction to HPLC. We encourage you to read the references below and to study the Appendix on HPLC Nomenclature.

Appendix: HPLC Nomenclature

*Indicates a definition adapted from: L.S. Ettre, Nomenclature for Chromatography, Pure Appl. Chem. 65: 819-872 [1993], 1993 IUPAC; an updated version of this comprehensive report is available in the Orange Book, Chapter 9: Separations [1997] at: <http://www.iupac.org/publications/analytical_compendium>.

Alumina A porous, particulate form of aluminum oxide [Al203] used as a stationary phase in normal-phase adsorption chromatography. Alumina has a highly active basic surface; the pH of a 10% aqueous slurry is about 10. It is successively washed with strong acid to make neutral and acidic grades [slurry pH 7.5 and 4, resp.]. Alumina is more hygroscopic than silica. Its activity is measured according to the Brockmann scale for water content; e.g., Activity Grade I contains 1% H2O. H. Brockmann and H. Schodder, Ber. 74: 73 (1941). Baseline* The portion of the chromatogram recording the detector response when only the mobile phase emerges from the column. Cartridge A type of column, without endfittings, that consists simply of an open tube wherein the packing material is retained by a frit at either end. SPE cartridges may be operated in parallel on a vacuummanifold. HPLC cartridges are placed into a cartridge holder that has fluid connections built into each end. Cartridge columns are easy to change, less expensive, and more convenient than conventional columns with integral endfittings. Chromatogram* A graphical or other presentation of detector response or other quantity used as a measure of the concentration of the analyte in the effluent versus effluent volume or time. In planar chromatography [e.g., thin-layer chromatography or paper chromatography], chromatogram may refer to the paper or layer containing the separated zones. Chromatography* A dynamic physicochemical method of separation in which the components to be separated are distributed between two phases, one of which is stationary [the stationary phase] while the other [the mobile phase] moves relative to the stationary phase. Column Volume* [Vc] The geometric volume of the part of the tube that contains the packing [internal cross-sectional area of the tube multiplied by the packed bed length, L]. The interparticle volume of the column, also called the interstitial volume, is the volume occupied by the mobile phase between the particles in the packed bed. The void volume [V0] is the total volume occupied by the mobile phase, i.e. the sum of the interstitial volume and the intraparticle volume [also called pore volume]. Detector* [see Sensitivity] A device that indicates a change in the composition of the eluent by measuring physical or chemical properties [e.g., UV/visible light absorbance, differential refractive index, fluorescence, or conductivity]. If the detectors response is linear with respect to sample concentration, then, by

suitable calibration with standards, the amount of a component may be quantitated. Often, it may be beneficial to use two different types of detectors in series. In this way, more corroboratory or specific information may be obtained about the sample analytes. Some detectors [e.g., electrochemical, mass spectrometric] are destructive; i.e., they effect a chemical change in the sample components. If a detector of this type is paired with a non-destructive detector, it is usually placed second in the flow path. Display A device that records the electrical response of a detector on a computer screen in the form of a chromatogram. Advanced data recording systems also perform calculations using sophisticated algorithms, e.g., to integrate peak areas, subtract baselines, match spectra, quantitate components, and identify unknowns by comparison to standard libraries. Efficiency [H, see Plate Number, Resolution, Sensitivity, Speed] A measure of a columns ability to resist the dispersion of a sample band as it passes through the packed bed. An efficient column minimizes band dispersion or bandspreading. Higher efficiency is important for effective separation, greater sensitivity, and/or identification of similar components in a complex sample mixture. Nobelists Martin and Synge, by analogy to distillation, introduced the concept of plate height [H, or H.E.T.P., height equivalent to a theoretical plate] as a measure of chromatographic efficiency and as a means to compare column performance. Presaging HPLC and UPLC technology, they recognized that a homogeneous bed packed with the smallest possible particle size [requiring higher pressure] was key to maximum efficiency. The relation between column and separation system parameters that affect bandspreading was later described in an equation by van Deemter. Chromatographers often refer to a quantity that they can calculate easily and directly from measurements made on a chromatogram, namely plate number [N], as efficiency. Plate height is then determined from the ratio of the length of the column bed to N [H = L/N; methods of calculating N from a chromatogram are shown in Figure U]. It is important to note that calculation of N or H using these methods is correct only for isocratic conditions and cannot be used for gradient separations. A.J.P. Martin and R.M. Synge, Biochem. J. 35: 1358-1368 [1941] J.J. van Deemter, F. J. Zuiderweg and A. Klinkenberg, Chem. Eng. Sci. 5: 271-289 [1956] Eluate The portion of the eluent that emerges from the column outlet containing analytes in solution. In analytical HPLC, the eluate is examined by the detector for the concentration or mass of analytes therein. In preparative HPLC, the eluate is collected continuously in aliquots at uniform time or volume intervals, or discontinuously only when a detector indicates the presence of a peak of interest. These fractions are subsequently processed to obtain purified compounds.

Eluent The mobile phase [see Elution Chromatography]. Eluotropic Series A list of solvents ordered by elution strength with reference to specified analytes on a standard sorbent. Such a series is useful when developing both isocratic and gradient elution methods. Trappe coined this term after showing that a sequence of solvents of increasing polarity could separate lipid fractions on alumina. Later, Snyder measured and tabulated solvent strength parameters for a large list of solvents on several normal-phase LC sorbents. Neher created a very useful nomogram by which equi-eluotropic [constant elution strength] mixtures of normal-phase solvents could be chosen to optimize the selectivity of TLC separations. A typical normal-phase eluotropic series would start at the weak end with non-polar aliphatic hydrocarbons, e.g., pentane or hexane, then progress successively to benzene [an aromatic hydrocarbon], dichloromethane [a chlorinated hydrocarbon], diethyl ether, ethyl acetate [an ester], acetone [a ketone], and, finally, methanol [an alcohol] at the strong end [see Figure R-1]. W. Trappe, Biochem. Z. 305: 150 [1940] L. R. Snyder, Principles of Adsorption Chromatography, Marcel Dekker [1968], pp. 192-197 R. Neher in G.B. Marini-Bettlo, ed., Thin-Layer Chromatography, Elsevier [1964] pp. 75-86. Elute* [verb] To chromatograph by elution chromatography. The process of elution may be stopped while all the sample components are still on the chromatographic bed [planar thin-layer or paper chromatography] or continued until the components have left the chromatographic bed [column chromatography]. Note: The term elute is preferred to develop [a term used in planar chromatography], to avoid confusion with the practice of method development, whereby a separation system [the combination of mobile and stationary phases] is optimized for a particular separation. Elution Chromatography* A procedure for chromatographic separation in which the mobile phase is continuously passed through the chromatographic bed. In HPLC, once the detector baseline has stabilized and the separation system has reached equilibrium, a finite slug of sample is introduced into the flowing mobile phase stream. Elution continues until all analytes of interest have passed through the detector. Elution Strength A measure of the affinity of a solvent relative to that of the analyte for the stationary phase. A weak solvent cannot displace the analyte, causing it to be strongly retained on the stationary phase. A strong solvent may totally displace all the analyte molecules and carry them through the column unretained. To achieve a proper balance of effective separation and reasonable elution volume,

solvents are often blended to set up an appropriate competition between the phases, thereby optimizing both selectivity and separation time for a given set of analytes [see Selectivity]. Dipole moment, dielectric constant, hydrogen bonding, molecular size and shape, and surface tension may give some indication of elution strength. Elution strength is also determined by the separation mode. An eluotropic series of solvents may be ordered by increasing strength in one direction under adsorption or normal-phase conditions; that order may be nearly opposite under reversed-phase partition conditions [see Figure R-1]. Fluorescence Detector Fluorescence detectors excite a sample with a specified wavelength of light. This causes certain compounds to fluoresce and emit light at a higher wavelength. A sensor, set to a specific emission wavelength and masked so as not to be blinded by the excitation source, collects only the emitted light. Often analytes that do not natively fluoresce may be derivatized to take advantage of the high sensitivity and selectivity of this form of detection, e.g., AccQTag derivatization of amino acids. Flow Rate* The volume of mobile phase passing through the column in unit time. In HPLC systems, the flow rate is set by the controller for the solvent delivery system [pump]. Flow rate accuracy can be checked by timed collection and measurement of the effluent at the column outlet. Since a solvents density varies with temperature, any calibration or flow rate measurement must take this variable into account. Most accurate determinations are made, when possible, by weight, not volume. Uniformity [precision] and reproducibility of flow rate is important to many LC techniques, especially in separations where retention times are key to analyte identification, or in gel-permeation chromatography where calibration and correlation of retention times are critical to accurate molecularweight-distribution measurements of polymers. Often, separation conditions are compared by means of linear velocity, not flow rate. The linear velocity is calculated by dividing the flow rate by the cross-sectional area of the column. While flow rate is expressed in volume/time [e.g., mL/min], linear velocity is measured in length/time [e.g., mm/sec]. Gel-Permeation Chromatography* Separation based mainly upon exclusion effects due to differences in molecular size and/or shape. Gelpermeation chromatography and gel filtration chromatography describe the process when the stationary phase is a swollen gel. Both are forms of size-exclusion chromatography. Porath and Flodin first described gel-filtration using dextran gels and aqueous mobile phases for the size-based separation of biomolecules. Moore applied similar principles to the separation of organic polymers by size in solution using organic-solvent mobile phases on porous polystyrene-divinylbenzene polymer gels.

J. Porath, P. Flodin, Nature 183: 1657-1659 [1959] J.C. Moore, U.S. Patent 3,326,875 [filed Jan. 1963; issued June 1967] Gradient The change over time in the relative concentrations of two [or more] miscible solvent components that form a mobile phase of increasing elution strength. A step gradient is typically used in solid-phase extraction; in each step, the eluent composition is changed abruptly from a weaker mobile phase to a stronger mobile phase. It is even possible, by drying the SPE sorbent bed in between steps, to change from one solvent to another immiscible solvent. A continuous gradient is typically generated by a low- or high-pressure mixing system [see Figures J-2 and J-3] according to a pre-determined curve [linear or non-linear] representing the concentration of the stronger solvent B in the initial solvent A over a fixed time period. A hold at a fixed isocratic solvent composition can be programmed at any time point within a continuous gradient. At the end of a separation, the gradient program can also be set to return to the initial mobile phase composition to re-equilibrate the column in preparation for the injection of the next sample. Sophisticated HPLC systems can blend as many as four or more solvents [or solvent mixtures] into a continuous gradient. Injector [Autosampler, Sample Manager] A mechanism for accurately and precisely introducing [injecting] a discrete, predetermined volume of a sample solution into the flowing mobile phase stream. The injector can be a simple manual device, or a sophisticated autosampler that can be programmed for unattended injections of many samples from an array of individual vials or wells in a predetermined sequence. Sample compartments in these systems may even be temperature controlled to maintain sample integrity over many hours of operation. Most modern injectors incorporate some form of syringe-filled sample loop that can be switched on- or offline by means of a multi-port valve. A well-designed, minimal-internal-volume injection system is situated as close to the column inlet as possible and minimizes the spreading of the sample band. Between sample injections, it is also capable of being flushed to waste by mobile phase, or a wash solvent, to prevent carryover [contamination of the present sample by a previous one]. Samples are best prepared for injection, if possible, by dissolving them in the mobile phase into which they will be injected; this may prevent issues with separation and/or detection. If another solvent must be used, it is desirable that its elution strength be equal to or less than that of the mobile phase. It is often wise to mix a bit of a sample solution with the mobile phase offline to test for precipitation or miscibility issues that might compromise a successful separation. Inlet The end of the column bed where the mobile phase stream and sample enter. A porous, inert frit retains the packing material and protects the sorbent bed inlet from particulate contamination. Good HPLC practice dictates that samples and mobile phases should be particulate-free; this becomes

imperative for small-particle columns whose inlets are much more easily plugged. If the column bed inlet becomes clogged and exhibits higher-than-normal backpressure, sometimes, reversing the flow direction while directing the effluent to waste may dislodge and flush out sample debris that sits atop the frit. If the debris has penetrated the frit and is lodged in the inlet end of the bed itself, then the column has most likely reached the end of its useful life. Ion-Exchange Chromatography* [see section: Separations Based on Charge] This separation mode is based mainly on differences in the ion-exchange affinities of the sample components. Separation of primarily inorganic ionic species in water or buffered aqueous mobile phases on small particle, superficially porous, high-efficiency, ion-exchange columns followed by conductometric or electrochemical detection is referred to as ion chromatography [IC]. Isocratic Elution* A procedure in which the composition of the mobile phase remains constant during the elution process. Liquid Chromatography* [LC] A separation technique in which the mobile phase is a liquid. Liquid chromatography can be carried out either in a column or on a plane [TLC or paper chromatography]. Modern liquid chromatography utilizing smaller particles and higher inlet pressure was termed high-performance (or high-pressure) liquid chromatography [HPLC] in 1970. In 2004, ultra-performance liquid chromatography dramatically raised the performance of LC to a new plateau [see UPLC Technology]. Mobile Phase* [see Eluate, Eluent] A fluid that percolates, in a definite direction, through the length of the stationary-phase sorbent bed. The mobile phase may be a liquid [liquid chromatography] or a gas [gas chromatography] or a supercritical fluid [supercritical-fluid chromatography]. In gas chromatography the expression carrier gas may be used for the mobile phase. In elution chromatography, the mobile phase may also be called the eluent, while the word eluate is defined as the portion of the mobile phase that has passed through the sorbent bed and contains the compounds of interest in solution. Normal-Phase Chromatography* An elution procedure in which the stationary phase is more polar than the mobile phase. This term is used in liquid chromatography to emphasize the contrast to reversed-phase chromatography. Peak* [see Plate Number] The portion of a differential chromatogram recording the detector response while a single component is eluted from the column. If separation is incomplete, two or more components may be eluted as one unresolved peak. Peaks eluted under optimal conditions from a well-packed, efficient column, operated in a system that minimizes bandspreading, approach the shape of a Gaussian distribution. Quantitation is usually done by measuring the peak area [enclosed by the baseline and the peak

curve]. Less often, peak height [the distance measured from the peak apex to the baseline] may be used for quantitation. This procedure requires that both the peak width and the peak shape remain constant. Plate Number* [N, see Efficiency] A number indicative of column performance [mechanical separation power or efficiency, also called plate count, number of theoretical plates, or theoretical plate number]. It relates the magnitude of a peaks retention to its width [variance or bandspread]. In order to calculate a plate count, it is assumed that a peak can be represented by a Gaussian distribution [a statistical bell curve]. At the inflection points [60.7% of peak height], the width of a Gaussian curve is twice the standard deviation [] about its mean [located at the peak apex]. As shown in Figure U, a Gaussian curves peak width measured at other fractions of peak height can be expressed in precisely defined multiples of . Peak retention [retention volume, VR, or retention time, tR] and peak width must be expressed in the same units, because method of calculating N is aN is a dimensionless number. Note that the 5 sigma more stringent measure of column homogeneity and performance, as it is more severely affected by peak asymmetry. Computer data stations can automatically delineate each resolved peak and calculate its corresponding plate number. Preparative Chromatography The process of using liquid chromatography to isolate a compound in a quantity and at a purity level sufficient for further experiments or uses. For pharmaceutical or biotechnological purification processes, columns several feet in diameter can be used for multiple kilograms of material. For isolating just a few micrograms of a valuable natural product, an analytical HPLC column is sufficient. Both are preparative chromatographic approaches, differing only in scale [see section on HPLC Scale and Table A]. Resolution* [Rs, see Selectivity] The separation of two peaks, expressed as the difference in their corresponding retention times, divided by their average peak width at the baseline. Rs = 1.25 indicates that two peaks of equal width are just separated at the baseline. When Rs = 0.6, the only visual indication of the presence of two peaks on a chromatogram is a small notch near the peak apex. Higher efficiency columns produce narrower peaks and improve resolution for difficult separations; however, resolution increases by only the square root of N. The most powerful method of increasing resolution is to increase selectivity by altering the mobile/stationary phase combination used for the chromatographic separation [see section on Chemical Separation Power]. Retention Factor* [k] A measure of the time the sample component resides in the stationary phase relative to the time it resides in the mobile phase; it expresses how much longer a sample component is retarded by the stationary phase than it would take to travel through the column with the velocity of the mobile phase. Mathematically, it is the ratio of the adjusted retention time [volume] and the hold-up time [volume]: k = tR'/tM [see Retention Time and Selectivity].

Note: In the past, this term has also been expressed as partition ratio, capacity ratio, capacity factor, or mass distribution ratio and symbolized by k'. Retention Time* [tR] The time between the start of elution [typically, in HPLC, the moment of injection or sample introduction] and the emergence of the peak maximum. The adjusted retention time, tR', is calculated by subtracting from tR the hold-up time [tM, the time from injection to the elution of the peak maximum of a totally unretained analyte]. Reversed-Phase Chromatography* An elution procedure used in liquid chromatography in which the mobile phase is significantly more polar than the stationary phase, e.g. a microporous silica-based material with alkyl chains chemically bonded to its accessible surface. Note: Avoid the incorrect term reverse phase. [See Reference 4 for some novel ideas on the mechanism of reversed-phase separations.] Selectivity [Separation Factor, ] A term used to describe the magnitude of the difference between the relative thermodynamic affinities of a pair of analytes for the specified mobile and stationary phases that comprise the separation system. The proper term is separation factor []. It equals the ratio of retention factors, k2/k1 = 1, then bothis always 1. If [see Retention Factor]; by definition, peaks co-elute, and no separation is obtained. It is important in preparative chromatography to maximize for highest sample loadability and throughput. [see section on Chemical Separation Power] Sensitivity* [S] The signal output per unit concentration or unit mass of a substance in the mobile phase entering the detector, e.g., the slope of a linear calibration curve [see Detector]. For concentration-sensitive detectors [e.g., UV/VIS absorbance], sensitivity is the ratio of peak height to analyte concentration in the peak. For mass-flow-sensitive detectors, it is the ratio of peak height to unit mass. If sensitivity is to be a unique performance characteristic, it must depend only on the chemical measurement process, not upon scale factors. The ability to detect [qualify] or measure [quantify] an analyte is governed by many instrumental and chemical factors. Well-resolved peaks [maximum selectivity] eluting from high efficiency columns [narrow peak width with good symmetry for maximum peak height] as well as good detector sensitivity and specificity are ideal. Both the separation system interference and electronic component noise should also be minimized to achieve maximum sensitivity. Solid-Phase Extraction [SPE] A sample preparation technique that uses LC principles to isolate, enrich, and/or purify analytes from a complex matrix applied to a miniature chromatographic bed. Offline SPE is done [manually or via automation] with larger particles in individual plastic cartridges or in micro-elution plate wells, using low positive pressure or vacuum to assist flow. Online SPE is done with smaller particles in miniature

HPLC columns using higher pressures and a valve to switch the SPE column online with the primary HPLC column, or offline to waste, as appropriate. SPE methods use step gradients [see Gradient] to accomplish bed conditioning, sample loading, washing, and elution steps. Samples are loaded typically under conditions where the k of important analytes is as high as possible, so that they are fully retained during loading and washing steps. Elution is then done by switching to a much stronger solvent mixture [see Elution Strength]. The goal is to remove matrix interferences and to isolate the analyte in a solution, and at a concentration, suitable for subsequent analysis. Speed [see Efficiency, Flow Rate, Resolution] A benefit of operating LC separations at higher linear velocities using smaller-volume, smaller-particle analytical columns, or larger-volume, larger-particle preparative columns. Order-of-magnitude advances in LC speed came in 1972 [with the use of 10 m particles and pumps capable of delivering accurate mobile-phase flow at 6000 psi], in 1976 [with 75-m preparative columns operated at a flow rate of 500 mL/min], and in 2004 [with the introduction of UPLC technology1.7 m-particle columns operated at 15,000 psi]. High-speed analytical LC systems must not only accommodate higher pressures throughout the fluidics; injector cycle time must be short; gradient mixers must be capable of rapid turnaround between samples; detector sensors must rapidly respond to tiny changes in eluate composition; and data systems must collect the dozens of points each second required to plot and to quantitate narrow peaks accurately. Together, higher resolution, higher speed, and higher efficiency typically deliver higher throughput. More samples can be analyzed in a workday. Larger quantities of compound can be purified per run or per process period. See #3 on list of References for Further Reading above. Stationary Phase* One of the two phases forming a chromatographic system. It may be a solid, a gel, or a liquid. If a liquid, it may be distributed on a solid. This solid may or may not contribute to the separation process. The liquid may also be chemically bonded to the solid [bonded phase] or immobilized onto it [immobilized phase]. The expression chromatographic bed or sorbent may be used as a general term to denote any of the different forms in which the stationary phase is used. The use of the term liquid phase to denote the mobile phase in LC is discouraged. This avoids confusion with gas chromatography where the stationary phase is called a liquid phase [most often a liquid coated on a solid support].

Open-column liquid-liquid partition chromatography [LLC] did not translate well to HPLC. It was supplanted by the use of bonded-phase packings. LLC proved incompatible with modern detectors because of problems with bleed of the stationary-phase-liquid coating off its solid support, thereby contaminating the immiscible liquid mobile phase. UPLC Technology The use of a high-efficiency LC system holistically designed to accommodate sub-2 m particles and very high operating pressure is termed ultra-performance liquid chromatography [UPLC technology]. The major benefits of this technology are significant improvements in resolution over HPLC, and/or faster run times while maintaining the resolution seen in an existing HPLC separation.

The growth of pharmaceutical industry is based on continuing success in producing new products whether they are used as therapeutic or prophylactic agents. The role of R&D is pivotal in this endeavor.
Pharmaceutical research is aimed at meeting the medical needs of the population for whom appropriate therapeutic remedies are not available or at those that are available are unsafe for prophylactic use for various disorders. While meeting medical needs, research also has to ensure that market needs for such exist and that the product will command sales and profits proportionate to investments. In cases where there are mismatches between these two, the products suffer the status of orphan drugs. The selection of an appropriate R&D portfolio is a strategic management exercise for a company, which should take into account apart from medical needs, innovative potential for success and available resources. WHO Guidelines for Quality Standardized Herbal Formulations a.Quality control of crude drugs material, plant preparations and finished products. b.Stability assessment and shelf life. c.Safety assessment; documentation of safety based on experience or toxicological studies. d. Assessment of efficacy by ethnomedical informations and biological activity evaluations. The bioactive extract should be standardized on the basis of active principles or major compounds along with the chromatographic fingerprints (TLC, HPTLC, HPLC and GC). The standardization of crude drug materials include the following steps: 1.Authentication (Stage of collection, parts of the plant collected, regional status, botanical identity like phytomorphology, microscopical and histological analysis, taxonomical identity, etc.) 2.Foreign matter (herbs collected should be free from soil, insect parts or animal excreta, etc.)

3.Organoleptic evaluation (sensory characters taste, appearance, odor, feel of the drug, etc.) 4.Tissues of diagnostic importance present in the drug powder. 5. Ash values and extractive values. 6.Volatile matter 7.Moisture content determination 8.Chromatographic and spectroscopic evaluation. TLC, HPTLC, HPLC methods will provide qualitative and semi quantitative information about the main active constituents present in the crude drug as chemical markers in the TLC fingerprint evaluation of herbals (FEH). The quality of the drug can also be assessed on the basis of the chromatographic fingerprint. 9.Determination of heavy metals e.g. cadmium, lead, arsenic, etc. 10.Pesticide residue WHO and FAO (Food and Agricultural Organization) set limits of pesticides, which are usually present in the herbs. These pesticides are mixed with the herbs during the time of cultivation. Mainly pesticides like DDT, BHC, toxaphene, aldrin cause serious side-effects in human beings if the crude drugs are mixed with these agents. 11.Microbial contamination usually medicinal plants containing bacteria and molds are coming from soil and atmosphere. Analysis of the limits of E. coli and molds clearly throws light towards the harvesting and production practices. The substance known as afflatoxins will produce serious side-effects if consumed along with the crude drugs. Limits for Microbial Contamination Microorganism E. coli Salmonella Total aerobic bacteria Enterobacteria Finished product 101 105 103 Raw materials 104 -

Afflatoxins should be completely removed or should not be present. 12.Radioactive contamination Microbial growth in herbals are usually avoided by irradiation. This process may sterilize the plant material but the radioactivity hazard should be taken into account. The radioactivity of the plant samples should be checked accordingly to the guidelines of International Atomic Energy (IAE) in Vienna and that of WHO. In order to obtain quality oriented herbal products care should be taken right from the proper identification of plants; season and area of collection, extraction, isolation and verification process.

Chemical and instrumental analyses are routinely used for analyzing synthetic drugs to confirm its authenticity. In the case of herbal drugs, however the scene is different especially for polyherbal formulation, as there is no chemical or analytical methods available. Therefore biological-screening methods can be adopted for routine checkup of herbal drugs and formulations. In the case of herbal drugs, the quality of raw materials and products can be furnished by regular pharmacognostic identifications and phytochemical analysis. The herbal formulations in general can be standardized schematically as to formulate the medicament using raw materials collected from different localities and a comparative chemical efficacy of different batches of formulation are to be observed. The preparation with better clinical efficacy are to be selected. After all the routine physical, chemical and pharmacological parameters are to be checked for all the batches to select the final finished product and to validate the whole manufacturing process. The stability parameters for the herbal formulations which includes physical parameters, chemical parameters, and microbiological parameters. Physical parameters include color, appearance, odor, clarity, viscosity, moisture content, pH, disintegration time, friability, hardness, flowability, flocculation, sedimentation, settling rate and ash values. Chemical parameters includes limit tests, extractive values, chemical assays, etc. Chromatographic analysis of herbals can be done using TLC, HPLC, HPTLC and GC, UV, Fluorimetry, GC-MS, etc. Microbiological parameters include total viable content, total mold count, total enterobacterial and their count. Limiters can be utilized as a quantitative or semiquantitative tool to ascertain and control the amount of impurities like the reagents used during abstraction of various herbs, impurities coming directly from the manufacturing vessels, impurities from the solvents, etc. Chemical decomposition of substances present in the formulation also produces several toxic or impure compounds during storage in undesirable conditions. Contaminants may come directly from the atmosphere also. This include mainly dust, sulfur dioxide, H2S, CO2, Arsenic, moisture, etc. The guidelines set by WHO can be summarized as follows: a.Reference to the identity of the drug. Botanical evaluation sensory characters, foreign organic matter, microscopical, histological, histochemical evaluation, quantitative measurements, etc. b.Reference to the physiochemical character of the drug. Chromatographic profiles, ash values, extractive values, refractive index, polarimetric readings, moisture content, volatile oil content, etc. c.Reference to the pharmacological parameters. Biological activity profiles, bitterness values, haemolytic index, astringency, swelling factor, foaming index, etc.

d.Toxicity details heavy metals like cadmium, lead, arsenic, mercury, etc. Pesticide residues. Maximum residue limits = Acceptable daily index x body weight x extraction factor --------------------------------------------------------------- x Therapeutic doses Mean daily intake of drug x safety factor x 100 e.Microbial contamination Total viable aerobic count, pathogenic bacteria like enterobacteria, E. coli, salmonella, Pseudomonous aeruginosa, Staphylococcus aureus, etc. and presence of afflatoxins etc. f. Radioactive contamination. Modern herbal ayurvedic monographs In the modern herbal ayurvedic monographs the standardization parameters are discussed in a comprehensive way. According to the modern ayurvedic monograph the quality control protocols include the following: Title, synonyms, publications related to that plant, constituents present, analytical methods. Descriptive evaluation: Description of the drug, phytomorphological, microscopical, organoleptic evaluations, foreign matter, foreign minerals, etc. Physicochemical parameters Identity: Physical and chemical identity, chromatographic finger prints, ash values, extractive values, moisture content. Strength: Ethanol and water extractive values, volatile oil and alkaloidal assays, quantitative estimation protocols, etc. Biological Activity Evaluation Bitterness values, astringency, swelling factor, form index, hemolytic index, etc. Toxicological evaluation Pesticide residues, heavy metals, microbial contamination like total viable aerobic count, pathogens like E. coli, Salmonella, P. aeruginosa, S. aureus, Enterobacteria, etc. Aflatoxins

The presence of aflatoxins can be determined by chromatographic methods using standard aflatoxins B1, B2, G1, G2 mixtures. Aflatoxin is a product of the microbial strain Aspergillus flavus. Radioactive Contaminants Therapeutic Evaluation Classical Evaluation as per Ayurvedic Literatures Classical therapeutical attributes like Rasna, Guna, Virya, Vipaka and Karma classical formulations, doses, storage conditions. The quality of the raw materials can be tested according to the following format: Name of the drug (English, Regional names, Exact botanical nomenclature) Part of the plant used Area of collection Distribution details Season of Crop Time and year of collection Pesticide and insecticides Condition of the drug (fresh or dry) Form of the drug (powdered or intact or cuttings like etc.) CONCLUSION The subject of herbal drug standardization is massively wide and deep. There is so much to know and so much seemingly contradictory theories on the subject of herbal medicines and its relationship with human physiology and mental function. For the purpose of research work on standardization of herbal formulations and neutraceuticals a profound knowledge of the important herbs found in India and widely used in Ayurvedic formulation is of utmost importance. India can emerge as the major country and play the lead role in production of standardized, therapeutically effective ayurvedic formulation. India needs to explore the medicinally important plants. This can be achieved only if the herbal products are evaluated and analyzed using

sophisticated modern techniques of standardization such as UV-visible, TLC, HPLC, HPTLC, GC-MS, spectrofluorimetric and other methods.
Herbal Drug Standardization and Evaluation:- In recent years, there has been great demand for plant derived products in developed countries. These products are increasingly being sought out as medicinal products, nutraceuticals and cosmetics. (1) There are around 6000 herbal manufacturers in India. More than 4000 units are producing Ayurveda medicines. Due to lack of infrastructures, skilled manpower reliable methods and stringent regulatory laws most of these manufacturers produce their product on very tentative basis. (2) In order to have a good coordination between the quality of raw materials, in process materials and the final products, it has become essential to develop reliable, specific and sensitive quality control methods using a combination of classical and modern instrumental method of analysis. Standardization is an essential measurement for ensuring the quality control of the herbal drugs. (3) "Standardization" expression is used to describe all measures, which are taken during the manufacturing process and quality control leading to a reproducible quality. It also encompasses the entire field of study from birth of a plant to its clinical application. It also means adjusting the herbal drug preparation to a defined content of a constituent or a group of substances with known therapeutic activity respectively by adding excipients or by mixing herbal drugs or herbal drug preparations.(4) "Evaluation" of a drug means confirmation of its identity and determination of its quality and purity and detection of its nature of adulteration.(5) Standardization of herbal drugs is not an easy task as numerous factors influence the bio efficacy and reproducible therapeutic effect. In order to obtain quality oriented herbal products, care should be taken right from the proper identification of plants, season and area of collection and their extraction and purification process and rationalizing the combination in case of polyherbal drugs.(3) The Standardization of crude drug materials includes the following steps:1. Authentication: - Each and every step has to be authenticated. a) b) c) d) Stage of collection. Parts of the plant collected. Regional status. Botanical identity like phytomorphology, microscopical and histological analysis

(characteristic of cell walls, cell contents, starch grains, calcium oxalate crystals, trichomes, fibers, vessels etc).(6) Various histological parameter studies are:1. Leaf constant: - Palisade ratio, Vein islet number, Vein termination, Stomatal number, and Stomatal index. 2. Trichomes. 3. Stomata. 4. Quantitative microscopy.

5. Taxonomical identity. 6. Foreign matter. 7. Organoleptic evaluation. 8. Ash values and extractive values. 9. Moisture content determination. 10. Chrometographic and spectroscopic evaluation. 11. Heavy metal determination. 12. Pesticide residue. 13. Microbial contamination. 14. Radioactive contamination. The herbal formulation in general can be standardize schematically as to formulate the medicament using raw materials collected from different localities and a comparative chemical efficacy of different batches of formulation are to be observed. The preparations with better clinical efficacy are to be selected. After all the routine physical, chemical and pharmacological parameters are to be checked for all the batches to select the final finished product and to validate the whole manufacturing process. (6) The stability parameters for the herbal formulations which include physical, chemical and microbiological parameters are as follow: 1. Physical parameters include color, odor, appearance, clarity, viscosity, moisture content, pH, disintegration time, friability, hardness, flow ability, flocculation, sedimentation, settling rate and ash values. 2. Chemical parameters include limit tests, chemical tests, chemical assays etc. 3. Chromatographic analysis of herbals can be done using TLC, HPLC, HPTLC, GC, UV, GC-MS, fluorimetry etc. 4. Microbiological parameters include total viable content, total mold count, total enterobacterial and their count. Limiters can be utilized as a quantitative or semi quantitative tool to ascertain and control the amount of impurities like the reagents used during abstraction of various herbs, impurities coming directly from the manufacturing vessels and from the solvents etc. GUIDELINES FOR HERBAL DRUG STANDARDISATION WHO Guidelines:The subject of herbal drug standardization is massively wide and deep. The guidelines set by WHO can be summarized as follows:-

Reference to the identity of the drug. Botanical evaluation- sensory characters, foreign organic matter, microscopical, histological, histochemical evaluation, quantitative measurements etc. Reference to the physicochemical character of the drug. Physical and chemical identity, chromatographic fingerprints, ash values, extractive values, moisture content, volatile oil and alkaloidal assays, quantitative estimation protocols etc.

Reference to the pharmacological parameters, biological activity profiles, bitterness values, hemolytic index, astringency, swelling factor, foaming index etc.

Toxicity details- pesticide residues, heavy metals, microbial contamination like total viable count, pathogens like E.coli, Salmonalla, P.aeroginosa, S. aureus, Enterobacteria etc. Microbial contamination. Radioactive contamination. Modern herbal ayurvedic monographs In the modern herbal ayurvedic monographs the standardization parameters are discussed in a comprehensive way. According to the modern ayurvedic monograph the quality control protocols include the following: The synonyms, publication related to the plant, constituents present, analytical methods. Descriptive evaluation: Description of the drug, phytomorphological, microscopical, organoleptic evaluation, foreign matter etc. WHO GUIDELINES MONOGRAPH TITLE (7)

Botanical: - Sensory evaluation, Foreign matter, Microscopy measurement. Physicochemical TLC: -Ash, Extractable matter, Water content and volatile matter, Volatile oils. Pharmacological: - Bitterness value, Haemolytic activity, Astringency, Sterling index, Foaming index. Toxicological: - Pesticide residue, Arsenic, Metals. Microbial contamination: - Total viable count, Pathogens, Aflatoxins, Radioactive contamination. STANDARDIZATION OF HERBAL DRUG/PRODUCTS Commercial production of herbal medicines and their trade are fast growing sector of industry today, due t6o increasing demand of medicinal plants; the supply line is adversely affected leading to the adulteration and substitution for genuine drugs. 1. Fluorescence quenching:- When a plant extract is spotted on a fluorescent silica gel layer and exposed to UV light, it appears as spot on a fluorescent background, thus causing quenching and is directly proportional to concentration of the extract. Silica gel GF plate was used as an adsorbent for fluorescence quenching. Solvents taken- hexane toluene, ether, ethyl acetate, butanol, methanol and water.(8) 2. Use of fingerprinting and marker compounds for identification and standardization of botanical drugs:- Chemical and chromatographic techniques may be used to aid in identification of an herbal material or extract. Chromatographic technique such as HPLC, TLC, GC and capillary electrophoresis and spectroscopic methods such as IR, NMR, and UV-may also be used for fingerprinting. DNA fingerprinting has been widely used in many species, e.g. DNA fingerprinting of Panax species and their adulterants.(9) Marker compounds may be used to help identify herbal materials, set specifications for raw materials, standardize botanical preparations during all aspects of manufacturing processes and obtain stability profiles.(10)

3. Densitometric thin layer chromatographic determination of aescin in an herbal medicinal product containing Asculum and Vitis dry extract:- A TLC method is developed to analyze the total saponin content, also referred to as the aescin content, in an herbal medicinal product containing two dry extract in capsules. After a purification step using C(18) solid phase extraction, the samples are analyzed on a silica gel HPTLC plate with the upper layer of a mixture of acetic acid/water/butanol(10/40/50v/v/v) as the mobile phase. Spots are visualized by spraying with anisaldehyde reagent and heating the plate for 5-10 min.(100-105oc) and measured at a wavelength of 535 nm.(11) 4. Determination of stigmasterol, beta-sitosterol and stigmastanol in oral dosage forms using HPLC with evaporative light scattering detection: - A validated and repeatable HPLC method with online evaporative light scattering was developed for the analysis of two sterols, stegmasterol, beta-sitosterol and a stanol found to be common in many herbal formulations and health care supplements. This method was used to assay commercially available products formulated as oral dosage forms purported to contain African potato and associated sterols and stanol. (12) Ads by Google

5. Elemental analysis of herbal preparations for traditional medicines by neutron activation analysis with the kO standardization method: - Medicinal herb preparations prescribed for specific treatment purposes were purchased from markets and were analysed by instrumental neutron activation analysis with kO standardization. 500-700 mg of each sample was palletized under a pressure of six tones and irradiated together with monitors for alpha and neutron flux ratio determination for about 6h in a thermal flux of 2.29 x 10(12) n/cm2/s.(13) 6. Liquid chromatography UV-determination and liquid chromatography-atmospheric pressure chemical ionization mass spectrometric characterization of sitosterol and stigmasterol in soyabean oil:- A narrow bore HPLC-UV method was developed for the analysis of two of the more abundant naturally occurring phytosterols in vegetable oils: sitosterol and stigmasterol. The method enabled detection of the compounds at a concentrationof0.42 /ml and quantization at concentration of 0.52 and 0.54 /ml for sitosterol and stigmasterol, respectively.(14)

7. Simultaneous determination of cinnamaldehyde, eugenol and paeonol in traditional Chinese medicinal preparations by capillary GC-FID: - A capillary GC method was established for simultaneous determination of cinnamaldehyde(CNMD), eugenol(EL) and paeonol(PL) in two traditional Chinese herbal medicinal preparations, Weitongding tablet (WTDT) and Guifu Dihuang pill (GDHP). The assays were based on a programmed temperature GC in a 30 m x 0.53 mm capillary column with nitrogen as carrier and FID detector. Good linearity were obtained over ranges of 0.450.452 mg/l CNMD, 0.31-0.625 mg/l EL, and 0.30-610 mg/l PL, respectively. (15) 8. HPTLC fingerprinting of marketed formulation containing Shankhpushpi: - These are the important Ayurveda formulations used for perinatal care of mother and child health. Standardization of churnas was carried out by organoleptic study, phytochemical analysis; qualitative organic and inorganic analysis, thin layer chromatography, UV- visible spectrophotometer and HPLC fingerprint studies. Qualitative organic analysis of both the churnas revealed the presence of alkaloids, steroids, phenols, tannins, glycosides, resins, saponins and flavonoids.(16) EVALUATION OF HERBAL DRUG/PRODUCTS 1. Biological parameter (bioassay):- It is well established that the biological potency of the herbal constituents is due to not one but a mixture of bioactive plant constituents and the relative properties of a single bioactive compound can vary from batch to batch while the biological activity remains within the desirable limits. (1) Some of the examples are:_ a. Evaluation of adaptogenic activity profile of herbal preparation: - Adaptogens help the body to come up with stress and enhance general health and performance. AVM is an herbal formulation. Composition- Emblica officinalis, Withania somnifera, Asparagus racemosus, Ocimum sanctum, Tribulus terrestris and Piper longum. AVM shows significant antistress, immunomodulatory and anabolic activities in different animal models there by proving a promising adaptogen. (17) b. Evaluation of antioxidant activity of herbal products: - A new test method for measuring the antioxidant power of herbal products, based on solid phase spectrophptometry using tetrabenzo-b, f, j, n, l, 5, 9, 13- tetraazacy- clohexadecin- Cu (II) complex immobilized on silica gel is proposed. The method represents an alternative to the mostly used scavenging capacity assays. The method was approved in the analysis of the most popular herbal beverages and drugs Echinacea determined spectrophotometrically.(18) c. Evaluation of microbial contamination reduction on plants through technological process of decoction and spray dry: - The technological process of raw material has many stages, generally, adverse to microbial growth, but its complete elimination depends on the initial and work condition utilized. The aim of this work was to verify the microbial contamination, such as extractive solution (SE) and spray dried extract (PSA) with the purpose of evaluating the decrease of contamination after the decoction and the spray dry. The microbiological analysis of the products was performed by total plate count and MPN coliform. (19) d. Evaluation of nitric oxide scavenging activity of selected medicinal plants used in inflammatory diseases: - Four traditional medicinal plants, namely Ventilago madraspatana Gaertn., Rubia cordifolia Linn., Lanatana camara Linn. And Morinda citrifolia Linn. Were selected for a study on

the inhibition of nitric oxide (NO), a key mediator in the phenomenon of inflammation, signifying the presence of effective anti-inflammatory constituents therein. Plant samples were extracted with different solvents for evaluation of their inhibitory activity on NO produced in vitro from sodium nitroprusside, and in LPS- activated murine peritoneal macrophages, ex-vivo.(20) e. The lipid peroxidation inhibitory activity:- The reaction mixture contained mice liver homogenate (0.2 ml, 10% w/v) in 0.15 KCl, KCl (0.1 ml, 150 m), Tris buffer (0.4 ml, Ph 7.5) and various concentration of test extracts. In vitro lipid peroxidation was initiated by addition of Feso4.7H2O (0.1 ml, 10 m). The reaction mixture was incubated at 37o for 1 h. After the incubation period, reaction was terminated by addition of thiobarbituric acid (TBA-2 ml, 0.8%) and by heating the contents for 15 min. for development of colored complex. The tubes were then centrifuged at 4000 rpm for 10 min. and cooled. The % inhibition of lipid peroxidation was determined by comparing the results of test compound with those of control not treated with extracts by monitoring the color intensity at 532 nm. Gallic acid was used as a positive control. (21) 2. Evaluation of marketed polyherbal antidiabetic formulatios using biomarker charantin: Charantin is one of the phytoconstituents present in Momordica charantia. It is well known to possess antihyperglycaemia, anticholesterol, immunosuppressive, antiulcerogenic, antispermatogenic and androgenic activities. HPTLC method is fast, precise, sensitive and reproducible with good recoveries for standardization of polyherbal formulations. The recovery values of charantin were found to be about 98.89%. (2) 3. In vivo and in vitro evaluation of hair growth potential of Shoe flower: - The leaves and flowers of Hibiscus rosa-sinensis are used as promoters of hair growth and as an aid in healing of ulcers. Petroleum ether extract of leaves and flowers of the plant was evaluated for the potential growth in vivo and in vitro methods. In vivo, 1% extract of leaves and flowers in liquid was applied topically over the shaved skin of albino rats and monitored and assessed for 30 days. The length of hair and different cyclic phases of hair follicles, like anagen and telogen phases were determined at different time periods. In vitro, the hair follicles from albino rat neonates were isolated and cultured in DMEM supplemented with 0.01 mg/ml petroleum ether extract of leaves and flowers. It is concluded that the leaf extract, when compared to flower extract, exhibits more potency on hair growth. (22) 4. Clinical evaluation to assess the safety and efficacy of coded herbal medicine "Dysmooff" versus allopathic medicine "Diclofenac sodium" for the treatment of primary dysmenorrhoea: - The clinical study on primary dysmenorrhoea to comparatively examine the coded herbal drug formulation "Dysmo-off" with authentic allopathic medicine "Diclofenac sodium". A random controlled clinical trial was conducted. These evaluations were based on verbal rating scale so as to ascertain the rate of analgesic effects on dysmenorrhoeic pain. The patients were randomly allocated with the ratio of 1:2 for controlled treatment with (NSAIDS) (n=40) received Diclofenac sodium tablets twice daily for 4 days (50 mg one day prior to and three days after the menstruation), and test treatment with Dysmo-off (n=80) received powdered Dysmo-off twice daily for 4 days (5 g one day prior to and three days after the menstruation). Treatment lasted for 4 consecutive menstrual cycles. Haemoglobin, ESR and ultrasound were measured at baseline during study. All subjects were clinically studied.(23)

5. Thermographic evaluation: - In the present study, the authors used thermography to evaluate the effects of herbal formulations based on "Sho" scientifically. In the cases that were suitable for Keishibukuryogan, the so called Keishibukuryogan Sho, a significant skin temperature rise was observed in the upper half of the body after the intake of Keishibukuryogan. In a case that was suitable for Hochuekkito, a marked elevation of skin temperature spread through the upper trunk. It suggested that thermography is useful for an objective evaluation of Sho in Kampo medicines, and for identification of the action site of the herbal formulation.(24) 6. Biochemical evaluation: - Most of the herbal drugs are a mixture of a number of ingredients. Their cumulative effect increases the efficacy of the drug in curing the diseases. Muthu Marunthu is an herbal formulation comprising of eight various plant ingredients, and has been claimed to possess anticancer effect. It was observed that the growth rate in rats was normal and there was no change in blood parameters such as glucose, urea, proteins, cholesterol and also in the activities of pathophysiological enzymes such as lactate dehydrogenase (LDH), gluconate oxaloacetate transaminase (GOT), glutamate pyruvate transaminase (GPT), alkaline and acid phosphatase after Muthu Marunthu administration. The tumor weight was found to be reduced in methylcholanthrene induced fibrosarcoma rats after Muthu Marunthu treatment. (25) 7. Evaluation of Kutaj-Ghanavati for alkaloidal principles:- Kutaj-Ghanavati is a reputed Ayurvedic preparation used in dysentery and diarrhea. It contains water extract of Kurchi bark and fine powder of aconite roots. It was evaluated quantitatively and qualitatively employing TLC and titrimetric method. In TLC study no interference of Kurchi and Aconite alkaloids with one another in their respective solvent systems. The formulation was found to contain all alkaloids of Kurchi and Aconite. (26) 8. Organoleptic evaluation: - Organoleptic evaluation of food products plays an important role in judging the censoring acceptability or rejection of food items in the market. Effect of various treatments (blanching, pricking, and lye treatment), sugar concentration (50%, 60%, 70%) and storage on the color scores; flavor scores; texture scores of intermediate moisture apricots. The overall acceptability of the products was significantly higher in 70% sugar syrup but these scores decreased as the storage period advanced. (27) CONCLUSION: - The subject of herbal drug standardization is massively wide and deep. There is so much to know and so much seemingly contradictory theories on the subject of herbal medicines and its relationship with human physiology and mental function. For the purpose of research work on standardization of herbal formulations, a profound knowledge of the important herbs found in India and widely used in Ayurvedic formulation is of utmost importance.(6) Even when the chemical composition of a plant extract is known, the pharmacologically active moiety may not be. Environment, climate, and growth conditions influence composition, as does the specific part of the plant and its maturity. Monographs detailing standardization of active ingredients would improve the marketplace. Even if an herbal product is standardized to, for example, 4% of a constituent, the remaining 96% of ingredients is not standardized and may affect the product's

solubility, bioavailability, stability, efficacy and toxicity. Just as controlled trials are necessary to establish safety and efficacy, manufacturing standards are required to ensure product quality.(28) Now a days newer and advanced methods are available for the standardization of herbal drugs like fluorescence quenching, combination of chromatographic and spectrophotometric methods, biological assays, use of biomarkers in fingerprinting etc. Bioassay can play an important role in the standardization of herbal drugs and can also become an important quality control method as well as for proper stability testing of the product.(4) India can emerge as the major country and play the lead role in the production of standardized, therapeutically effective ayurvedic formulation. India needs to explore the medicinally important plants. This can be achieved only if the herbal products are evaluated and analyzed using sophisticated modern techniques of standardization such as UV- visible, TLC, HPLC, HPTLC, GC-MS, spectrofluorimetric and other methods.(6) References 1. Sagar Bhanu P.S., Zafar R., Panwar R., "Herbal drug standardization", The Indian Pharmacist, vol. 4(35), May 2005, 2005, pp.19-22. 2. Patel P.M., Patel N.M., Goyal R.K., "Evaluation of marketed polyherbal antidiabetic formulations uses biomarker charantin", The Pharma Review, vol.4 (22), June 2006, pp.113. 3. Patel P.M., Patel N.M., Goyal R.K., "Quality control of herbal products", The Indian Pharmacist, vol.5(45), March 2006, pp.26-30. 4. Bhutani K.K., "Herbal medicines an enigma and challenge to science and directions for new initiatives", Indian Journal of Natural Products, vol.19 (1), March 2003, pp.3-8. 5. Kokate C.K., Purohit A.P., Gokhale S.B., "Analytical pharmacognosy", Pharmacognosy, 30th edition, Feb. 2005, pp.1,99. 6. Shrikumar S., Maheshwari U., Sughanti A., Ravi T.K., "WHO guidelines for herbal drug standardization", 2006. 7. Ansari S.H., "Standardization of crude drugs", Essentials of Pharmacognosy, Ist edition, 2005-06, pp.14, 581. 8. Gokhale S.B., Surana S.J., "Fluorescence quenching as a tool for identification and quality control of crude drugs", Planta indica, vol 2 (3), July 2006, pp.47. 9. Shaw P.C., Pui-Hat Butt P., "Authentication of Panax species and their adulterants by random primed polymerase chain reaction", Planta Medica, vol. 61, 1995, pp.466-469. 10. Lazarowych N.J., Pekos P., "Use of fingerprinting and marker compounds for identification and standardization of botanical drugs: Strategies for applying pharmaceutical HPLC analysis to herbal products", Drug Information Journal, Vol.32, 1998, pp.497-512. 11. Apers S., Naessens T., Pieters L., Vlietinck A., "Densitometric thin-layer chromatographic determination of aescin in an herbal medicinal product containing Aesculus and Vitis dry extract", Jr. of Chromatographic Analysis, vol.1112(1-2), April 2006, pp.165-170. 12. Nair V.D., Kanfer I., Hoogmartens J., "Determination of stigmasterol, beta-sitosterol and stigmastanol in oral dosage forms using HPLC with evaporative light scattering detection", Journal of pharmaceutical and biomedical analysis, vol. 41(3), June 2006, pp. 731-737.

13. Sarmani S.B., Abugassa I., Hamzah A., Yahya M.D., "Elemental analysis of herbal preparations for traditional medicines by neutron activation analysis with the kO standardization method", Biological trace element research, 1999, pp. 365-376. 14. Careri M., Elviri L., Mangia A., "Liquid chromatography-UV determination and liquid chromatographyatmospheric pressure chemical ionization mass spectrometric characterization of sitosterol and stigmasterol in soyabean oil", Jr. of Chromatographic Analysis, vol. 935(1-2), Nov.2001, pp.249257. 15. Yu B.S., Lai S.G., Tan QL, "Simultaneous determination of cinnamaldehyde, eugenol and paeonol in traditional Chinese medicinal preparations by capillary GC-FID", Chemical and pharmaceutical bulletin, vol. 54(1), Jan 2006, pp. 114-116. 16. Santosh M.K., Shaila D., Sanjeeva Rao I., "Standardization study of dadimastaka and pushyannga churnas", Asian Jr. of Chemistry, vol. 16(34), 2004, pp. 1735-1741. 17. Azamathulla Shaik, Hule Amolkumar, "Evaluation of adaptogenic activity profile of herbal preparation", Indian Jr. of Experimental Biology, vol. 44, July 2006, pp.574-579. 18. Zaporozhets O.A., Lipkovska N.A., "A new test method for the evaluation of total antioxidant activity of herbal products", Jr. of Agricultural and Food Chemistry, vol. 52(1), 2004, pp.21-25. 19. De Souza T.P., Zulian Lionzo M.I., "Evaluation of microbial contamination reduction on plants through technological process of decoction and spray dry", Brazilian Jr. of Pharmacognosy, vol. 16(1), 2006, pp.94-98. 20. Basu S., Hazra B., "Evaluation of nitricoxide scavenging activity of selected medicinal plants used in inflammatory diseases", Phytotherapy research, vol. 20(10), 2006, pp. 896-900. 21. Shinde A.D., Bhise S.B., "Evaluation of wound healing activity of herbal drug combination of Tridax Procumbens, Azadirachta indica, Curcuma longa and Apis mellifera", Indian Drugs, vol. 41(6), June 2004, pp.376-378. 22. Adhiraj N., Ravikumar T., Shanmugasundaram N., "In vivo and in vitro evaluation of hair growth potential of Shoe flower", Jr. of ethanopharmacology, vol. 88(2-3), 2003, pp. 235-239. 23. Nazar H., Usmanghani K., "Clinical evaluation toi assess the safety and efficacy of coded herbal medicine "Dysmo-off" versus allopathic medicine "Diclofenac sodium" for the treatment of primary dysmenorrhoea", Jr. Herb Pharmacotherapy, vol. 6(1), 2006, pp.21-39. 24. Inokawa M., Iguchi K., Kohda H., "Thermographic evaluation of the efficacy of Kampo medicine", Hiroshima Jr. Med Sci., vol. 55(1), March 2006, pp.1-8. 25. Palani V., Senthilkumaran R.K., "Biochemical evaluation in antitumour effect of Muthu Marunthu on experimental fibrosarcoma in rats", Jr. of Ethanopharmacology, vol. 65(3), 1999, pp.257-265. 26. Bhavasar G.C., Pundarikakshudu K., "Evaluation of Kutaj-Ghanvati for alkaloidal principles", Indian Jr. Natural Products, vol. 20(1), 2003, pp.33. 27. Sharma H.R., Verma P., "Organoleptic and chemical evaluation of osmotically processed Apricot wholes and halves", Natural Product Radiance, vol.5 (3), Sep-Oct 2006, pp.350-356. 28. Boullata I.J., Nace M.A., "Safety issue with herbal medicine", Pharmacotherapy, vol. 20(3), 2000, pp.257-269

Ayurveda (Sanskrit: ; yurveda, "the knowledge for long life"; /a.rved/[1]) or ayurvedic medicine is a system of traditional medicine native to India and a form of alternative medicine.[2][3] The earliest literature on Indian medical practice appeared during the Vedic period in India,[3] i.e., in the mid-second millennium BCE. The Suruta Sahit and the Charaka Sahit, encyclopedias of medicine compiled from various sources from the mid-first millennium BCE to about 500 CE,[4] are among the foundational works of Ayurveda. Over the

following centuries, ayurvedic practitioners developed a number of medicinal preparations and surgical procedures for the treatment of various ailments.[5] Current practices derived (or reportedly derived) from Ayurvedic medicine are regarded as part of complementary and alternative medicine.[6] Safety concerns have been raised about Ayurveda, with two U.S. studies finding about 20% of Ayurvedic treatments contained toxic levels of heavy metals such as lead, mercury and arsenic. Other concerns include the use of herbs containing toxic compounds and the lack of quality control in Ayurvedic facilities.[7][8]

Contents
[hide]

1 Approach 2 Practices o 2.1 Balance o 2.2 Diagnosis o 2.3 Hygiene o 2.4 Treatments o 2.5 Srotas 3 History 4 Current status o 4.1 India o 4.2 Sri Lanka o 4.3 Outside South Asia o 4.4 Journals 5 Scientific evidence 6 Safety 7 References 8 Further reading 9 External links

[edit] Approach

The three doas and the 5 elements from which they are composed.

At an early period, Ayurveda adopted the physics of the "five elements" (Devangar: [

); Pthv (earth), Jala(water), Agni (fire), Vyu (air) and ka (Sky)) that compose

the universe, including the human body.[2] Chyle or plasma (called rasa dhtu), blood (rakta dhtu), flesh (msa dhtu), fat (medha dhtu), bone (asthi dhtu), marrow (majja dhtu), and semen or female reproductive tissue (ukra dhtu) are held to be the seven primary constituent elements saptadhtu (Devangar: ) of the body.[9] Ayurvedic literature deals elaborately with measures of healthful living during the entire span of life and its various phases. Ayurveda stresses a balance of three elemental energies or humors: Vyu vta (air & space "wind"), pitta (fire & water "bile") and kapha (water & earth "phlegm"). According to ayurvedic medical theory, these three substances doas (literally that which deteriorates Devangar: )are important for health, because when they exist in equal quantities, the body will be healthy, and when they are not in equal amounts, the body will be unhealthy in various ways. One ayurvedic theory asserts that each human possesses a unique combination of doas that define that person's temperament and characteristics. Another view, also present in the ancient literature, asserts that humoral equality is identical to health, and that persons with preponderances of humours are proportionately unhealthy, and that this is not their natural temperament. In ayurveda, unlike the Skhya philosophical system, there are 20 fundamental qualities, gua (Devangar: , meaning qualities) inherent in all substances.[10] Surgery and surgical instruments were employed from a very early period,[10] Ayurvedic theory asserts that building a healthy metabolic system, attaining good digestion, and proper excretion leads to vitality.[10] Ayurveda also focuses on exercise, yoga, and meditation[11] The practice of panchakarma (Devangar: elements from the body.[12] ) is a therapeutic way of eliminating toxic

As early as the Mahbhrata, ayurveda was called "the science of eight components" (Skt. aga, Devangar: ), a classification that became canonical for ayurveda. They are:[13]

Internal medicine (Kya-cikits) Paediatrics (Kaumrabhtyam) Surgery (alya-cikits) Eye and ENT (lkya tantra) Bhta vidy has been called psychiatry.[3] Toxicology (Agadatantram) Prevention of diseases and improving immunity and rejuvenation (rasayana) Aphrodisiacs and improving health of progeny (Vajikaranam)

In Hindu mythology, the origin of ayurvedic medicine is attributed to Dhanvantari, the physician of the gods.[14]

[edit] Practices

Several philosophers in India combined religion and traditional medicinenotable examples being that of Hinduism and ayurveda. Shown in the image is the philosopher Nagarjunaknown chiefly for his doctrine of the Madhyamaka (middle path)who wrote medical works The Hundred Prescriptions and The Precious Collection, among others.[15]

[edit] Balance
Hinduism and Buddhism have been an influence on the development of many of ayurveda's central ideas particularly its fascination with balance, known in Buddhism as Madhyamaka (Devangar: ).[16] Balance is emphasized; suppressing natural urges is seen to be unhealthy, and doing so claimed to lead to illness.[16] However, people are cautioned to stay within the limits of reasonable balance and measure.[16] For example, emphasis is placed on moderation of food intake,[2] sleep, sexual intercourse.[16]

[edit] Diagnosis
The Charaka Samhita recommends a tenfold examination of the patient.[17]

constitution abnormality essence stability body measurements diet suitability psychic strength

digestive capacity physical fitness age

In addition, Chopra (2003) identifies five influential criteria for diagnosis:[17]

origin of the disease prodrominal (precursory) symptoms typical symptoms of the fully developed disease observing the effect of therapeutic procedures the pathological process'

Ayurvedic practitioners approach diagnosis by using all five senses.[17] Hearing is used to observe the condition of breathing and speech.[9] The study of the lethal points or marman marma is of special importance.[10] Ayurvedic doctors regard physical and mental existence together with personality as a unit, each element having the capacity to influence the others. One of the fundamental aspects of ayurvedic medicine is to take this into account during diagnosis and therapy.

[edit] Hygiene
Hygiene is an Indian cultural value and a central practice of ayurvedic medicine. Hygienic living involves regular bathing, cleansing of teeth, skin care, and eye washing. Daily anointing of the body with oil is also prescribed.[9]

[edit] Treatments

Head massage is used to apply oils.

Ayurveda stresses the use of plant-based medicines and treatments. Hundreds of plant-based medicines are employed, including cardamom and cinnamon. Some animal products may also be used, for example milk, bones, and gallstones. In addition, fats are used both for consumption and for external use. Minerals, including sulfur, arsenic, lead, copper sulfate and gold are also consumed as prescribed.[9] This practice of adding minerals to herbal medicine is known as rasa shastra. In some cases, alcohol was used as a narcotic for the patient undergoing an operation. The advent of Islam introduced opium as a narcotic.[13] Both oil and tar were used to stop bleeding.[9] Traumatic bleeding was said to be stopped by four different methods ligation of the blood vessel; cauterisation by heat; using different herbal or animal preparations locally which could facilitate clotting; and different medical preparations which could constrict the bleeding or oozing vessels. Various oils could be used in a number of ways, including regular consumption as a part of food, anointing, smearing, head massage, and prescribed application to infected areas.[18][page needed]

[edit] Srotas
Ensuring the proper functions of channels (srotas) that transport fluids from one point to another is a vital goal of ayurvedic medicine, because the lack of healthy srotas is thought to cause rheumatism, epilepsy, autism, paralysis, convulsions, and insanity. Practitioners induce sweating and prescribe steam-based treatments as a means to open up the channels and dilute the doshas that cause the blockages and lead to disease.[19]

[edit] History

The mantra Om mani padme hum written on rocks. Chanting mantras has been a feature of ayurveda since the Atharvaveda, the vedic spiritual text, was compiled.[20]

One view of the early history of ayurveda asserts that around 1500 BC, ayurveda's fundamental and applied principles got organized and enunciated. In this historical construction, Ayurveda traces its origins to the Vedas, Atharvaveda in particular, and is connected to Hindu religion. Atharvaveda (one of the four most ancient books of Indian knowledge, wisdom and culture) contains 114 hymns or formulations for the treatment of diseases. Ayurveda originated in and developed from these hymns. In this sense, ayurveda is considered by some to have divine

origin. Indian medicine has a long history, and is one of the oldest organised systems of medicine. Its earliest concepts are set out in the sacred writings called the Vedas, especially in the metrical passages of the Atharvaveda, which may possibly date as far back as the 2nd millennium BC. According to a later writer, the system of medicine was received by Dhanvantari from Brahma, and Dhanvantari was deified as the god of medicine. In later times his status was gradually reduced, until he was credited with having been an earthly king[9] named Divodasa.[21]

Cataract in human eye magnified view seen on examination with a slit lamp. Cataract surgery was known to the physician Sushruta in the early centuries of the first millennium AD, and was performed with a special tool called the jabamukhi salaka, a curved needle used to loosen the obstructing phlegm and push it out of the field of vision. The eye would later be soaked with warm butter and then bandaged.[22]

Underwood & Rhodes (2008) hold that this early phase of traditional Indian medicine identified "fever (takman), cough, consumption, diarrhea, dropsy, abscesses, seizures, tumours, and skin diseases (including leprosy)".[9] Treatment of complex ailments, including angina pectoris, diabetes, hypertension, and stones, also ensued during this period.[5][23] Plastic surgery, couching (a form of cataract surgery), puncturing to release fluids in the abdomen, extraction of foreign elements, treatment of anal fistulas, treating fractures, amputations, cesarean sections, and stitching of wounds were known.[9] The use of herbs and surgical instruments became widespread.[9] The Charaka Samhita text is arguably the principal classic reference. It gives emphasis to the triune nature of each person: body care, mental regulation, and spiritual/consciousness refinement. Other early works of ayurveda include the Charaka Samhita, attributed to Charaka.[9] The earliest surviving excavated written material which contains references to the works of Sushruta is the Bower Manuscript, dated to the 6th century AD. The Bower manuscript is of special interest to historians due to the presence of Indian medicine and its concepts in Central Asia.[24] Vagbhata, the son of a senior doctor by the name of Simhagupta,[25] also compiled his works on traditional medicine.[9] Early ayurveda had a school of physicians and a school of surgeons.[3] Tradition holds that the text Agnivesh tantra, written by the sage Agnivesh, a student of the sage Bharadwaja, influenced the writings of ayurveda.[26]

The Chinese pilgrim Fa Hsien (ca. 337422 AD) wrote about the health care system of the Gupta empire (320550) and described the institutional approach of Indian medicine, also visible in the works of Charaka, who mentions a clinic and how it should be equipped.[27] Madhava (fl. 700), Sarngadhara (fl. 1300), and Bhavamisra (fl. 1500) compiled works on Indian medicine.[24] The medical works of both Sushruta and Charaka were translated into the Arabic language during the Abbasid Caliphate (ca. 750).[28] These Arabic works made their way into Europe via intermediaries.[28] In Italy, the Branca family of Sicily and Gaspare Tagliacozzi (Bologna) became familiar with the techniques of Sushruta.[28] British physicians traveled to India to see rhinoplasty being performed by native methods.[29] Reports on Indian rhinoplasty were published in the Gentleman's Magazine in 1794.[29] Joseph Constantine Carpue spent 20 years in India studying local plastic surgery methods.[29] Carpue was able to perform the first major surgery in the western world in 1815.[30] Instruments described in the Sushruta Samhita were further modified in the Western World.[30]

[edit] Current status

A typical ayurvedic Pharmacy, Rishikesh.

[edit] India
Up to 80% of people in India use either Ayurveda or other traditional medicines.[31] In 1970, the Indian Medical Central Council Act which aims to standardize qualifications for ayurveda and provide accredited institutions for its study and research was passed by the Parliament of India.[32] In India, over 100 colleges offer degrees in traditional ayurvedic medicine.[11] The Indian government supports research and teaching in ayurveda through many channels at both the national and state levels, and helps institutionalize traditional medicine so that it can be studied in major towns and cities.[33] The state-sponsored Central Council for Research in Ayurvedic Sciences (CCRAS) has been set up to research the subject.[34] To fight biopiracy and unethical patents, the Government of India, in 2001, set up the Traditional Knowledge Digital Library as repository of 1200 formulations of various systems of Indian medicine, such as ayurveda, unani and siddha.[35][36] The library also has 50 traditional ayurveda books digitized and available online.[37]

Central Council of Indian Medicine (CCIM) a statutory body established in 1971, under Department of Ayurveda, Yoga and Naturopathy, Unani, Siddha and Homoeopathy (AYUSH), Ministry of Health and Family Welfare, Government of India, monitors higher education in ayurveda.[38] Many clinics in urban and rural areas are run by professionals who qualify from these institutes.[32]

[edit] Sri Lanka

The Sri Lankan tradition of Ayurveda is very similar to the Indian tradition. Practitioners of Ayurveda in Sri Lanka refer to texts on the subject written in Sanskrit, which are common to both countries. However, they do differ in some aspects, particularly in the herbs used. The Sri Lankan government has established a Ministry of Indigenous Medicine (established in 1980) to revive and regulate the practice of this indigenous medical science within the country [39] The Institute of Indigenous Medicine (affiliated to the University of Colombo currently offers undergraduate, postgraduate, and MD degrees in the practice of Ayurveda Medicine and Surgery, and similar degrees in unani medicine. [40] There are currently 62 Ayurvedic Hospitals and 208 central dispensaries in the public system, and they served almost 3 million people (approximately 11% of Sri Lanka's total population) in 2010. In total there are currently approximately 20,000 registered practitioners of Ayurveda in the country.[41][42] Many Sri Lankan hotels and resorts offer Ayurveda themed packages, where guests are treated to a wide array of Ayurveda treatments during their stay.

[edit] Outside South Asia

Due to different laws and medical regulations in the rest of the world, the unregulated practice and commercialization of ayurvedic medicine has raised ethical and legal issues; in some cases, this damages the reputation of ayurvedic medicine outside India.[43][44][45]

[edit] Journals
There are two PubMed-indexed journals focusing on Ayurveda, the Journal of Ayurveda and Integrative Medicine (JAIM),[46] and The International Journal for Ayurveda Research (IJAR)[47]

[edit] Scientific evidence

In studies in mice, the leaves of Terminalia arjuna have been shown to have analgesic and antiinflammatory properties.[48]

As a traditional medicine, many ayurveda products have not been tested in rigorous scientific studies and clinical trials. In India, research in ayurveda is largely undertaken by the statutory body of the Central Government, the Central Council for Research in Ayurveda and Siddha (CCRAS), through a national network of research institutes.[49] A systematic review of ayurveda treatments for rheumatoid arthritis concluded that there was insufficient evidence, as most of the trials were not done properly, and the one high-quality trial showed no benefits.[50] A review of ayurveda and cardiovascular disease concluded that the evidence for ayurveda was not convincing, though some herbs seemed promising.[51] Two varieties of Salvia have been tested in small trials; one trial provided evidence that Salvia lavandulifolia (Spanish sage) may improve word recall in young adults,[52] and another provided evidence that Salvia officinalis (Common sage) may improve symptoms in Alzheimer's patients.[53] Many plants used as rasayana (rejuvenation) medications are potent antioxidants.[54] Neem appears to have beneficial pharmacological properties.[55]

[edit] Safety
Rasa shastra, the practice of adding metals, minerals or gems to herbs, is a source of toxic heavy metals such as lead, mercury and arsenic.[7] Adverse reactions to herbs due to their pharmacology are described in traditional ayurvedic texts, but ayurvedic practitioners are reluctant to admit that herbs could be toxic and the reliable information on herbal toxicity is not readily available.[56] According to a 1990 study on ayurvedic medicines in India, 41% of the products tested contained arsenic, and 64% contained lead and mercury.[31] A 2004 study found toxic levels of heavy metals in 20% of ayurvedic preparations made in South Asia and sold in the Boston area, and concluded that ayurvedic products posed serious health risks and should be tested for heavymetal contamination.[57] A 2008 study of more than 230 products found that approximately 20%

of remedies (and 40% of rasa shastra medicines) purchased over the Internet from both US and Indian suppliers contained lead, mercury or arsenic.[7][58][59] Ayruvedic proponents believe that the toxicity of these materials is reduced through purification processes such as samskaras or shodhanas (for metals), similar to the Chinese pao zhi, although the ayurvedic technique is more complex and may involve prayers as well as physical pharmacy techniques. However, these products have nonetheless caused severe lead poisoning and other toxic effects.[7][58] Due to these concerns, the Government of India ruled that ayurvedic products must specify their metallic content directly on the labels of the product,[8] but, writing on the subject for Current Science, a publication of the Indian Academy of Sciences, M. S. Valiathan noted that "the absence of post-market surveillance and the paucity of test laboratory facilities [in India] make the quality control of Ayurvedic medicines exceedingly difficult at this time.[8]

Indian herbal market to grow by 20%

ASHOK B SHARMA Posted: Friday, Apr 04, 2008 at 1834 hrs IST Tags:

New Delhi, April 4:: Indian herbal market is registering an extremely significant growth and is likely to reach Rs.14,500 crore (Rs 145,000 million) by 2012 and exports to Rs.9,000 crore (Rs 90,000 million) with a CAGR of 20% and 25% respectively, according to findings of the Associated Chambers of Commerce and Industry of India (Assocham). In a Chamber Study on `Herbal Industry Biz Potential' has revealed that currently, the Indian herbal market size is estimated at Rs.7000 crore (Rs 70000 mn) and over Rs.3600 crore (Rs 36000 mn ) of herbal raw materials and medicines are exported by India. Assocham has organized an International Herbal Expo in Delhi on Friday in which 50 international buyers are participating The reasons cited for the herbal industry experimental growth comprises setting up of Herbal farm clusters by the government for improving quality of drugs and promotion of exports, doubling the cultivation of medicinal plants by converting existing farmland, continuous focus for R&D on product and process development and effective marketing of herbal products, the study said. The study also revealed that out of 700 plant species commonly used in India, only 20% were earlier being cultivated on commercial scale and 90% of medicinal plant used by the industries are collected from the wild. On the whole, India is stated to have 45,000 plant species (nearly 20% of the global species) occurs in the Indian sub-continent. Out of these, about 4,500 species of both higher and lower plant groups are of medicinal value. The study, however, said that the major hurdle for cultivating medicinal and aromatic plants as a sustainable agricultural profession were the lack of organized and regulated markets in India. The regulated production on

scientific lines, effective enforcement of licensing system and setting up of Export Promotion Zones (EPZ) in select states will push up exports of herbal material and medicines. Apart from that, the Indian herbal drug exporters face the stringent quality norms imposed by the EU through the Traditional Herbal Medicinal Products Directive (THMPD), Food Supplement Directive (FSD) and these directives also encouraged the high quality products and subsequently the unorganized sectors sub-standard products are rejected by them. India followed by China is the largest producer of medicinal plants, having more than 40% of global diversity. The states which are major producer of herbal plants having the highest medicinal value include Gujarat, Rajasthan, Haryana, Tamil Nadu, Andhra and the Himalayan Range. According to Assocham estimates, over 70% of the plant collections involve destructive harvesting because of the use of parts like roots, bark, wood, stem and the whole plant in case of herbs. This poses a definite threat to the genetic stocks and to the diversity of medicinal plants if biodiversity is not sustainably used. Around 70% of India's medicinal plants are found in tropical areas mostly in the various forest types spread across the Western and Eastern ghats, the Vindhyas, Chotta Nagpur plateau, Aravalis and Himalayas. Although less than 30% of the medicinal plants are found in the temperate and alpine areas and higher altitudes they include species of high medicinal value. Macro studies show that a larger percentage of the known medicinal plant occur in the dry and moist deciduous vegetation as compared to the evergreen or temperate habitats. "This will be particularly so because in the identified countries, urge for swadeshi (indigenous) herbal medicines has been rising due to their quality ingredients, availability factor and price competitiveness with virtually little side effects. Secondly, swadeshi (indigenous) herbs and medicines meet all the WHO prescribed standards and norms and thus encounter no restrictions in overseas markets to have instant acceptability from its takers", the study said. The medicines that have established export demand in economies of scale and produced with international quality norms include psyllium husk, sema leaves & pods, sandalwood chips and dust, Jojoba seeds, psyllium seeds, pyrethrum, basil, hyasop, rosemary safe, svory, galangal rhizonmes and roots. The application of these medicines is multifaceted and cure even serious aliments with little precautions and that's why are in great demand. India's share in medicinal plant export in global trade is just about 2.5% against 13% of China. The paper highlights that India has 15 Agroclimatic zones, 4700 different plant species and 15000 medicinal plants The Indian Systems of Medicine have identified 1500 medicinal plants, of which 500 species are mostly used in the preparation of drugs.