How do I improve my peak shapes? Some or all of my peaks are not gaussian shaped, but have a long leading edge and a sharp, almost vertical tailing edge.
 

Fronting is column overload. It can occur when one or more of the compounds injected on the column exceeds the capacity of the liquid phase of the column. The thinner the liquid phase film, the less of each compound can be retained by the column. This includes both the injection volume and the concentration of each peak in the injection. Reduce the volume by injecting less, splitting the sample, or injecting a less concentrated sample.

Why are some of my peaks tailing?
 

This could be a dirty injector or column, or a miss-cut column. Cool the injector down, turn off the flows, and replace or clean the injector parts, including the inlet liner and gold seal. Remove the column. Trim a length off the column to eliminate non- volatile residue, septum material and pieces of ferrule. This length may be from 1 inch to 1 meter or more as needed. Cut the column with a proper cutting tool. If the cut is poorly made, sample adsorption can occur. Consider scrubbing the steel inside walls of the injection port with a brass brush or mild abrasive such as aluminum oxide powder. Be sure to rinse the injection port well before re-installing any parts. Removing the split vent line and rinsing it with solvent is a good idea for your 5890 ( See the "How do I clean the split vent line on my 5890? question). When analyzing in the splitless mode, a too long splitless time can cause tailing. Common times are in the 0.5 to 1 minute range. Tailing can also occur due to unswept (dead) volumes. Verify the column is installed properly in the injector and detector. If losing a portion of the column is of concern, consider using an un-coated retention gap (pre-column) before the column. This can be cut or replaced without losing any column efficiency, and will lengthen the life of the column. Remember that the union between the retention gap and the analytical column is a source for leaks and sample adsorption.

 
Why are all of my peaks missing?
 

Check to see that the correct signal is assigned. You can assign not only detector outputs, but detector, injector and oven temperatures, as well as pressures to a signal. When you verify the signal assignment, check the signal level. You want some number greater than 0-1. This check is important especially for a TCD or FID, to ensure the wire is intact or the flame is lit. Check the column head pressure. If it is lower than expected, check for leaks.

 
How come my peaks are larger and eluting earlier than they used to?
 

Faster, larger peaks are a result of less gas flow going out the split and septum purge vents and more going on the column; thereby increasing the head pressure and decreasing the split ratio. Check the flows out of the vents, and adjust if possible. If the problem persists, remove and clean the split vent. The problem could also indicate faulty flow controllers.

 
Why are my peaks of interest showing up in blanks?
 
This can be a sample preparation or system cleanliness problem. Try new solvents, both the ones for sample and blank preparation, as well as the solvents in the sample wash bottles. Try a new syringe and septum. Remove and clean the split vent line. Make a run without any injection, to see if the peaks show up just by heating the column.
 
What is the best way to troubleshoot column bleed?
 

The best way to diagnose whether or not you are having a problem with column bleed is by generating a bleed profile when you first install a column under your method conditions, then comparing a bleed profile from a more recent run. If there is a substantial increase, the column might be beyond it's prime, and this could be due to a variety of problems including oxygen in the carrier gas, and more likely sample residues. If you have a GC-MS, typical bleed ions for low polarity columns (DB/HP-1 or 5 for example) will be m/z 207, 73, 281, 355, etc…, mostly cyclic siloxanes.

 
What is gas chromatography?
 

Chromatography is the separation of a mixture of compounds (solutes) into separate components. By separating the sample into individual components, it is easier to identify (qualitate) and measure the amount (quantitate) of the various sample components. There are numerous chromatographic techniques and corresponding instruments. Gas chromatography (GC) is one of these techniques. It is estimated that 10-20% of the known compounds can be analyzed by GC. To be suitable for GC analysis, a compound must have sufficient volatility and thermal stability. If all or some of a compound�s molecules are in the gas or vapor phase at 400-450�C or below, and they do not decompose at these temperatures, the compound can probably be analyzed by GC.

The main parts of a basic GC system are shown in Figure 1. One or more high purity gases are supplied to the GC. One of the gases (called the carrier gas) flows into the injector, through the column and then into the detector. A sample is introduced into the injector usually with a syringe or an exterior sampling device. The injector is usually heated to 150-250�C which causes the volatile sample solutes to vaporize. The vaporized solutes are transported into the column by the carrier gas. The column is maintained in a temperature controlled oven. The solutes travel through the column at a rate primarily determined by their physical properties, and the temperature and composition of the column. The various solutes travel through the column at different rates. The fastest moving solute exits (elutes) the column first then is followed by the remaining solutes in corresponding order. As each solute elutes from the column, it enters the heated detector. An electronic signal is generated upon interaction of the solute with the detector. The size of the signal is recorded by a data system and is plotted against elapsed time to produce a chromatogram.

Figure 1. The Basic Components of a GC System

 
 

The ideal chromatogram has closely spaced peaks with no overlap of the peaks. Any peaks that overlap are called coeluting. The time and size of a peak are important in that they are used to identify and measure the amount of the compound in the sample. The size of the resulting peak corresponds to the amount of the compound in the sample. A larger peak is obtained as the concentration of the corresponding compound increases. If the column and all of operating conditions are kept the same, a given compound always travels through the column at the same rate. Thus, a compound can be identified by the time required for it to travel through the column (called the retention time). The identity of a compound cannot be determined solely by its retention time. A known amount of an authentic, pure sample of the compound has to be analyzed and its retention time and peak size determined. This value can be compared to the results from an unknown sample to determine whether the target compound is present (by comparing retention times) and its amount (by comparing peak sizes). If any of the peaks overlap, accurate measurement of these peaks is not possible. If two peaks have the same retention time, accurate identification is not possible. Thus, it is desirable to have no peak overlap or co-elution.

Inside a Capillary GC Column
capillary GC column is comprised of two major parts - tubing and stationary phase. A thin film (0.1-10.0 �m) of a high molecular weight, thermally stable polymer is coated onto the inner wall of small diameter (0.05-0.53 mm I.D.) tubing. This polymer coating is called the stationary phase. Gas flows through the tubing and is called the carrier gas or mobile phase.Upon introduction into the column, solute molecules distribute between the stationary and mobile phases. The molecules in the mobile phase are carried down the column; the molecules in the stationary phase are temporarily immobile and do not move down the column. As the molecules in the mobile phase move through the column, some of them eventually collide with and re-enter the stationary phase. During the same time span, some of the solute molecules leave the stationary phase and enter the mobile phase. This occurs thousands of times for each solute molecule as it passes through the column. All of the molecules corresponding to a specific compound travel through the column at nearly the same rate and appear as a band of molecules (called the sample band). The goal is to have no overlap between adjacent sample bands as they exit the column. This is accomplished by making each sample band travel at a different rate and by minimizing the width of the sample band. The rate at which each sample band moves through the column depends on the structure of the compound, the chemical structure of the stationary phase and the column temperature. The width of the sample band depends on the operating conditions and the dimensions of the column. The proper column and operating conditions are critical in obtaining no, or the least amount of, peak co-elution.

 
What are some possible cause of baseline instability and disturbances?
 

Evaluating The Problem
The first step in any troubleshooting effort is to step back and evaluate the situation. Rushing to solve the problem often results in a critical piece of important information being overlooked or neglected. In addition to the problem, look for any other changes or differences in the chromatogram. Many problems are accompanied by other symptoms. Retention time shifts, altered baseline noise or drift, or peak shape changes are only a few of the other clues that often point to or narrow the list of possible causes. Finally, make note of any changes or differences involving the sample. Solvents, vials, pipettes, storage conditions, sample age, extraction or preparation techniques, or any other factor influencing the sample environment can be responsible.

Checking The Obvious
A surprising number of problems involve fairly simple and often overlooked components of the GC system or analysis. Many of these items are transparent in the daily operation of the GC and are often taken for granted (set it and forget it). The areas and items to check include:

• Gases - pressures, carrier gas average linear velocity, and flow rates (detector, split vent, septum purge).
• Temperatures - column, injector, detector and transfer lines.
• System parameters - purge activation times, detector attenuation and range, mass ranges, etc.
• Gas lines and traps - cleanliness, leaks, expiration.
• Injector consumables - septa, liners, O-rings and ferrules.
• Sample integrity - concentration, degradation, solvent, storage.
• Syringes - handling technique, leaks, needle sharpness, cleanliness.
• Data system - settings and connections.

Ghost Peaks or Carryover
System contamination is responsible for most ghost peaks or carryover problems. If the extra ghost peaks are similar in width to the sample peaks (with similar retention times), the contaminants were most likely introduced into the column at the same time as the sample. The extra compounds may be present in the injector (i.e., contamination) or in the sample itself. Impurities in solvents, vials, caps and syringes are only some of the possible sources. Injecting sample and solvent blanks may help to find possible sources of the contaminants. If the ghost peaks are much broader than the sample peaks, the contaminants were most likely already in the column when the injection was made. These compounds were still in the column when a previous GC run was terminated. They elute during a later run and are often very broad. Sometimes numerous ghost peaks from multiple injections overlap and elute as a hump or blob. This often takes on the appearance of baseline drift or wander.Increasing the final temperature or time in the temperature program is one method to minimize or eliminate a ghost peak problem. Alternatively, a short bake-out after each run or series of runs may remove the highly retained compounds from the column before they cause a problem. Performing a condensation test is a good method to determine whether a contaminated injector is the source of the carryover or ghost peaks.

 
Excessive Baseline Noise
 
Possible Cause Solution Comments
Injector contamination Clean the injector Try a condensation test; gas lines may also need cleaning
Column contamination Bake-out the column Limit the bake-out to 1-2 hours
Column contamination Solvent rinse the column Only for bonded and corss-linked phases
Detector contamination Clean the detector Usually the noise increases over time and not suddenly
Contaminated or low quality gases Use better grade gases; also check for expired gas traps or leaks Usually occurs after changing a gas cylinder
Column inserted too far into detector Reinstall the column Consult GC manual for the proper insertion distance
Incorrect detector gas flow rates Adjust the flow rates to the recommended values Consult GC manual for the proper flow rates
Leak when using an MS, ECD or TCD Find and eliminate the leak Usually at the column fittings or injector
Old detector filament , lamp or electron multiplier Replace appropriate part  
 
Baseline Instability or Disturbances
 
Possible Cause Solution Comments
Injector contamination Clean the injector Try a condensation test; gas lines may also need cleaning
Column contamination Bake-out the column Limit bake-out to 1-2 hours
Unequilibrated detector Allow the detector to stabilized Since detectors may require up to 24 hours to fully stabilize
Incompletely conditioned column Fully condition the column More critical for trace level analysis
Change in carrier gas flow rate during the temperature program Normal in many cases MS, TCD and ECD respond to changes in carrier gas flow rate
 
Tailing Peaks
 
Possible Cause Solution Comments
Column Contamination Trim the column Remove 1/2-1 meter from the front of the column
Column Contamination Solvent rinse the column Only for bonded and cross-linked phases
Column activity Irreversible Only affects active compounds
Solvent-phase polarity mismatch Change sample solvent More tailing for the early eluting peaks or those closest to solvent front
Solvent-phase polarity mismatch Install a retention gap 3-5 meter retention gap is sufficient
Solvent effect violation for splitless or on-column injections Decrease the initial column tempterature Peak tailing decreases with retention
Too low of a split ratio Increase the split ratio Flow from split vent should be 20 mL/min or higher
Poor column installation Reinstall the column More tailing for the early eluting peaks
Some active compounds always tail None Most common for amines and carboxylic acids
 
Split Peaks
 
Possible Cause Solution Comments
Injection technique Change technique Usually related to erratic plunger depression or  having sample in the syringe needle
Mixed sample solvent Change the sample solvent to a single  solvent Worse for solvents with large differences in polarity or boiling points
Poor column installation Reinstall the column in the injector Usually a large error in the insertion distance
Sample degradation in the injector Reduce the injector temperature Peak broadening or tailing may occur if the temperature is too low
Sample degradation in the injector Change to an on-column injector Requires an on-column injector
 
Retention Time
 
Possible Cause Solution Comments
Change in carrier gas velocity Check the carrier gas velocity All peaks will shift in the same direction by approximately the same amount
Change in column temperature Check the column temperature Not all peaks will shift by the same amount
Change in column dimension Verify column identity  
Large change in compound concentration Try a different sample concentration May also affect adjacent peaks
Leak in the injector Leak check the injector A change in peak size also usually occurs.
Blockage in a gas line Clean or replace the plugged line More common for the split line; also check flow controllers and solenoids
 
Change in Peak Size
 
Possible Cause Solution Comments
Change in detector response Check gas flows, temperatures and settings All peaks may not be equally affected
Change in detector response Check background level or noise May be caused by system contamination and not the detector
Change in the split ratio Check split ratio All peaks will not by equally affected
Change in the purge activation time Check the purge activation time For splitless injectors
Change in injector volume Check the injection technique Injection volumes are not linear
Change in sample concentration Check and verify sample concentration Changes may also be caused by degradation,evaporation, or variances in sample temperature or pH
Leak in the syringe Use a different syringe Sample leaks passed the plunger or around the needle; leaks are often not readily visible
Column contamination Trim the column Remove 1/2-1 meter from the front of the column
Column contamination Solvent rinse the column Only for bonded and cross-linked phases
Column activity Irreversible Only affects active compounds
 
Loss of Resolution
 
Possible Cause Solution Comments
Decrease in Separation
Different column temperature Check column temperature Differences in other peaks will be visible
Different column dimensions or phase Verify column identity Differences in other peaks will be visible
Coelution with other peak Change the column temperature Decrease column temperature and check for the appearance of a peak shoulder or tail
Increase in peak width
Change in carrier gas velocity Check carrier gas velocity A change in retention time also occurs
Column contamination Trim the column Remove 1/2 to 1 meter from the front of the column
Column contamination Solvent rinse the column Only for bonded and cross-linked phases
Column contamination Trim the column Remove 1/2-1 meter from the front of the column
Column contamination Solvent rinse the column Only for bonded and cross-linked phases
Change in the injector Check the injector settings Typical areas: split ratio, liner, temperature, injection volume
Change in sample concentration or solvent Try a different sample concentration Peak widths increase at higher concentrations
 

Condensation Test
Use this test whenever injector or carrier gas contamination problems are suspected (e.g., ghost peaks or erratic baselines).

• Leave the GC at 40-50�C for 8 or more hours.
• Run a blank analysis (i.e., start the GC, but with no injection) using the normal temperature conditions and instrument settings.
• Collect the chromatogram for this blank run.
• Immediately repeat the blank run as soon as the first one is completed. Do not allow more than 5 minutes to elapse before starting the second    blank run.
• Collect the chromatogram for the second blank run and compare it to the first chromatogram.
• If the FIRST chromatogram contains a substantially larger amount of peaks and baseline instability, then that is an indication that there is    contamination upstream of the capillary column (ie. contaminated inlet, dirty carrier gas, etc.).
• If BOTH chromatograms contain few peaks or very little baseline drift, it can be assumed that the carrier gas and/or inlet are relatively clean.
• If BOTH chromatograms contain a significant amount of noise and/or baseline drift, then that usually is an indication that the detector or detector   gases are contaminated.

"Low-Bleed" Columns - Fact or Fiction?
Prof. Walt Jennings
Cofounder, J&W Scientific Incorporated

Several manufacturers offer "low bleed" columns. In some cases, these are merely selected from the standard production process, but in other cases the columns are actually "synthesized" for low bleed. In recent years, it has been established that where functional groups (i.e. phenyl) are inserted into the polysiloxane chain as aryl inclusions, as opposed to being attached to the chain as pendant groups, the resultant phase possesses increased thermal and oxidative resistance. Columns coated with such phases emit lower levels of bleed signal and are capable of going to higher temperatures. The increased thermal resistance is apparent only at temperatures above ca. 300 degrees. While some users can reap the benefits of these developments, others find little or no improvement.. their bleed signals are still too high.

True column bleed, of course, comes only from the column. What the user perceives as bleed is usually the total signal reaching the detector, which is the summation of signal from the septum (this gives a typical silicone mass spectrum), the injector, and the detector, all of which is usually blamed on the column.

It is good procedure to first check the detector. Disconnect and remove the column, and place an undrilled cap on the column attachment fitting. Activate the detector, and note the signal at 50 degrees. Increase the oven temperature to 320 degrees, and again note the signal. On a pristine detector, the FID signal will increase by one to two picoamps. If the increase exceeds this level, attention should be directed to cleaning the detector, make-up gas and hydrogen lines. Once the detector signal falls to an acceptable level at 320 degrees, attention should be directed to the injector. If the injector liner is visibly soiled, the injector should be cooled, dissembled and interior cavities scrubbed with solvent and natural bristle brushes or cotton swabs. After assembling the injector, a "jumper tube" (one to three meters of uncoated fused silica or steel tubing) is then used to connect the injector directly to the detector. The injector heater should be energized, and the oven set at 320 degrees. Any increase in "bleed" signal over that observed with the detector alone must come from the front end of the instrument, and may originate with the septum, the carrier gas line, in-line regulators, valves, or flow controllers.

Wrap a new septum in aluminum foil, ensuring that one face is smooth, and install this, smooth side down. If the signal emanating from the jumper tube is decreased, it indicates a need for better quality septa. If the signal is still high, materials entrained in the carrier gas may have deposited in lines, valves, or regulators, which should be dissembled and cleaned or replaced.

When the combined signal from the injector and detector falls to an acceptable level (one to two picoamps @ 320 degrees on an FID), the user is ready to install and reap the benefits of a true low-bleed column. The bleed rate of conventional columns is normally high enough to mask signal from the injector and detector unless these latter are heavily contaminated. With low bleed columns, the signal from the injector and detector assumes increased importance. This spurious signal is not infrequently limiting, and is usually (and incorrectly) perceived as "column bleed".

 
Our lab routinely injects samples with an aqueous matrix, and we commonly have problems getting reproducible results. Can we improve this?
 

PID/FID

Question:
I've noticed an interesting phenomenon when running a diesel standard on my tandem PID/FID; hydrocarbons eluting after C16 start to significantly decrease and also tail very badly. What causes this?

Answer:
This is a symptom of condensation in the PID. As the relative volatility of solutes decreases, the effect becomes more pronounced. Normally, this phenomenon can be minimized by increasing the temperature of the detector, up to the upper temperature limit (about 250�C). The life of the PID lamp is greatly reduced with higher temperatures, so compounds should be restricted to the volatile range.

Calibration Curve

Question:
EPA Method 8270 (GC/MS of semivolatile organics via capillary column techniques) requires a five-point calibration curve, typically 20, 40, 80, 120 and 160 ppm. Because the EPA recommends a 30 meter, 0.25 mm I.D. capillary column with a 1.0 �m film, my chromatograms exhibit all of the symptoms of overload for the 120 and 160 ppm standards. What can I do?

Answer:
0.25 mm I.D. capillary columns with a 1.0 �m film can handle approximately 125 to 175 ng1 of each individual analyte in a matrix; this means that if a 1.0 �L sample mix has 100 ppm each of two components, for a total of 200 ng, the column will not overload. Faced with column overload, the analyst may select a column with greater capacity (e.g., a 0.32 mm I.D. column with comparable b). Unfortunately, not all benchtop GC/MS systems can handle the greater flow rates of 0.32 mm I.D. columns. Another approach is to position the top of the column within the injector so that a smaller amount of sample is introduced to the column. The calibration curve remains linear because the injector discrimination is constant for all concentrations. This might be the simplest solution for most analysts. Alternatively, split injection could reduce the amount of analyte on column (e.g., 25:1). Many of the late eluting and trace concentration compounds will become difficult to detect.

Water Injections

Question:
Our lab routinely injects samples with an aqueous matrix, and we commonly have problems getting reproducible results. Can we improve this?

Answer:
Water has one of the largest vapor volumes of the common laboratory solvents. For water injections, the injector liner may be too small to accommodate the vaporized mixture, so the excess vapor will "backflash" outside of the injection port. This vapor mixture condenses on cooler surfaces resulting in a loss of sample. In the case of water, losses can be minimized by setting the injection port temperature to between 150 and 220�C (lowering the expansion volume) or using a smaller injection volume.

Guard Columns

Question:
How long should a guard column be?

Answer:
Guard columns are typically from 0.5 to 10 meters long. Although there are no definitive lengths that are good for all samples, the following guidelines can be used.

If the sample matrix is relatively "clean" (a small concentration of non-volatile compounds) and the solutes are active, the guard column should be 0.5 meter to 1 meter in length. If the sample matrix is dirty, the guard column should be longer (to collect the nonvolatile compounds). Five to ten meters help simplify system maintenance. With use a guard columns saturates and it becomes necessary to replace it. The longer guard column allows the user to simply cut off the first meter or so and reinstall it into the injector instead of replacing the entire guard column.

Flow on dual column assemblies

Question:
I'm having a problem matching flows on my homemade dual-column assemblies and resolving some of my anlaytes. Help!

Answer:
Even though a column manufacturer specifies particular dimensions, the dimensions are not exact. This can cause problems when coupling columns in a single injection, dual-column analysis. J&W offers a column connection service, but if you want to do it yourself, try the following.

Connect the columns with the Y splitter and guard column. Verify the integrity of the connection. Heat the columns to a temperature at which you can inject a detectable, unretained compound.1 I like 150�C. Note the elution time of the compound. If it elutes more than 0.1 minute apart, cut 10-15 cm from the column with the later time. Repeat the process until the nonretained compound elutes within 0.1 minute on both columns, making the column's flow rates nearly the same.

Run a standard under typical run conditions until resolution criteria are met. Pick a member of a pair of compounds that is difficult to resolve on one or both columns. Raise the column oven temperature high enough so that it will elute the compound between 5 and 10 minutes, inject it and note the elution times on both columns.

When installing new dual columns for the same analysis, repeat the steps in paragraph two, inject the chosen compound and adjust the head pressure until the retention time for both columns is within a percentage or two of the previously recorded times.

For a list of compounds for different detectors, refer to: Rood, D. A Practical Guide to the Care, Maintenance, and Trouble Shooting of Capillary Gas Chromatography Systems; Huthig, Heidelberg, 1991.

Conditioning New Columns

Question:
I've heard conflicting opinions about conditioning new columns. Some of my coworkers say it isn't necessary, some say you should bake the thing overnight, and others say you should ramp the column slowly. So what's the deal? Is it necessary to condition a new column? If so, how?

Answer:
Condition a new capillary column at approximately twenty degrees higher than the final temperature of your oven program without exceeding the upper temperature limit of the column. If a temperature higher than the isothermal temperature limit of the column is needed for your analysis, recondition the column at that higher temperature, but, again, don't exceed the upper program limit.

When you install your column, purge it with at least three volumes of carrier gas prior to ramping it to the conditioning temperature. The total column conditioning time will depend on the type of application you're running and how much bleed is acceptable. The lower the detection limit that's needed, the longer the column will need to be conditioned. (Column bleed is closely related to the polarity and the film thickness of the stationary phase.) Polar and thick film columns bleed more and require more conditioning. For most applications, 30-60 minutes of conditioning is usually sufficient.

But how can you really determine when a column is sufficiently conditioned?

A flame ionization detector (FID) works best for monitoring the baseline during conditioning. Toward the end of the temperature ramp (i.e., 30-40�C below the isothermal upper temperature limit), the baseline will rise, then come down and level off, at which time you may consider the column conditioned. There are those that report detector fouling during conditioning when using other types of detectors (e.g., ECD, MS), but it's generally considered a safe practice to condition the column while connected to these detectors.

One more thing: don't condition a column overnight. Column life expectancy is greatly reduced when the column is stored at high temperatures. If you're experiencing an excessive amount of bleed for more than two hours, bring the oven down to room temperature and locate the source of the problem (usually oxygen entering the column from loose fittings or a leaky septum). Baseline signals that mimic column bleed can also originate from residues present in the GC itself.

One more note: if the column has not been in use for a while, a mild conditioning step may be needed to drive off contamination which may have condensed inside the column during storage. Also, there is nothing to suggest a limit to the ramp rate of the oven when conditioning a column.

Inlet activity problems

Question:
How do you have chemically deactivate injection liners? How do I know when I have an activity problem in my inlet?

Answer:
Most inlet liners are made of borosilicate glass (e.g., Pyrex). Borosilicate glass exhibits characteristics advantageous to gas chromatography. It has a low coefficient of thermal expansion and is resistant to thermal shock. Most glasses contain Lewis acid sites, and in the borosilicates, these are in the form of boron, metal oxides, and surface silanols. These sites can interact with solutes in the sample, resulting in tailing peaks. These sites may also contribute to solute degradation. Thus, when you experience tailing peaks or loss in sensitivity for chromatographically active solutes, you may be experiencing an activity problem in the inlet liner.

The deactivation process entails two basic steps: a leaching step to remove metal oxides at the glass surface and a derivatization step to deactivate surface silanols. Leaching involves soaking the inlet liner in a 25% mineral acid solution (e.g., hydrochloric, nitric, and sulfuric acids, but not chromic acid), usually overnight at room temperature. This portion of the deactivation process can be shortened to several hours if the acid solution is mildly heated (65 ? C).

The derivatization step is more involved. After leaching, the liner is heated to remove free and bound water from the surface of the glass2), and then it is derivatized with a chemical agent to deactivate the surface silanol groups The choices for derivatizing agents are numerous, and methods are just as varied.

Although simple deactivation procedures exist, and are fairly effective (40-50%), the procedure is a very thorough deactivation procedure, which produces a more chemically inert liner than is commonly commercially available. This procedure is especially effective for very active compounds.

For more information on the properties of glass and chemical deactivation, we recommend two books by Walt Jennings: Analytical Gas Chromatography, Academic Press, and Comparisons of Fused Silica and Other Glass Columns in Gas Chromatography , H ? thig. Also, Dean Rood's book, A Practical Guide to the Care, Maintenance, and Troubleshooting of Capillary Gas Chromatographic Systems , offers discussions on poor peak shape and activity phenomena.

Flowmeter readings

Question:
I am getting a different reading on my flowmeter than I get if I inject an unretained compound and calculate the flow. The unretained compound elutes in 1.04 min on my 30 meter, 0.32 mm I.D. DB -5, which gives me a calculated 2.31 mL/min flow rate. My flowmeter says the flow rate is 4.59 mL/min. Am I doing the calculation wrong, or is my flowmeter wrong?

Answer:
Both answers are correct, but they answer different questions. The flowmeter is measuring the flow rate at the exit end of the column, whereas the calculated flow rate is a measure of the average flow rate through the column. The calculation should look something like this.
 
During a chromatographic run, standards and samples show widening peaks over time. Is this normal?
 
Assuming the retention times are not significantly different and the widening peaks are tailing, active sites may be indicated. If the widening peaks are symmetrical, then it may be normal column “wear and tear”. If the peaks are fronting, then the column is overloaded.
 
When should one replace either the septum or liner?
 
Typically modern septa can last 100 injections or more before problems start to occur. Liners need to be changed when chromatographic symptoms indicate a problem. Factors that affect the septa lifetime are syringe size, inlet temperature and to a lesser extent, pressure. Factors that affect liner lifetime are usually due to sample cleanliness. You should rely on the history of the instrument maintenance to tailor your specific program around chromatographic needs.
 
How do you clean the GC injection port liner?
 
Cleaning the injection port liners can cause expensive instrument down time when done incorrectly, or incompletely. Liner quality is specific to each application and there is no general method for cleaning liners. We cannot recommend a general method.
 
Does analyte pyrolysis become a problem with increasing injector temp?
 
Pyrolysis of an analyte can be a problem if the compounds are thermally labile. This is common in the pharmaceutical industry. If the drug or intermediate thermally decomposes at or below the injection port temperature, the result may be extra peaks, or reaction with the analytes of interest.
 
What prevention tools can we use to assist with the minimization of Endrin & DDT breakdown?
 
njection port maintenance is the first line of defense in any problem with active sites and sample degradation. One should use clean/deactivated liners, and be sure to change the (gold) inlet seal on the split/splitless injection port on a routine basis, or when degradation of performance dictates. Clean samples are always preferred but may not be realistic in every laboratory setting. Injection port maintenance is even more critical if the samples are “dirty”. Aggressive inlet cleaning may be necessary as most problems with Endrin and DDT breakdown occur in the inlet Explained Here. Agilent GCs equipped with Electronic Pneumatic Control offer the availability of pulsed splitless or ramped flow injection techniques. These techniques have been used successfully to minimize sample residence time in the inlet, thus minimizing degradation.
 
How does one effectively use a pressure control program vs. a temperature program?
 
The benefits of pressure programming, and electronic pressure control available on Agilent (5890 and) 6890 GCs are myriad, and more than is appropriate for this forum. In one example, pressure programming can take the place of temperature programming at the end of a run. Many analysts include a ramp to an elevated temperature after all analytes of interest have eluted. This can help to insure that any unknown material from the samples is eluted so as not to interfere with subsequent injections. A similar process may accomplish the same thing, at lower temperatures, by increasing the flow rate at the end of the run, depending on the capacity of the column and detector. (This may not be appropriate to detectors such as the MSD where high flow may tax the vacuum system.) The advantage is that cool-down time is shortened (decreasing cycle time), and column lifetime may be lengthened. Another advantage of pressure programming is described in Ramping the 6890 Inlet Pressure OR Ramping the 5890 Inlet Pressure .
 
How do you know that the volume injected in a split/splitless injection port will not overfill the reaction chamber of the injector liner?
 

This question does not usually arise unless there is a precision problem with multiple injections of the same solution. Precision problems can often originate with inappropriate injection volumes. Because of the high inlet flows in split injections, the sample moves in and out of the injector much more quickly than in splitless. As such, split mode injection volumes are not as critical, as the solvent does not have time to expand beyond the boundary of the liner. Practically, 1 microliter is appropriate, as decreasing the split ratio is more effective at increasing peak size than increasing the injection volume. Splitless injections, however, are much more critical and it is recommended that a vapor volume calculator be used to approximate the resulting expansion volume

 
What are problems with water injections on capillary column? How to minimize them?
 
How can I know if my sample is causing damage to my capillary column?
 

There is a quick and easy test to determine if your sample contains potentially damaging residues. Deposit about 20 µl of the sample onto a microscope slide. Set it over the heated injection port or on a hotplate until dry. If you can see any residue where the sample was deposited, that residue will likely cause chromatography problems at a minimum, and will potentially damage the column stationary phase.

 
How do I know when it’s time to change my gas purifiers?
 
What are problems with water injections on capillary column? How to minimize them?
 

Agilent offers a variety of deactivated inlet liners to increase inlet inertness and a number of “ms” columns such as the HP-5ms and DB-5ms that are manufactured and stringently tested to ensure maximum inertness.
Note: For a thorough evaluation of liner inertness,

 
Why does there appear to be a greater loss of active analytes in the lower levels with GC trace analysis?
 
Most likely, close to the same number of analyte molecules are lost on each injection, regardless of level. At low standard levels, a greater percentage of the active analyte molecules are being lost, resulting in the lower relative response factors.
 
I've noticed an interesting phenomenon when running a diesel standard on my tandem PID/FID. The hydrocarbons that elute after C16 are starting to significantly decrease and also tail very badly. What causes this?
 

This is a symptom of condensation in the PID. As the relative volatility of solutes decreases, the effect becomes more pronounced. Normally, this phenomenon can be minimized by increasing the temperature of the detector, up to the upper temperature limit (about 250°C). The life of the PID lamp is greatly reduced with higher temperatures, so compounds should be restricted to the volatile range.

 
Our Method requires a five-point calibration curve. Because the EPA recommends a 30 meter, 0.25 mm I.D. capillary column with a 1.0 µm film, my chromatograms exhibit all of the symptoms of overload for the 120 and 160 ppm standards. What can I do?
 

0.25 mm I.D. capillary columns with a 1.0 µm film can handle approximately 125 to 175 ng1 of each individual analyte in a matrix; this means that if a 1.0 µL sample mix has 100 ppm each of two components, for a total of 200 ng, the column will not overload. Faced with column overload, the analyst may select a column with greater capacity (e.g., a 0.32 mm I.D. column.).
Unfortunately, not all benchtop GC/MS systems can handle the greater flow rates of 0.32 mm I.D. columns. Another approach is to position the top of the column within the injector so that a smaller amount of sample is introduced to the column. The calibration curve remains linear because the injector discrimination is constant for all concentrations. This might be the simplest solution for most analysts. Alternatively, split injection could reduce the amount of analyte on column (e.g., 25:1). Many of the late eluting and trace concentration compounds will become difficult to detect

 
Our lab routinely injects samples with an aqueous matrix, we commonly have problems getting reproducible results. How do we improve this?
 

Water has one of the largest vapor volumes of the common laboratory solvents. For water injections, the injector liner may be too small to accommodate the vaporized mixture, so the excess vapor will "backflash" outside of the injection port. This vapor mixture condenses on cooler surfaces resulting in a loss of sample. In the case of water, losses can be minimized by setting the injection port temperature to between 150 and 220 degree Ccentigrade (lowering the expansion volume) or using a smaller injection volume.

 
How to choose right column for my GC analysis?
 

The heart of Gas chromatography is the column. There are many varieties and deciding between packed and capillary, polar and non-polar, and other variables including film thickness and stationary phase, is often confusing. Agilents technical Support Team is always available to answer any question related to columns and most applications. Please refer the attached document for some of the details.

 
How to troubleshoot peak shape problems?
 
How do you chemically deactivate injection liners? How do I know when I have an activity problem in my inlet?
 
Most inlet liners are made of borosilicate glass (e.g., Pyrex). Borosilicate glass exhibits characteristics advantageous to gas chromatography. It has a low coefficient of thermal expansion and is resistant to thermal shock. Most glasses contain Lewis acid sites, and in the borosilicates, these are in the form of boron, metal oxides, and surface silanols. These sites can interact with solutes in the sample, resulting in tailing peaks. These sites may also contribute to solute degradation. Thus, when you experience tailing peaks or loss in sensitivity for chromatographically active solutes, you may be experiencing an activity problem in the inlet liner.
The deactivation process entails two basic steps: a leaching step to remove metal oxides at the glass surface and a derivatization step to deactivate surface silanols. Leaching involves soaking the inlet liner in a 25% mineral acid solution (e.g., hydrochloric, nitric, and sulfuric acids, but not chromic acid), usually overnight at room temperature. This portion of the deactivation process can be shortened to several hours if the acid solution is mildly heated (65?C).
The derivatization step is more involved. After leaching, the liner is heated to remove free and bound water from the surface of the glass2), and then it is derivatized with a chemical agent to deactivate the surface silanol groups The choices for derivatizing agents are numerous, and methods are just as varied.
Although simple deactivation procedures exist, and are fairly effective (40-50%), the procedure is a very thorough deactivation procedure, which produces a more chemically inert liner than is commonly commercially available. This procedure is especially effective for very active compounds.
For more information on the properties of glass and chemical deactivation, we recommend two books:
1. by Walt Jennings: Analytical Gas Chromatography, Academic Press, and Comparisons of Fused Silica and Other Glass Columns in Gas Chromatography .
2. Also, Dean Rood's book, A Practical Guide to the Care, Maintenance, and Troubleshooting of Capillary Gas Chromatographic Systems , offers discussions on poor peak shape and activity phenomena.
 
What is the best way to condition a capillary column?
 

You should condition a new capillary column at approximately twenty degrees higher than the final temperature of your oven program without exceeding the upper temperature limit of the column. If a temperature higher than the isothermal temperature limit of the column is needed for your analysis, recondition the column at that higher temperature, but, again, don't exceed the upper program limit.
When you install your column, purge it with at least three volumes of carrier gas prior to ramping it to the conditioning temperature. The total column conditioning time will depend on the type of application you're running and how much bleed is acceptable. The lower the detection limit that's needed, the longer the column will need to be conditioned. (Column bleed is closely related to the polarity and the film thickness of the stationary phase.) Polar and thick film columns bleed more and require more conditioning. For most applications, 30-60 minutes of conditioning is usually sufficient.
But how can you really determine when a column is sufficiently conditioned?
A flame ionization detector (FID) works best for monitoring the baseline during conditioning. Toward the end of the temperature ramp (i.e., 30-40?C below the isothermal upper temperature limit), the baseline will rise, then come down and level off, at which time you may consider the column conditioned. There are those that report detector fouling during conditioning when using other types of detectors (e.g., ECD , MS ), but it's generally considered a safe practice to condition the column while connected to these detectors.
Conditioning a column overnight is not recommended. Column life expectancy is greatly reduced when the column is stored at high temperatures. If you're experiencing an excessive amount of bleed for more than two hours, bring the oven down to room temperature and locate the source of the problem (usually oxygen entering the column from loose fittings or a leaky septum). Baseline signals that mimic column bleed can also originate from residues present in the GC itself.
Additional note: if the column has not been in use for a while, a mild conditioning step may be needed to drive off contamination which may have condensed inside the column during storage. Also, there is nothing to suggest a limit to the ramp rate of the oven when conditioning a column.

 
How can we improve our 6890 GC’s detection limit with better consumables?
 
Copyright © LCGC. All Right Reserved.