INTRODUCTION

The Chromatographic technique that being used is Gas chromatography (GC) and High performance liquid chromatography (HPLC). Chromatography which is a Latin’s means colored drawing was first used by Russia botanist, Mikhail Tswett in 1906 to describe the separation that occurred when solutions of plant pigment pass through column of calcium carbonate/alumina using petroleum ether.

Tswett conduct it by filled an open glass column with particles. Two specific materials that he found useful were calcium carbonate and alumina. By pouring his sample into the column, allowed it to pass into the column that the particle was placed and followed by pouring 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 compound’s chemical attraction to the particles. The compounds that were more strongly attracted to the particles will slow 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.

In 1941, Martin and Synge predicted that liquid partition chromatography mobile phase can also be vapor not only liquid. In 1951, Martin and his co-worker A.T. James bring the concept into reality practical when they published their paper describing the first GC and demonstrated the technique by separating and quantitatively determining the component of C1-C12 fatty acid mixture. Due to that, the importance of GC was recognized almost immediately by petrochemical laboratory, which faced the challenge of analyzing complex hydrocarbon mixture.

GC is conduct by placing a small amount of the sample into a hot injector port using a syringe. The injector is set to a temperature higher than the components’ boiling points.  So, components of the mixture evaporate into the gas phase inside the injector.  A carrier gas, such as helium (inert gas), flows through the injector and push the gas components of the sample onto the GC column.  It is within the column that separation of the components takes place.  Molecules partition between the carrier gas (mobile phase) and the high boiling liquid (the stationary phase) within the GC column. After components of the mixture move through the GC column, they reach a detector.  Ideally, components of the mixture will reach the detector at varying times due to differences in the partitioning between mobile and stationary phases.  The detector sends a signal to the chart recorder which results in a peak on the chart paper.  The area of the peak is proportional to the number of molecules generating the signal. That is basically how HPLC and GC was conducted.

Uses of HPLCThis technique is used for chemistry and biochemistry research analyzing complex

mixtures, purifying chemical compounds, developing processes for synthesizing chemical compounds, isolating natural products, or predicting physical properties. It is also used in quality control to ensure the purity of raw materials, to control and improve process yields, to quantify assays of final products, or to evaluate product stability and monitor degradation.

In addition, it is used for analyzing air and water pollutants, for monitoring materials that may jeopardize occupational safety or health, and for monitoring pesticide levels in the environment. Federal and state regulatory agencies use HPLC to survey food and drug products, for identifying confiscated narcotics or to check for adherence to label claims.

Principle of HPLCThe components of a basic high-performance liquid chromatography [HPLC] system are shown in the simple diagram in figure.

High-Performance Liquid Chromatography [HPLC] System

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 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 elute containing that purified compound for further study. This is called preparative chromatography

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. 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. 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.

 

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 below

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.

 

Understanding How a Chromatographic Column Works – Bands

 

The function of main component

Injector and the path

The function of the injector is to place the sample into the high-pressure flow in as narrow volume as possible so that the sample enters the column as a homogeneous, low-volume plug. To minimize spreading of the injected volume during transport to the column, the shortest possible length of tubing should be used from the injector to the column. When an injection is started, an air actuator rotates the valve: solvent goes directly to the column; and the injector needle is connected to the syringe. The air pressure lifts the needle and the vial is moved into position beneath the needle. Then, the needle is lowered to the vial.

Column

The column is one of the most important components of the HPLC chromatograph because the separation of the sample components is achieved when those components pass through the column. The High performance liquid chromatography apparatus is made out of stainless steel tubes with a diameter of 3 to 5mm and a length ranging from 10 to 30cm. Normally, columns are filled with silica gel because its particle shape, surface properties, and pore structure help to get a good separation. Silica is wetted by nearly every potential mobile phase, is inert to most compounds and has a high surface activity which can be modified easily with water and other agents. Silica can be used to separate a wide variety of chemical compounds, and its chromatographic behavior is generally predictable and reproducible.

Pump

The role of the pump is to propel (force) a liquid (the mobile phase) through the chromatograph at a specific flow rate, expressed in ml/min. Normal flow rates in HPLC are 1-2 ml/min and typical pumps can reach pressures in the range of 2000 – 9000 psi but in applications covered under UHPLC mode operating pressure can be as high as 15000-18000 psi. The normal flow rate stability must be < 1%.

An ideal pump should have the following characteristics:

Solvent compatibility and resistance to corrosion 

Constant flow delivery independent of back pressure 

Low dead volume for minimum problems on solvent changeover

The main types of pumps used in HPLC (or in LC) are the following:

Constant Pressure Pumps: Provide consistent continuous flow rate through the column with the use of pressure from a gas cylinder.

Constant Flow Pumps: a) Reciprocating Piston pumps deliver solvents through reciprocating motion of a piston in a hydraulic chamber. The main drawback of a reciprocating pump is that it produces a pulsing flow. With a flow-sensitive detector, such as micro-adsorption detector, a pulse damping system must be used and detector sensitivity is reduced. |

b) Syringe type pumps are suitable for small bore columns. Constant flow rate is delivered to column by a motorized screw arrangement.

The pump should be inert to solvents, buffer salts and solutes. They are made of stainless steel, titanium and resistant minerals.

Function of GC

Gas chromatography - specifically gas-liquid chromatography - involves a sample being vaporized and injected onto the head of the chromatographic column. The sample is transported through the column by the flow of inert, gaseous mobile phase. The column itself contains a liquid stationary phase which is adsorbed onto the surface of an inert solid.

Principle of Gas ChromatographyThe components of a basic Gas Chromatography (GC) system are shown in the simple diagram in figure.

Gas chromatography (GC), is a common type of chromatography used in analytical chemistry for separating and analyzing compounds that can be vaporized without decomposition.

Typical uses of GC include testing the purity of a particular substance, or separating the different components of a mixture (the relative amounts of such components can also be determined). In some situations, GC may help in identifying a compound. In preparative chromatography, GC can be used to prepare pure compounds from a mixture.

In gas chromatography, the mobile phase (or "moving phase") is a carrier gas, usually an inert gas such as helium or an unreactive gas such as nitrogen. The stationary phase is a microscopic layer of liquid or polymer on an inert solid support, inside a piece of glass or metal tubing called a column (a homage to the fractionating column used in distillation). The instrument used to perform gas chromatography is called a gas chromatograph (or "aerograph", "gas separator").

How does gas chromatography work?

Like for all other chromatographic techniques, a mobile and a stationary phase are required for this technique. The mobile phase (=carrier gas) is comprised of an inert gas i.e., helium, argon, or nitrogen. The stationary phase consists of a packed column where the packing or solid support itself acts as stationary phase, or is coated with the liquid stationary phase (=high boiling polymer). Most analytical gas chromatographs use capillary columns, where the stationary phase coats the walls of a small-diameter tube directly (i.e., 0.25 m film in a 0.32 mm tube).

The separation of compounds is based on the different strengths of interaction of the compounds with the stationary phase (“like-dissolves-like”-rule). The stronger the interaction is, the longer the compound interacts with the stationary phase, and the more time it takes to migrate through the column (=longer retention time). In the example above, compound X interacts stronger with the stationary phase, and therefore lacks behind compound O in its movement through the column. As a result, compound O has a much shorter retention time than compound X.

The function of main componentCarrier gas

The carrier gas must be chemically inert. Commonly used gases include nitrogen, helium, argon, and carbon dioxide. The choice of carrier gas is often dependant upon the type of detector which is used. The carrier gas system also contains a molecular sieve to remove water and other impurities.

Sample injection port

For optimum column efficiency, the sample should not be too large, and should be introduced onto the column as a "plug" of vapour - slow injection of large samples causes band broadening and loss of resolution. The most common injection method is where a microsyringe is used to inject sample through a rubber septum into a flash vapouriser port at the head of the column. The temperature of the sample port is usually about 50°C higher than the boiling point of the least volatile component of the sample. For packed columns, sample size ranges from tenths of a microliter up to 20 microliters. Capillary columns, on the other hand, need much less sample, typically around 10-3 mL. For capillary GC, split/splitless injection is used. Have a look at this diagram of a split/splitless injector;

The injector can be used in one of two modes; split or splitless. The injector contains a heated chamber containing a glass liner into which the sample is injected through the septum. The carrier gas enters the chamber and can leave by three routes (when the injector is in split mode). The sample vapourises to form a mixture of carrier gas, vapourised solvent and vapourised solutes. A proportion of this mixture passes onto the column, but most exits through the split outlet. The septum purge outlet prevents septum bleed components from entering the column.

Columns

There are two general types of column, packed and capillary (also known as open tubular). Packed columns contain a finely divided, inert, solid support material (commonly based on diatomaceous earth) coated with liquid stationary phase. Most packed columns are 1.5 - 10m in length and have an internal diameter of 2 - 4mm.

Capillary columns have an internal diameter of a few tenths of a millimeter. They can be one of two types; wall-coated open tubular (WCOT) or support-coated open tubular (SCOT). Wall-coated columns consist of a capillary tube whose walls are coated with liquid stationary phase. In support-coated columns, the inner wall of the capillary is lined with a thin layer of support material such as diatomaceous earth, onto which the stationary phase has been adsorbed. SCOT columns are generally less efficient than WCOT columns. Both types of capillary column are more efficient than packed columns.

Column temperature

For precise work, column temperature must be controlled to within tenths of a degree. The optimum column temperature is dependant upon the boiling point of the sample. As a rule of thumb, a temperature slightly above the average boiling point of the sample results in an elution

time of 2 - 30 minutes. Minimal temperatures give good resolution, but increase elution times. If a sample has a wide boiling range, then temperature programming can be useful. The column temperature is increased (either continuously or in steps) as separation proceeds.

Detectors

There are many detectors which can be used in gas chromatography. Different detectors will give different types of selectivity. A non-selective detector responds to all compounds except the carrier gas, aselective detector responds to a range of compounds with a common physical or chemical property and a specific detector responds to a single chemical compound. Detectors can also be grouped intoconcentration dependant detectors and mass flow dependant detectors. The signal from a concentration dependant detector is related to the concentration of solute in the detector, and does not usually destroy the sample Dilution of with make-up gas will lower the detectors response. Mass flow dependant detectors usually destroy the sample, and the signal is related to the rate at which solute molecules enter the detector. The response of a mass flow dependant detector is unaffected by make-up gas.

Sample PreparationFundamental theory

The sample should be dissolved in the eluent to be used and filtrated with 0.45μm disposable filter. Then, it should be injected into HPLC/GC system about 10 to 50μL. When the sample is dissolved in the eluent, good separation can be expected even for the sample whose peaks appear near Vo because the system peak will not appear near Vo. (When the column size is 4.6mmI.D. x 150 to 250mm length). Avoid overloading, not too much sample should not be injected. If peak shape changes by diluting the sample 10 to 100 folds or by decreasing the injection volume to one fifth, there is a possibility of sample overloading.

The injection volume of standards and that of actual sample should be the same. If they are different, retention times may be different. Some troubles may happen when the actual sample is analyzed though the troubles never happen when standards are analyzed. In most cases, they are caused by inappropriate selection of the solvent in which the sample is dissolved.

Troubles

(a) Leading or tailing of peaks.

(b) Instability of retention time.

(c) Split of peak top.

(d) Very broad ghost peak.

(e) Increase of column pressure.

(f) The above troubles happen just for a specific sample (or peak).

The sample preparation in general

Reversed phase

Consider the situation that acetonitrle/water = 50/50 is used as the eluent and sample is easy to be dissolved in acetonitrile. Of course, the best way is to dissolve the sample in the eluent. However, the sample cannot be dissolved in the eluent, what solvent should be selected to dissolve the sample? 100% acetonitrile? Or, acetonitrile solution less than 50%? In this case, the first choice should be "acetonitrile solution less than 50%". The second choice is "100% water" and it can be used up to 500μL. The worst choice is "100% acetonitrile". When the sample is dissolved in 100% actonitrile, there is a possibility that the sample educe in the column. Even if the sample is completely dissolved in 100% acetonitrile, it may educe in the column when the acetonitrile concentration decrease in the column. If such eduction takes place, first, trouble (a) and/or (b), and then, trouble (c) and/or (d) may happen. In the worst case, (f) may happen and it may damage the column. In case that the sample is a mixture, (g) may happen.In order to judge whether sample eduction is taking place in the column, reduce the concentration of acetonitrile and increase the injection volume. For example, instead of injecting 25μL of sample dissolved in 100% actonitrile, inject 125μL of sample dissolved in 20% acetonitrile. If the trouble is solved, it is considered that sample eduction is taking place in the column. And, in such case, the selection 100% acetonitrile is inappropreate.

Normal phase

Consider the situation that 15% dichloromethane/hexsane is used as the eluent and sample is easy to be dissolved in dichloromethane. The best way is to dissolve the sample in the eluent.

If the sample cannot be dissolved in the eluent and 100% dichloromethane is selected as the solvent in which sample is dissolved, some trouble may happen.

In reversed phase or normal phase chromatography, usually, a mixture of some solvents is used as the eluent. The solvent in which the sample dissolves more easily advances the elution time. Before selecting a solvent in which sample is dissolved, it is recommended to change the eluent composition by 5 to 10% and check the elution time. Of course, it is neccesssary that the solvent can be dissolved in the eluent.

Sample preparation for GC and HPLC

Usually, a sample must be in a liquid state prior to HPLC or GC analysis. Some insoluble solids contain soluble analytes such as additives in a polymer, fats in food, and poly-aromatic hydrocarbons in soil. Contacting the sample with solvent allows the extraction of analytes into the solvent, following which the solvent is separated from the solid residue by decanting, filtration, or centrifugation. The solution is further treated, if necessary, prior to HPLC or GC analysis. No one solvent extraction technique can be used for all samples. Most of these technique have been used for more than 100 years, are time tested, and accepted by most scientists. Regulatory agencies such as the United States environmental Protection Agency (U.S. EPA), the Food and Drug Administration (FDA), and their equivalents in other countries readily approve these classical methods for extracting solid samples. However, these methods often use large amounts of organic solvents, which have encouraged a trend toward miniaturization in recent years. In addition, some of the older extraction techniques require more glassware and labor.

Sample Preparation Methods for Solid Samples

Traditional Extraction Methods

Solvent extraction can assume many forms. The shake-flask method, which involves addition of a solvent to the sample followed by agitation, works well when the analytes is highly soluble in the extraction solvent and the sample is quite porous. For fast extraction, the sample should be finely divided. Heating or refluxing the sample in the solvent can speed up extraction. For faster and more complete extraction, ultrasonic agitation (sonication) often allows more effective solid-liquid contact, plus a gentle heating which aids extraction.

Modern Extraction Method

Most of these newer methods are based on performing solid extractions at increased temperatures and pressures. Table below provides background information on how these parameters accelerate extractions.

1974, Edward Randall made a major improvement in the extraction technique that reduced the extraction time dramatically. In his method, the sample was totally immersed in the boiling solvent. Compared to the classical method where the condensed extracting solvent’s temperature is slightly below the boiling point, the Randall method was faster because analytes are more soluble in hot solvent than in warm solvent. First, the thimble is lowered into the boiling solvent until the appropriate extraction takes place. Then, to flush residual extract from the sample, a rinse step follows. In this second stage, the thimble is raised above the boiling solvent for a period of time until residual extract is removed from the solid material by the condensed solvent, just as is performed in the original method. Finally, the drying step removes the solvent from the solvent flask and concentrates the analytes for further processing. In this step, by closing a solvent return valve, the condensed solvent is redirected away from the sample and boiling solvent and collected in a reservoir for possible re-use or disposal. In some systems, there is a fourth step where the sample cup is lifted from the heat source and allowed to evaporate further without the chance of sample overheating, boiling dry, or potential oxidation.

Sample Preparation Methods for Volatile Samples

Gaseous samples comprise a great number of the samples encountered in typical gas chromatography analyses. Volatile organics are among the compounds most often analyzed in the petroleum, petrochemical, food, flavor and fragrances, and environmental segments. In this chapter, an overview is provided of the methods that are used to collect and prepare such samples for introduction into the gas chromatographic instrument and occasionally into HPLC systems.

A sample preparation procedure would be a modification or treatment of the sample prior to sample introduction for the purpose of improving the introduction process into the GC. For example, performing a dynamic headspace process with an intermediate cold adsorbent trap to concentrate the analytes of interest prior to sample introduction into the GC would constitute a sample preparation procedure. Derivatizing an adsorbed polar compound on a solid material, thereby releasing it into vapor phase for subsequent sampling, would be a sample preparation process. Whatever you call it, it is most important that the components of interest be transferred from the point of collection to the GC column without being lost or modified.

Headspace Technique

Headspace refers to the vapors that form over liquids and solids. If the sample is in thermodynamic equilibrium with the gas phase in a closed thermostatic vessel, this method of analysis is referred to static headspace sampling. If an inert gas is passed through or over the sample and the stripped sample volatiles accumulated in an adsorbent trap or cryogenic trap, then the method is referred to as dynamic headspace or purge and trap. Since only the volatiles are sampled, headspace analysis is ideal for dirty samples, solid materials, samples with high boilers of no interest, samples with high water content, or samples that are difficult to handle by conventional chromatographic methods. These matrices remain behind since only the volatile portion of the sample is in the headspace. In headspace sampling, calibrations are performed by preparing a solution of the analytes in a volatile solvent then injecting a small known amount into the closed headspace container so that the entire solution is vaporized.

Dynamic headspace sampling continuously removes headspace vapors above a liquid or solid sample. The flow of gas over the sample (purging) will further volatilize analytes that can be trapped by an adsorbent or by cryogenic means. The trapping process will refocus (concentrate) the volatiles which are then re-volatilized into the gas chromatograph by thermal desorption. The dynamic process of purge and trap is particularly useful for analytes that are too low in concentration to be measured by static headspace methods. Purge and trap, or gas phase stripping, generally refers to the process where urging gas is bubbled below the surface of a liquid sample using a fritted orifice to produce finely dispersed bubbles.

The proper selection of a trap is an important consideration in ensuring that the desired analytes are quantitatively recovered. An adsorbent trap (typically a glass tube) is filled with porous sorbent material (typically 50 mg to one gram) that could be a polymer, carbon, silica gel, or alumina. Polymeric materials sometimes need to be cleaned prior to use in order to remove residual monomer. Tenax is especially useful since it is hydrophobic, water is un-retained, and it has a high sample capacity. Carbon materials are excellent, high capacity traps, but sometimes irreversibly adsorb certain classes of analytes. Silica gel and alumina have high capacity but take up water. These traps containing adsorbent may also be used off-line to collect volatile organic samples from air and then transported back to the laboratory for subsequent analysis. As mentioned above, analytes are transferred to the GC by thermal desorption, perhaps coupled with cold trapping via cryogenics to refocus the analytes. An especially important parameter of a particular trap is the breakthrough capacity, which determines how much organic material is trapped from the gaseous sample before it elutes from the opposite end of the trap and is no longer retained. Volatile organics may pass through a trap very quickly while semi- or non-volatile organics may adsorb strongly and never elute. For volatile organics, the trap may be cryogenically cooled or a different adsorbent material may be used that has a higher breakthrough volume for the analytes.