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High-performance liquid chromatography:

High-performance liquid chromatography (or high-pressure liquid chromatography, HPLC) is a chromatographic technique that can separate a mixture of compounds and is used in biochemistry and analytical chemistry to identify, quantify and purify the individual components of the mixture. HPLC typically utili es different types of stationary phases, a pump that moves the mobile phase(s) and analyte through the column, and a detector to provide a characteristic retention time for the analyte. !he detector may also provide additional information related to the analyte, (i.e. "#$#is spectroscopic data for analyte if so equipped). %nalyte retention time varies depending on the strength of its interactions &ith the stationary phase, the ratio$composition of solvent(s) used, and the flo& rate of the mobile phase. 't is a form of liquid chromatography that utili es smaller column si e, smaller media inside the column, and higher mobile phase pressures. (ith HPLC, a pump (rather than gravity) provides the higher pressure required to move the mobile phase and analyte through the densely pac)ed column. !he increased density arises from smaller particle si es. !his allo&s for a better separation on columns of shorter length &hen compared to ordinary column chromatography.

Operation
!he sample to be analy ed is introduced, in small volumes, into the stream of mobile phase. !he solution moved through the column is slo&ed by specific chemical or physical interactions &ith the stationary phase present &ithin the column. !he velocity of the solution depends on the nature of the sample and on the compositions of the stationary (column) phase. !he time at &hich a specific sample elutes (comes out of the end of the column) is called the retention time* the retention time under particular conditions is considered an identifying characteristic of a given sample. !he use of smaller particle si e column pac)ing (&hich creates higher bac)pressure) increases the linear velocity giving the components less time to diffuse &ithin the column, improving the chromatogram resolution. Common solvents used include any miscible combination of &ater or various organic liquids (the most common are methanol and acetonitrile). (ater may contain buffers or salts to assist in the separation of the sample components, or compounds such as trifluoroacetic acid &hich acts as an ion pairing agent. % further refinement of HPLC is to vary the mobile phase composition during the analysis* gradient elution. % normal gradient for reversed phase chromatography might start at +, methanol and progress linearly to +-, methanol over .+ minutes* the gradient depends on ho& hydrophobic the sample is. !he gradient separates the sample mixtures as a function of the affinity. !his partitioning process is similar to that &hich occurs during a liquid/liquid extraction but is continuous, not step/&ise. 'n this example, using a &ater$methanol gradient, more hydrophobic components &ill elute (come off the column) &hen the mobile phase consists mostly of methanol (giving a relatively hydrophobic mobile phase).

!he choice of solvents, additives and gradient depend on the nature of the column and sample. 0ften a series of tests are performed on the sample together &ith a number of trial runs in order to find the HPLC method &hich gives the best pea) separation.

Parameters
Internal diameter
!he internal diameter ('1) of an HPLC column is an important parameter that influences the detection sensitivity and separation selectivity in gradient elution. 't also determines the quantity of analyte that can be loaded onto the column. Larger columns are usually seen in industrial applications, such as the purification of a drug product for later use. Lo&/'1 columns have improved sensitivity and lo&er solvent consumption at the expense of loading capacity.

Larger '1 columns (over 2- mm) are used to purify usable amounts of material because of their large loading capacity. %nalytical scale columns (3.4 mm) have been the most common type of columns, though smaller columns are rapidly gaining in popularity. !hey are used in traditional quantitative analysis of samples and often use a "#/#is absorbance detector. 5arro&/bore columns (26. mm) are used for applications &hen more sensitivity is desired either &ith special "#/vis detectors, fluorescence detection or &ith other detection methods li)e liquid chromatography/mass spectrometry Capillary columns (under -.7 mm) are used almost exclusively &ith alternative detection means such as mass spectrometry. !hey are usually made from fused silica capillaries, rather than the stainless steel tubing that larger columns employ.

Particle size
8ost traditional HPLC is performed &ith the stationary phase attached to the outside of small spherical silica particles (very small beads). !hese particles come in a variety of si es &ith + 9m beads being the most common. :maller particles generally provide more surface area and better separations, but the pressure required for optimum linear velocity increases by the inverse of the particle diameter squared.;7<;3<;+< !his means that changing to particles that are half as big, )eeping the si e of the column the same, &ill double the performance, but increase the required pressure by a factor of four. Larger particles are used in preparative HPLC (column diameters + cm up to =7- cm) and for non/HPLC applications such as solid/phase extraction.

Pore size
8any stationary phases are porous to provide greater surface area. :mall pores provide greater surface area &hile larger pore si e has better )inetics, especially for larger analytes. >or example, a protein &hich is only slightly smaller than a pore might enter the pore but does not easily leave once inside.

Pump pressure
Pumps vary in pressure capacity, but their performance is measured on their ability to yield a consistent and reproducible flo& rate. Pressure may reach as high as 3- 8Pa (4--- lbf$in.), or about 3-- atmospheres. 8odern HPLC systems have been improved to &or) at much higher pressures, and therefore are able to use much smaller particle si es in the columns (?. 9m). !hese @"ltra High Performance Liquid Chromatography@ systems or A:LC$"HPLCs can &or) at up to 2-- 8Pa (2+,--- lbf$inB), or about 2--- atmospheres. !he term @"PLC@ is a trademar) of the (aters Corporation, but is sometimes used to refer to the more general technique.

HPLC APPLICATION SYSTEMS


High Performance Liquid Chromatograph (HPLC) is widely used for pharmaceutical industry, food and beverage industry, research and development, quality control, production process control, environmental analyses and clinical tests. himad!u LC"#P series modular concept enables fle$ible system configuration to meet analytical purposes. %he followings are e$ample of application dedicated systems. &on Chromatography ystems Preparative Chromatography ystems 'rganic (cid (nalysis ystem

The applications are divided in the fields of: Drugs Biological and natural compounds Food analysis Environmental analysis Organic compounds

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY - HPLC


High performance liquid chromatography is a powerful tool in analysis. This page looks at how it is carried out and shows how it uses the same principles as in thin layer chromatography and column chromatography.

Carrying out HPLC


Introduction High performance liquid chromatography is basically a highly improved form of column chromatography. Instead of a solvent being allowed to drip through a column under gravity, it is forced through under high pressures of up to 400 atmospheres. That makes it much faster. It also allows you to use a very much smaller particle size for the column packing material which gives a much greater surface area for interactions between the stationary phase and the molecules flowing past it. This allows a much better separation of the components of the mixture. The other major improvement over column chromatography concerns the detection methods which can be used. These methods are highly automated and extremely sensitive.

The column and the solvent Confusingly, there are two variants in use in HPLC depending on the relative polarity of the solvent and the stationary phase. Normal phase HPLC This is essentially just the same as you will already have read about in thin layer chromatography or column chromatography. Although it is described as "normal", it isn't the most commonly used form of HPLC. The column is filled with tiny silica particles, and the solvent is nonpolar - hexane, for example. A typical column has an internal diameter of 4.6 mm (and may be less than that), and a length of 150 to 250 mm. Polar compounds in the mixture being passed through the column will stick longer to the polar silica than non-polar compounds will. The non-polar ones will therefore pass more quickly through the column. Reversed phase HPLC

In this case, the column size is the same, but the silica is modified to make it non-polar by attaching long hydrocarbon chains to its surface - typically with either 8 or 18 carbon atoms in them. A polar solvent is used - for example, a mixture of water and an alcohol such as methanol. In this case, there will be a strong attraction between the polar solvent and polar molecules in the mixture being passed through the column. There won't be as much attraction between the hydrocarbon chains attached to the silica (the stationary phase) and the polar molecules in the solution. Polar molecules in the mixture will therefore spend most of their time moving with the solvent. Non-polar compounds in the mixture will tend to form attractions with the hydrocarbon groups because of van der Waals dispersion forces. They will also be less soluble in the solvent because of the need to break hydrogen bonds as they squeeze in between the water or methanol molecules, for example. They therefore spend less time in solution in the solvent and this will slow them down on their way through the column. That means that now it is the polar molecules that will travel through the column more quickly. Reversed phase HPLC is the most commonly used form of HPLC. Looking at the whole process A flow scheme for HPLC

Injection of the sample Injection of the sample is entirely automated, and you wouldn't be expected to know how this is done at this introductory level. Because of the pressures involved, it is not the same as in gas chromatography (if you have already studied that).

Retention time The time taken for a particular compound to travel through the column to the detector is known as its retention time. This time is measured from the time at which the sample is injected to the point at which the display shows a maximum peak height for that compound. Different compounds have different retention times. For a particular compound, the retention time will vary depending on:

the pressure used (because that affects the flow rate of the solvent) the nature of the stationary phase (not only what material it is made of, but also particle size) the exact composition of the solvent the temperature of the column

That means that conditions have to be carefully controlled if you are using retention times as a way of identifying compounds.

The detector There are several ways of detecting when a substance has passed through the column. A common method which is easy to explain uses ultra-violet absorption. Many organic compounds absorb UV light of various wavelengths. If you have a beam of UV light shining through the stream of liquid coming out of the column, and a UV detector on the opposite side of the stream, you can get a direct reading of how much of the light

is absorbed. The amount of light absorbed will depend on the amount of a particular compound that is passing through the beam at the time.

You might wonder why the solvents used don't absorb UV light. They do! But different compounds absorb most strongly in different parts of the UV spectrum. Methanol, for example, absorbs at wavelengths below 205 nm, and water below 190 nm. If you were using a methanol-water mixture as the solvent, you would therefore have to use a wavelength greater than 205 nm to avoid false readings from the solvent. Interpreting the output from the detector %he output will be recorded as a series of pea)s " each one representing a compound in the mi$ture passing through the detector and absorbing *# light. (s long as you were careful to control the conditions on the column, you could use the retention times to help to identify the compounds present " provided, of course, that you (or somebody else) had already measured them for pure samples of the various compounds under those identical conditions. +ut you can also use the pea)s as a way of measuring the quantities of the compounds present. Let,s suppose that you are interested in a particular compound, -. &f you in.ected a solution containing a )nown amount of pure - into the machine, not only could you record its retention time, but you could also relate the amount of - to the pea) that was formed. %he area under the pea) is proportional to the amount of - which has passed the detector, and this area can be calculated automatically by the computer lin)ed to the display. %he area it

would measure is shown in green in the (very simplified) diagram.

&f the solution of - was less concentrated, the area under the pea) would be less " although the retention time will still be the same. /or e$ample0

%his means that it is possible to calibrate the machine so that it can be used to find how much of a substance is present " even in very small quantities. +e careful, though1 &f you had two different substances in the mi$ture (- and 2) could you say anything about their relative amounts3 Not if you were using UV absorption as your detection method.

&n the diagram, the area under the pea) for 2 is less than that for -. %hat may be because there is less 2 than -, but it could equally well be because 2 absorbs *# light at the wavelength you are using less than - does. %here might be large quantities of 2 present, but if it only absorbed wea)ly, it would only give a small pea). Coupling HPLC to a mass spectrometer %his is where it gets really clever1 4hen the detector is showing a pea), some of what is passing through the detector at that time can be diverted to a mass spectrometer. %here it will give a fragmentation pattern which can be compared against a computer database of )nown patterns. %hat means that the identity of a huge range of compounds can be found without having to )now their

retention times.

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