Common chromatography techniques.

Three types of chromatography are routinely used in the organic chemistry teaching labs:

Column Chromatography

Thin Layer Chromatography (TLC)

Gas Chromatography (GC)

In these (and all types of) chromatographies, a mixture is separated by distributing the components between a stationary phase and a mobile phase. The mixture is first placed on the stationary phase (a solid or a liquid) and then the mobile phase (a gas or a liquid) is allowed to pass through the system.

Column chromatography: The stationary phase is a powdered adsorbent which is placed in a vertical glass column. The mixture to be analysed is loaded on top of this column. The mobile phase is a solvent poured on top of the loaded column. The solvent flows down the column, causing the components of the mixture to distribute between the powdered adsorbent and the solvent, thus (hopefully) separating the components of the mixture so that as the solvent flows out of the bottom of the column, some components elute with early collections and other components elute with late fractions.

Thin Layer Chromatorgraphy: The stationary phase is a powdered adorbent which is fixed to a aluminum, glass, or plastic plate. The mixture to be analyzed is loaded near the bottom of the plate. The plate is placed in a reservoir of solvent so that only the bottom of the plate is submerged. This solvent is the mobile phase; it moves up the plate causing the components of the mixture to distribute between the adsorbent on the plate and the moving solvent, thus separating the components of the mixture so that the components are separated into separate "spots" appearing from the bottom to the top of the plate.

Gas Chromatography: The stationary phase is a high-boiling liquid. (Think of it as a viscous oil, or waxy substance.) This high-boiliing liquid is packed into a long, narrow glass or metal column. The mixture to be analyzed is loaded by syringe into the beginning of this column. The mobile phase is an inert gas which continuously flows through the column. The components of the mixture distribute between the stationary high-boiling liquid (these components are either condensed or absorbed on the high-boiling liquid) and mobile gas (vapor) phase moving through the column. The gaseous mixture flows through a detector at the end of the column and if it has been successfully separated, the components show as different 'blips' or peaks on a recorder.

In all three of these chromatographies, separation of chemical components of a mixture is achieved due to the selective interaction of chemicals with both the stationary and mobile phases:

In Gas Chromatography, the determining factor in how fast a component travels is usually (but not always) the boiling point of the compound. (If a polar high-boiling liquid adsorbent is used in the GC column, the polarity of the components determines the elution order.)

In Column and Thin Layer chromatographies, the stationary phase (the adsorbent: silica gel or alumina) is polar, and the polarities of both the component of the mixture and the solvent used as the mobile phase are the determining factors in how fast the compound travels.

Column chromatography is used to separate and purify components of a mixture. TLC and GC are usually (but not always!) used only to analyze mixtures: to determine the number of components and to see if a desired component is present. TLC is often used to determine the "ideal system" for a column chromatography procedure (as explained in the following paragraphs).

Determining solvent systems for TLC and Column Chromatography

When you need to determine the best system (a "system" means the eluting solvent, itself often a mixture of solvents) to develop a TLC plate or chromatography column loaded with an unknown mixture, vary the polarity of the solvent in several trial runs -- a process of trial and error. Carefully observe and record the results of the chromatography in each solvent system. You will find that as you increase the polarity of the solvent system, all the components of the mixture move faster (and visa versa with lowering the polarity). The ideal solvent system is simply: the system that separates the components.

TLC elution patterns usually extrapolate to column chromatography elution patterns. Since TLC is a much faster procedure than column chromatography, TLC is often used to determine the best solvent system for column chromatography. For instance, in determining the solvent system for a flash chromatography procedure, the ideal system is the one that moves the desired component of the mixture to a TLC Rf of 0.25-0.35 and will separate this component from its nearest neighbor by difference in TLC Rf values of at least 0.20. Therefore a mixture is analyzed by TLC to determine the ideal solvent(s) for a flash chromatography procedure.

Beginners often do not know where to start: What solvents should they pull off the shelf to use to elute a TLC plate? Because of toxicity, cost, and flammability concerns, the common solvents are hexanes (or petroleum ethers, ligroin) and ethyl acetate (an ester). Diethyl ether can be used, but it is very flammable and volatile. Alcohols (methanol, ethanol) can be used. Acetic acid (a carboxylic acid) can be used, usually as a small percentage component of the system, since it is corrosive, non-volatile, very polar, and has irritating vapors. Acetone (a ketone) can be used. Methylene chloride (halogenated hydrocarbon) is a good solvent, but it is toxic and should be avoided whenever possible. If two solvents are equal in performance and toxicity, the more volatile solvent is preferred in column chromatography because it will be easier to remove from the desired compound after isolation from a column chromatography procedure.

Ask the lab instructor what solvents are available and advisable. Then, mix a non-polar solvent (hexanes, a mixture of 6-carbon alkanes) with a polar solvent (ethyl acetate or acetone) in varying percent combinations to make solvent systems of greater and lesser polarity. The charts below should help you in your solvent selection. Download the pdf file (linked below the charts) for a printable version to keep for ready reference.

Column Chromatography

In column chromatography, the stationary phase, a solid adsorbent, is placed in a vertical glass (usually) column and the mobile phase, a liquid, is added to the top and flows down through the column (by either gravity or external pressure). Column chromatography is generally used as a purification technique: it isolates desired compounds from a mixture.

The mixture to be analyzed by column chromatrography is applied to the top of the column. The liquid solvent (the eluent) is passed through the column by gravity or by the application of air pressure. An equilibrium is established between the solute adsorbed on the adsorbent and the eluting solvent flowing down through the column. Because the different components in the mixture have different interactions with the stationary and mobile phases, they will be carried along with the mobile phase to varying degrees and a separation will be achieved. The individual components, or elutants, are collected as the solvent drips from the bottom of the column.

Column chromatography is separated into two categories, depending on how the solvent flows down the column. If the solvent is allowed to flow down the column by gravity, or percolation, it is called gravity column chromatography. If the solvent is forced down the column by positive air pressure, it is called flash chromatography, a "state of the art" method currently used in organic chemistry research laboratories The term "flash chromatography" was coined by Professor W. Clark Still because it can be done in a “flash."

The Adsorbent

Silica gel (SiO2) and alumina (Al2O3) are two adsorbents commonly used by the organic chemist for column chromatography. These adsorbents are sold in different mesh sizes, as indicated by a

number on the bottle label: "silica gel 60" or "silica gel 230-400" are a couple examples. This number refers to the mesh of the sieve used to size the silica, specifically, the number of holes in the mesh or sieve through which the crude silica particle mixture is passed in the manufacturing process. If there are more holes per unit area, those holes are smaller, thus allowing only smaller silica particles go through the sieve. The relationship is: the larger the mesh size, the smaller the adsorbent particles.

Adsorbent particle size affects how the solvent flows through the column. Smaller particles (higher mesh values) are used for flash chromatography, larger particles (lower mesh values) are used for gravity chromatography. For example, 70–230 silica gel is used for gravity columns and 230–400 mesh for flash columns.

Alumina is used more frequently in column chromatography than it is in TLC. Alumina is quite sensitive to the amount of water which is bound to it: the higher its water content, the less polar sites it has to bind organic compounds, and thus the less “sticky” it is. This stickiness or activity is designated as I, II, or III, with I being the most active. Alumina is usually purchased as activity I and deactivated with water before use according to specific procedures. Alumina comes in three forms: acidic, neutral, and basic. The neutral form of activity II or III, 150 mesh, is most commonly employed.

Silica gel and alumina are the only column chromatography adsorbents used in the CU organic chemistry teaching labs; please refer to the references for information on other column chromatography adsorbents.

The Solvent

The polarity of the solvent which is passed through the column affects the relative rates at which compounds move through the column. Polar solvents can more effectively compete with the polar molecules of a mixture for the polar sites on the adsorbent surface and will also better solvate the polar constituents. Consequently, a highly polar solvent will move even highly polar molecules rapidly through the column. If a solvent is too polar, movement becomes too rapid, and little or no separation of the components of a mixture will result. If a solvent is not polar enough, no compounds will elute from the column. Proper choice of an eluting solvent is thus crucial to the successful application of column chromatography as a separation technique. TLC is generally used to determine the system for a column chromatography separation. The choice of a solvent for the elution of compounds by column chromatography is covered in the Chromatography Overview section.

Often a series of increasingly polar solvent systems are used to elute a column. A non-polar solvent is first used to elute a less-polar compound. Once the less-polar compound is off the column, a more-polar solvent is added to the column to elute the more-polar compound.

Interactions of the Compound and the Adsorbent

Compounds interact with the silica or alumina largely due to polar interactions. These interactions are discussed in the section on TLC.

Analysis of Column Eluants

If the compounds separated in a column chromatography procedure are colored, the progress of the separation can simply be monitored visually. More commonly, the compounds to be isolated from column chromatography are colorless. In this case, small fractions of the eluent are collected sequentially in labeled tubes and the composition of each fractions is analyzed by thin layer chromatography. (Other methods of analysis are available; this is the most common method and the one used in the organic chemistry teaching labs.)

Column Chromatography Procedures

Columns for chromatography can be small or big, according to the amount of material which needs to be loaded onto the column. Pictured below are three glass columns, two of which are used in the organic chemistry teaching labs at CU.

The "column" on the far left in the photo is actually a Pasteur pipet. This size of column is suitable for 10-125 mg of material. The middle column is a 10 mL disposable glass pipet. You can load about a gram of material on this size column. The column on the right would be used for many grams of material.

Of the three columns pictured, only the column on the right is actually manufactured as a chromatography column. Note the stopcock at the bottom of the column. This is to control the flow of solvent through the column, important for gravity column chromatography applications.

The middle column is used for gravity column chromatography in a few of the chemistry majors' laboratory courses (chem 3361 and 3381). Note the piece of flexible tubing which has been added to the bottom of the column.To control the flow of solvent, a pinch clamp would be placed on the flexible tubing at the bottom.

The Pasteur pipet column is used for microscale gravity and microscale flash chromatrography procedures; these procedures (usually) do not require a means of control of gravity-induced solvent flow through the column.

Much larger chromatography columns are available than the one on the right. The size employed depends on the amount of material which needs to be separated. Large-scale flash columns look like this column but have a standard taper connection at the top so they can be connected to a source of pressurized air.

In the Organic Chemistry teaching labs at CU, the most frequently used column is the Pasteur pipet. They work well in microscale flash column chromatography procedures because a pipet bulb fits conveniently on top of them to serve as a source of pressurized air (when you press on the bulb!). Microscale procedures are used at CU Boulder whenever feasible to cut down on waste chemical production.

Here is a picture of a packed column of the type on the right.

Procedure for Gravity Column Chromatography

Gravity columns are not used in any of the non-major organic lab courses at CU Boulder (chem 3321/3341). The majors (chem 3361/3381) do use this type of chromatography. Gravity columns are a lot slower to run than microscale flash columns. They also are more difficult to set up or "pack" with adsorbent.

detailed gravity column chromatography procedure

Procedure for Microscale Flash Column Chromatography

Microscale flash chromatography is the method used almost solely in the organic chemistry teaching labs because it is both easy and environmentally friendly. The method is only limited by the fact that it can separate only small amounts of sample. It works best for 25 mg amounts, although we have pushed it to separate 125 mg mixtures if the TLC Rf's of the components of the mixture differ by at least 0.20.

Procedure for Gravity Column Chromatography

There are (at least) two ways to pack a gravity column: the slurry method and the dry pack method.

Slurry Method for Packing

In the slurry method of column packing, you mix the adsorbent with the solvent and then pour this slurry into the prepared column. The nature of the slurry is a bit different whether you use silica gel or alumina; some slurries are easier to work with than others. This procedure was written for alumina slurries. The advantage of slurry methods is that they eliminate air bubbles from forming in the column as it packs.

Your TA will have 10 mL disposable glass pipets available for your use. The tops of these columns have been broken off (because as purchased, they have an interfering indent near the top) and they have a small length of plastic tubing attached to the bottom. Place a piece of glass wool in the bottom of the column, and gently tamp the glass wool down with a glass rod. Attach the column to a ring stand and make sure that the column is securely fastened in a vertical position. Add a pinch clamp to the bottom of the column and close the clamp.

Fill the column about half-way with a non-polar solvent, such as hexanes. Weigh 8 g of alumina into a beaker. Place 15 mL of hexanes in a 125 mL Erlenmeyer flask and slowly add the alumina powder, a little at a time, while swirling. Use a Pasteur pipet to mix the slurry, then quickly pipet the slurry onto the column (you can pour it instead if you prefer). Place an Erlenmeyer flask under the column, open the screw clamp, and allow the liquid to drain into it. Continue to transfer the slurry to the column until all the alumina is added. Add more hexanes as necessary; the hexanes collected in the Erlenmeyer flask can be re-used to add more alumina to the column. When finished packing, drain the excess solvent until it just reaches the top level of the alumina. Close the screw clamp. Your column is now "packed."

Sometimes a small amount of sand is added to the top of the column to prevent it from being disturbed when fresh solvent is added.

Dry-pack Method for Packing

This method is easier, but can lead to bubbles in the column. Obtain an empty column, plug it with a small piece of glass wool, and affix a pinchclamp to the bottom of the column. Clamp the column in a vertical position, close the pinchclamp, and fill the column with solvent. Using a dry funnel, sprinkle 8 g of alumina into the solvent, and allow solvent to drain from the column to prevent overflowing. Let the alumina settle and gently tap the column with a pencil so that the alumina will pack tightly into the column. Drain the solvent until the solvent level is just even with the surface of the alumina.

Loading and Eluting Gravity Chromatography Columns

Briefly, the sample to be analyzed is dissolved in a very small amount of solvent and added to the top of the column. The pinch clamp is opened and the solvent is allowed to drain just to the top of the column. A couple small portions of the eluting solvent are added and allowed to drain in until the mixture is a little ways into the adsorbent, then the column is filled to the top with eluting solvent. The column is now ready to run -- continue adding solvent at the top and collecting fractions at the bottom until the compounds elute at the bottom. If applicable, change the eluting solvent to a more polar solvent during the eluting process. Never let the solvent level drop below the top of the adsorbent. The process is discontinued when the compound(s) desired is (are) off the column.

Procedure for Microscale Flash Column Chromatography

In microscale flash chromatography, the column does not need either a pinchclamp or a stopcock at the bottom of the column to control the flow, nor does it need air-pressure connections at the top of the column. Instead, the solvent flows very slowly through the column by gravity until you apply air pressure at the top of the column with an ordinary Pasteur pipet bulb.

For more pictures of this process, see the ID Unknowns experimental procedure.

(1) Prepare the column.The column is packed using a simple dry-pack method.

Plug a Pasteur pipet with a small amount of cotton; use a wood applicator stick to tamp it down lightly. Take care that you do not use either too much cotton or pack it too tightly. You just need enough to prevent the adsorbent from leaking out.

Add dry silica gel adsorbent, 230-400 mesh -- usually the jar is labeled "for flash chromatography." One way to fill the column is to invert it into the jar of silica gel and scoop it out . . .

. . . then tamp it down before scooping more out. Another way to fill the column is to pour the gel into the column using a 10 mL beaker.

When properly packed, the silica gel fills the column to just below the indent on the pipet. This leaves a space of 4–5 cm on top of the adsorbent for the addition of solvent. Clamp the filled column securely to a ring stand using a small 3-pronged clamp.

Whichever method you use to fill the column, you must tamp it down on the bench top to pack the silica gel. You can also use a pipet bulb to force air into the column and pack the silica gel.

(2) Pre-elute the column.

The procedure for the experiment that you are doing will probably specify which solvent to use to pre-elute the column. A non-polar solvent such as hexanes is a common choice.

Add hexanes (or other specified solvent) to the top of the silica gel. The solvent flows slowly down the column; on the column above, it has flowed down to the point marked by the arrow.

Monitor the solvent level, both as it flows through the silica gel and the level at the top. If you are not in a hurry (or busy doing something else), you can let the top level drop by gravity, but make sure it does not go below the top of the silica. Again, the arrow marks how far the solvent has flowed down the column.

Speed up the process by using a pipet bulb to force the solvent through the silica gel - this puts the flash in microscale flash chromatography. Place the pipet bulb on top of the column, squeeze the bulb, and then remove the bulb while it is still squeezed. You must be careful not to allow the pipet bulb to expand before you remove it from the column, or you will draw solvent and silica gel into the bulb.

When the bottom solvent level is at the bottom of the column, the pre-elution process is completed and the column is ready to load.

If you are not ready to load your sample onto the column, it is okay to leave the column at this point. Just make sure that it does not go dry -- keep the top solvent level above the top of the silica (as shown in the picture to the left) by adding solvent as necessary.

(3) Load the sample onto the silica gel column.

Two different methods are used to load the column: the wet method and the dry method: wet and dry. Below are illustrations of both methods of loading a crude sample of ferrocene onto a column.

In the wet method, the sample to be purified (or separated into components) is dissolved in a small amount of solvent, such as hexanes, acetone, or other solvent. This solution is loaded onto the column.

Wet loading method

The column at the left is being loaded by the wet method. Follow the thumbnails below to see close-up details of the sample as it is allowed to sink into the column. Once it's in the column, fresh eluting solvent is added to the top and you are ready to begin the elution process (see step 4).

Sometimes the solvent of choice to load the sample onto the column is more polar than the eluting solvents. In this case, if you use the wet method of column loading, it is critical that you only use a few drops of solvent to load the sample. If you use too much solvent, the loading solvent will interfere with the elution and hence the purification or separation of the mixture. In such cases, the dry method of column loading is recommended.

Dry loading method

First dissolve the sample to be analyzed in the minimum amount of solvent and add about 100 mg of silica gel. Swirl the mixture until the solvent evaporates and only a dry powder remains. Place the dry powder on a folded piece of weighing paper and transfer it to the top of the prepared column. Add fresh eluting solvent to the top -- now you are ready to begin the elution process (see step 4). Follow the thumbnails below to see close-up details of this process.

(4) Elute the column.

Force the solvent through the column by pressing on the top of the Pasteur pipet with a pipet bulb. Only force the solvent to the very top of the silica: do not let the silica go dry. Add fresh solvent as necessary.

The photo at the left shows the solvent being forced through the column with a pipet bulb.

The series of 5 photos below show the colored compound as it moves through the column after successive applications of the pipet bulb process.

The last two photos illustrate collection of the colored sample. Note that the collection beaker is changed as soon as the colored compound begins to elute.

The process is complicated if the compound is not colored. In such experiments, equal sized fractions are collected sequentially and carefully labeled for later analysis.

(5) Elute the column with the second elution solvent.

If you are separating a mixture of one or more compounds, at this point you would change the eluting solvent to a more polar system, as previously determined by TLC. Elution would proceed as in step (4).

(6) Analyze the fractions.

If the fractions are colored, you can simply combine like-colored fractions, although TLC before combination is usually advisable. If the fractions are not colored, they are analyzed by TLC (usually). Once the composition of each fraction is known, the fractions containing the desired compound(s) are combined.

Gas Chromatography

Study Questions/Answers from the Handbook for Organic Chemistry Lab

In gas chromatography (GC), the stationary phase is a high-boiling liquid and the mobile phase is an inert gas. In the organic chemistry teaching labs at CU Boulder, GC is used as an analytical tool to find out how many components are in a mixture. It can also be used to separate small amounts of material.

Movie on how to run a GC. On GoogleVideo - choose "smoothing" and "original size" from the lower right pull-down menu for best video.

The GC Instrument

The process of gas chromatography is carried out in a specially designed instrument. A very small amount of liquid mixture is injected into the instrument and is volatilized in a hot injection chamber. Then, it is swept by a stream of inert carrier gas through a heated column which contains the stationary, high-boiling liquid. As the mixture travels through this column, its components go back and forth at different rates between the gas phase and dissolution in the high-boiling liquid, and thus separate into pure components. Just before each compound exits the instrument, it passes through a detector. When the detector "sees" a compound, it sends an electronic message to the recorder, which responds by printing a peak on a piece of paper.

The type of GC used in the organic chemistry teaching labs is shown below: Gow-Mac series 350/400. Click on the photo for a detailed enlargement.

The GC consists of an injection block, a column, and a detector. An inert gas flows through the system. The injection chamber is a heated cavity which serves to volatilize the compounds. The sample is injected by syringe into this chamber through a port which is covered by a rubber septum. Once inside, the sample becomes vaporized and is carried out of the chamber and onto the column by the carrier gas.

The large photo below is of the inside of one of the older GC models, but it still shows useful information. It shows the column in the oven and the insulated chamber that houses the detector. Click on the thumbnails to see larger photos of the column and detector, as well as the inside of the injector port (showing the septum).

inside of the injector port

the septum

the column

the detector inside the housing

On the Gow Mac 350 chromatographs, detection of the compounds is achieved with a thermal conductivity (TC or hot wire) detector.

The column (see the photo above) is an integral part of the GC system. On the outside, all you see is a long stainless steel tube, 1/8 to 1/4 inch in diameter and 4-5 feet long, which is coiled to fit inside the instrument. Inside the column is the important component: the stationary phase composed of the high-boiling liquid. The liquid is usually impregnated on a high surface area solid support like diatomaceous earth, crushed firebrick, or alumina. The liquid can be applied in various concentrations: the more liquid, the more sites it has to interact with the compounds.

All of our GCs have columns which are five feet long and 1/8" or 1/4" in diameter and contain a methyl silicone polymer liquid phase (OV-101, 1.5%) on a diatomaceous earth support (chromosorb G). Methyl silicone is a liquid phase of intermediate polarity, and non-polar compounds will separate according to their respective boiling points.

The carrier gas is an inert gas, helium. The flow rate of the gas influences how fast a compound will travel through the column; the faster the flowrate, the lower the retention time. Generally, the flow rate is held constant throughout a run. (The GCs at CU Boulder are set at a flow rate of 55 mL/min.)

This is where the carrier gas enters the Varian GCs and where the gas flow rate can be adjusted. Click on the photo above for details.

In a professional laboratory, the GC conditions would be critical for another experimenter trying to duplicate your observations. All of our GCs have the same columns (1.5% OV-101 on Chromasorb G) and the same flow rate (55 mL/minute) and detector bridge current (150 mAmps). Each instrument will have a different setting for:

column temperature injection port temperature detector temperature

It is a good practice to write down some of the settings on the instrument. The values for these temperatures are viewed by turning the knob on the instrument below the gauge -- click on the thumbnail below to see detailed photos of how to do this.

reading temperatures on the Gow-Mac

Recorders

Two devices are used to record the GC traces/areas under peaks:

integrating recorders computer program

Each type of device records the messages sent to them by the detector as peaks, calculates the retention time, and calculates the area under each peak; all of this information is included in the printout. For similar compounds, the area under a GC peak is roughly proportional to the amount of compound injected. If a two-component mixture gives relative areas of 75:25, you may conclude that the mixture contains approximately 75% of one component and 25% of the other.

An integrating recorder is pictured below. Click on the photo for a detailed picture and the location of the start button (press when you inject), the stop button (press when you have seen your peaks, it tells the recorder to do the calculations and to print), and the enter button (paper feed).

The screen of one of the computers is pictured below. A "Shortcut to GasChrom" is on the desktop - double click to launch the program. Once inside the program, press Start, Stop, and Print as appropriate.

Retention Time (RT)

The retention time, RT, is the time it takes for a compound to travel from the injection port to the detector; it is reported in minutes on our GCs. The retention time is measured by the recorder as the time between the moment you press start and the time the detector sees a peak. If you do not press start at the same time you inject your sample, the RT values will not be consistent from run to run.

Factors that affect GC separations

Efficient separation of compounds in GC is dependent on the compounds traveling through the column at different rates. The rate at which a compound travels through a particular GC system depends on the factors listed below:

Volatility of compound: Low boiling (volatile) components will travel faster through the column than will high boiling components

Polarity of compounds: Polar compounds will move more slowly, especially if the column is polar.

Column temperature: Raising the column temperature speeds up all the compounds in a mixture.

Column packing polarity: Usually, all compounds will move slower on polar columns, but polar compounds will show a larger effect.

Flow rate of the gas through the column: Speeding up the carrier gas flow increases the speed with which all compounds move through the column.

Length of the column: The longer the column, the longer it will take all compounds to elute. Longer columns are employed to obtain better separation.

Generally the number one factor to consider in separation of compounds on the GCs in the teaching labs is the boiling points of the different components. Differences in polarity of the compounds is only important if you are separating a mixture of compounds which have widely different polarities. Column temperature, the polarity of the column, flow rate, and length of a column are constant in GC runs in the Organic Chemistry Teaching Labs. For each planned GC experiment, these factors have been optimized to separate your compounds and the instrument set up by the staff.

Gas Chromatography: Procedure

(1) Add the sample to be injected to the syringe.

A 25µL glass Hamilton syringe is used to inject the GC samples. Only 2-4 µL of sample is injected onto the column, which means that you fill only a small part of the barrel with sample. Examine the syringe carefully before you fill it. The divisions are marked "5 - 10 - 15 - 20 - 25".

This is a 25 µL glass Hamilton syringe. You only inject 2.5 µL, so it will NOT be filled to the top.

Click on the photo to see an enlargement.

Place the tip of the needle in the liquid. Slowly draw up a small amount of liquid by raising the plunger, then press on the plunger to expel the liquid back into the liquid. This serves to “rinse” the syringe with your sample, ensuring that what you will measure in the GC run is the composition of your mixture. Repeat the rinse process one or two times. Then, draw up the plunger slowly again while the needle is in the liquid and carefully fill the syringe with liquid about halfway to the “5”.

It is often hard to see the liquid in the syringe. If the syringe is clogged, the plunger will be in the correct position but the barrel of the syringe will be filled with only air, as in the bottom syringe in the photo to the left.

The best thing to do is to carefully examing the syringe after you think that you have filled it. Hold it up to the light to get a better view.

Small air bubbles in the syringe will not affect the GC run (middle syringe in the photo to the left). As long as there is enough liquid in the syringe, the GC run will work fine. If you keep getting bubbles, just pull the plunger up a bit past the "halfway to the 5" mark to compensate.

If you have a VERY large air bubble, you will not have enough liquid to show a reading on the GC (e.g., the bottom syringe in the photo).

(2) Inject the sample into the injector port.

You are need to do two things sequentially and quickly, so make sure you know where the injection port is and where the start button on the recorder is.

Push the needle of the syringe through the injection port and immediately press the plunger to inject the sample, then immediately press the start button on the recorder.

You will feel a bit of resistance from the rubber septum in the injection port; this is to be expected and you should be prepared to apply some pressure to the syringe as you force the needle into the instrument all the way to the base of the needle.

(Click on each of the photos below for a larger view.)

Push the needle of the filled syringe through the injector (as far as it will go) and quickly push the plunger.

Remove the syringe immediately . . .

. . . and quickly press the start button on the integrating recorder or the start recording button on the computer (ask your TA which device is connected to the GC that you are using)

Here's a close-up of the integrating recorder.

(3) Sit back and wait.

Observe the recorder. Within several minutes, it should record several peaks.

(4) End the GC run.

When you have seen all of the peaks which you suspect are in the mixture, or when the recorder has shown a flat baseline for a few minutes or so, press stop on the recorder.

When you press stop, the recorder will print out the peaks, the retention times, and the areas under the peaks. When it is done printing, you can press “enter” a couple times to advance the paper.

Carefully tear the paper off the recorder. The paper is not perforated, so do not try to pull up and expect it to pop out of the recorder. Instead, pull it down to start a tear from one edge, and then continue the tear until the paper is cut and free.

This may seem trivial -- showing you how to tear the paper. But too many times a student has tried to yank the paper out instead of starting a tear and tearing it neatly. Yanking the paper can result in the paper being torn below the plastic cutting surface on the recorder, and the paper gets jammed down inside the recorder.

If this happens, the entire recorder has to be dis-assembled, a process which takes about 15 minutes, thus putting the entire GC out of service until it can be fixed.