Solve Carryover Problems in Gas Chromatography

Let’s first properly define carryover in the context that I’d like to discuss here. An injection is made and a chromatogram obtained. On injecting a “blank” as the next injection, one or more of the components of the previous injection appear in the “blank” chromatogram.

This definition needs further clarification:

  1. Blank injection can be a pure solvent (or solvent mixture), or, if contamination of the solvent(s) is suspected, then the “blank” may be an injection of air (for example, a 0 mL injection).
  2. While the “blank” may contain components of the previous injection, depending upon the solvent used, one may observe carryover from several injections previously. Here, I refer to the often puzzling issue where several injections are made without evidence of carryover then “out of the blue” a component will appear in the chromatogram that was present in a sample from several injections ago. I will explain this more fully subsequently.

In summary, carryover in this context is related to the instrument rather than solvent contamination. However, while the contamination may not arise from the sample solvent injected, the nature of the solvents used within the system are intrinsically linked to the majority of carryover problems in gas chromatography (GC). So, why is this and what can be done about it? To help with the visualization of the concepts discussed below, Figure 1 is a schematic of a typical split–splitless inlet with the various gas flows noted.

Injection Volume Related Carryover

In split–splitless injection, the injected liquid expands rapidly to form a gas plasma containing our analytes, hopefully also in the gas phase. The space within the inlet, into which this expansion occurs, is primarily dependent upon the internal volume of the inlet liner used, and the available volume may vary significantly depending upon the design of the liner used. Most manufacturers will publish the internal volume of their various liner styles or this information will be available through one of the many online vapour volume calculators: CHROMacademy Calculator (1), Agilent GC Calculator (2), and Restek Backflash Calculator (3).

The inlet pressure (determined by the total flow into the inlet), inlet temperature, and sample volume injected all influence the volume of gas created from the injected sample. If the volume of gas created by the sample exceeds the available volume within the liner, gas may overflow from the liner and will typically end up in the septum purge and carrier gas lines (yes, overflowing sample vapour may overcome the forward pressure of the inlet gas supply and flow back up the carrier gas inlet lines). As these lines are typically unheated, higher boiling and more polar sample components may condense and “coat” the lines. Any subsequent overloaded injection (typically known as “backflash”) may then flow through the unheated lines and re‑solubilize the condensed components. As the inlet pressure equalizes, or as the split line is opened in the case of splitless injection, then this re-dissolved component is drawn back into the inlet and may ultimately enter the column, therefore causing carryover.

One should note that this may not occur every time a backflash injection is made, and in some cases the polarity of the sample vapour relative to the condensed contaminant will determine how well the contaminant is re-dissolved, and therefore whether it is seen within the subsequent chromatogram. This can lead to the situation in which the contaminant may not become obvious until several injections later, if the solubility of the contaminant is not high in the intermediate injections. So, a polar contaminant may not appear in backflashed injections of, say, hexane, but may then become apparent when a backflashed injection of methanol is made several injections later. Not only is the problem of backflash rather insidious, it can also be very confusing because the appearance of the carryover may appear to be random and not apparent in the injections immediately following.

As splitless injections have inherently lower total gas pressure in the inlet (only the carrier flow is passing through the liner) and as the residence time of the sample solvent within the inlet is higher, splitless injection is considered to have a higher risk in terms of injection backflash and inlet contamination.

In order to overcome backflash issues there are three choices:

  1. Use pressure pulsed injection—in which the inlet pressure (total flow into the inlet) is increased during the injection phase and then reset to the desired pressure (and column flow) post injection. In splitless injection the pressure pulse time is usually matched to the splitless time.
  2. Reduce the amount injected
  3. Use a small split to increase the inlet pressure during injection

Obviously choices 2 and 3 need to be evaluated against any loss in sensitivity unless the sample concentration can be increased prior to injection, whereas choice 1 will
not lead to any reduction in analytical sensitivity.

The calculators mentioned above can all be used to assess the likelihood of backflash within the inlet and therefore will help to mitigate the issue.

Contamination of the Split Line

You may have noted that the split line on your gas chromatography instrument is also unheated. So whatever issues occur with the deposition of components into the carrier and septum purge lines may also occur within the split line. However, most inlet designs have a split line lower within the inlet than the split line (Figure 1), and the sample gas typically follows a more tortuous path through the liner prior to passing out of the split line. This being said, it is perfectly possible for less volatile sample components to condense within the split line and the charcoal trap, which is also included in the split line on most instrument designs. If the concentration of this contamination is high or builds up over a period of time, it is possible for carryover to occur in a very similar fashion to the backflash injection. This can be confirmed, and indeed mitigated, by “steam cleaning the split line” with several large volume injections (typically 5 mL) of water at very high split flows. If the contamination is nonpolar, then ethyl acetate can also be injected in a similar fashion until the carryover is eliminated.

Contamination of Inlet Components

Inlet components such as the liner may become “active” over time, as a result of exposure of silanol groups on the quartz glass from which the liner is made or from any quartz wool packing within the liner or from active metal sites on the inner metal surfaces of the inlet body. This is typically associated with peak tailing phenomena for polar analytes because of unwanted secondary interactions between the analyte and the active inlet site. However, if this interaction is strong, sample components may be irreversibly adsorbed until the following injection. However, again, if the solvent used for the next injection does not readily dissolve the contaminant, the carryover may not occur until an injection of a solvent of the same polarity is made.

To avoid these issues, ensure that deactivated liners are used, avoid the use of glass wool and packing materials within the liner if possible (check the impact on analytical sensitivity and reproducibility and discrimination effects before moving to a liner with no packing), and ensure that the inlet body is regularly cleaned. Liners should be regularly replaced and should be changed as a matter of priority when carryover problems are being investigated.

Carryover from the underside of the septum can also occur if the septum purge flow is not sufficient. This gas flow is designed to flush away septum outgassing products and to avoid sample deposition on the underside of the septum. Many instrument designs have automated control of septum purge flow and so this variable is rarely considered or the flow manually measured. Septum purge flow should be manually measured as part of preventative maintenance routines.

Contamination of Autosampler Components

Finally, the syringe and wash solvents should be considered in any investigation into carryover. Contamination may be carried on the inside and outside surfaces of the syringe needle and this can be somewhat mitigated by using rapid plunger depression and very short residence times for the syringe needle within the inlet. Most manufacturers ensure that this is built into the autosampler routine, however if your instrument provides the option for “slow” or “fast” injection, be sure to choose the fast injection option, especially with splitless injection.

Further, the syringe wash solvents should be matched to the polarity of the potentially contaminating analytes. While most users match the wash solvent with the sample solvent, one needs to carefully consider the solubility–polarity of the components involved in carryover when selecting the wash solvent. Further ensure that waste solvent bottles and the bottle tops are kept clean and regularly emptied. When the autosampler offers more than one wash solvent, one should experiment with the wash solvent routine to minimize carryover—use of one solvent post injection and one solvent pre injection, or the use of both solvents in turn both before and after injection, and so on.

The number of sample washes and syringe primes prior to injection can also be optimized to reduce carryover to the minimum levels. I have known methods that will be carryover free only after five sample washes and five sample primes prior to injection.

Hopefully you will now have a more thorough understanding of the potential sources for carryover that are instrument related and that some or all of this advice will help you to overcome issues with quantitative reproducibility or contaminants in qualitative analysis.



Akinbuli Opeyemi,


Simple Steps for Clearing a Blocked Injector in Your ICP-OES Torch

Remove blockages to ICP-OES productivity

Deposition of the sample matrix, salts or even carbon build-up can lead to injector blockage in the torch. How quickly blockages occur varies, depending on sample type, sample workload, torch type, and even the method parameters. A blocked injector can restrict the flow of sample aerosol into the plasma, decreasing sensitivity and
degrading accuracy and precision.
Prevention is the best cure to reduce injector blockage and extend the operating time. Make sure that you are using the recommended torch type and check that you have the recommended instrument parameters for your application. Filter all samples to ensure you remove large particulates. Regular rinsing between samples and at the end of the run can also help to keep the injector clear. However, improper cleaning techniques can permanently damage the
torch. Follow the steps outlined in this technical overview to safely clean your torch, and to remove blockages if or when they occur.

Simple Steps for Clearing a Blocked Injector in Your ICP-OES Torch

Routine cleaning

5100/5110 ICP-OES
– Prepare a 50% aqua regia solution (1 part deionized water to 1 part aqua regia [three parts hydrochloric acid and one part nitric acid]) in a wide diameter tall form beaker.
– Place the beaker under the torch cleaning stand (P/N G8010-68021). This suspends the torch (or injector/base assembly) in the cleaning solution, reducing the risk of spills and damage to the quartz outer tube.
– Invert the torch and position this on the torch cleaning stand so that the quartz outer tube and injector is immersed in the aqua regia solution.
– Pipette some of the aqua regia through the ball joint of the injector to remove buildup from the lower part of the injector.
– Soak the torch for at least 1 hour.
– If deposits remain, repeat the cleaning process using a higher concentration of aqua regia.
– Thoroughly flush the inside and outside of the torch with deionized water (18 MΩ cm) using a wash bottle. Invert the torch and flush deionized water through the quartz tubes so that the water flows out of the gas entry ports and ball joint connector for at least 30 s.
– Invert the torch and dry by blowing clean compressed air or nitrogen through the gas ports on the base and through the opening of the ball joint to remove moisture.

Caution: Do not place the torch in a drying oven. It is not as effective at removing moisture as using compressed air or nitrogen, and may damage the torch.

One-piece quartz torch for 700, Vista, and Liberty Series ICP-OES

– Soak the torch overnight in concentrated aqua regia (three parts hydrochloric acid and one part nitric acid).
– If necessary, use a pipe cleaner dipped in aqua regia to gently remove persistent deposits from the injector tube.
– Rinse with deionized water and allow to dry.

Removing salt deposits
– Rinse the torch with water.
– Soak overnight in a 25% detergent solution.
– Rinse the torch with deionized water and allow to dry.
Important: The torch must be completely dry before re-installing. Replace the torch if chipped, cracked,
or distorted.

Re-installing the torch for 700, Vista, and Liberty Series ICP-OES

– Position the torch in the center of the RF coil, resting on the torch stand.
– Close the torch clamp and turn the locking knob.
– Gently attach the transfer tube to the base of the torch.
– Align the torch so the intermediate tube is about 2 to 3 mm away from the RF induction coil.
– Connect the auxiliary and plasma gas hoses to the appropriate inlets on the torch.
– Complete the torch alignment procedure to ensure the optics are viewing the highest emission signal from the plasma.

– Never place a torch in an ultrasonic bath, or use a wire to clean the injector.
– Do not use hydrofluoric acid with glass or quartz sample introduction components.
– Always use care when handling or installing a torch. Excessive force can break the torch.
– Do not touch quartz torches with bare hands. This can
reduce torch life.

– A one-piece quartz torch is simple to install and use and delivers great performance for most applications.
– For organic solvents, use a torch with a smaller ID injector. For volatile organic solvents, use a torch with a narrow-bore (0.8 mm ID) injector.
– For greater flexibility and reduced running costs, choose a semi-demountable torch. The injector and/or outer tube
is removable and replaceable separately.
– For fusions and HF digests, use a torch with an alumina injector.
– A fully demountable torch allows you to replace components individually, which can help lower your operating costs.

Learn how to achieve better sensitivity and precision, and improve tolerance to samples with high levels of total dissolved solids (TDS) by switching to the Agilent OneNeb Series 2

written by Ayodeji Ogunlowo

A Guide to Transporting Materials

It’s one thing to ensure your lab is operating safely, but the regulations and processes surrounding the transportation of hazardous materials are especially stringent. Whichever method these materials are being transported by, their packing and transportation must adhere to international regulations.

Safety is of the utmost importance in this process. In this guide, we’ll talk you through the differing material classifications, the regulations surrounding materials and modes of transport, as well as any necessary training required for transporting chemicals.

Types of materials and classifications

When processing, packaging or transporting dangerous goods, you will need to be able to classify them correctly so everyone involved in the process of transportation is aware of the potential hazards.

Based on their main hazards, goods are assigned to different numbered classes, as decreed by the UN and are as follows:

UN Class

Dangerous Goods







Flammable gas; non-flammable, non-toxic gas


Flammable liquids

Flammable liquid


Flammable solids

Flammable solid; spontaneously combustible substance; substance which emits flammable gas in contact with water.


Oxidisers and organic peroxides

Oxidising substance; organic peroxide


Toxic and infectious substances

Toxic substance; infectious substance


Radioactive materials

Radioactive material


Corrosive substances

Corrosive substance


Miscellaneous dangerous substances

Miscellaneous dangerous substances

Regulations covering different materials and types of transport

Whether you’re transporting materials in the UK or throughout the European Union, your legal obligations are broadly similar and must adhere to the respective regulations governing transport by road, rail, inland waterway, sea and air.

By road: If you’re transporting by road, you must adhere to the International Carriage of Dangerous Goods by Road Regulations, while domestic transportation must stick to the Carriage of Dangerous Goods and Use of Transportable Pressure Equipment Regulations.

By rail: Governed by Appendix C of the Convention Covering International Carriage by Rail, train transport must adhere to the regulations covered within.

By sea: When transporting by sea, you must adhere to guidance provided by the International Maritime Dangerous Goods code, which is used by operators transporting dangerous goods that need to travel across seas.

By air: The International Civil Aviation Organisation’s Technical Instructions are an internationally-agreed set of provisions that dictate the requirements for transporting dangerous goods by air.

Required training for transporting chemicals

If your business is frequently involved with the handling, processing or transporting of dangerous goods then you must appoint a Dangerous Goods Safety Adviser as per the Health and Safety at Work Act 1974. A DGSA is not required if you transport smaller quantities of dangerous goods than specified in the legislation, or your business is only occasionally involved in the transportation of dangerous materials.

The DGSA is required to monitor your business’ compliance with the rules that govern transportation, provide advice on said transportation and prepare annual reports on the business’ activities in material transportation. They are also responsible for monitoring safety measures, investigating accidents and advising on the potential security aspects of transport.

It is mandatory for DGSAs to obtain a vocational training certificate upon completing the correct training, which involves passing a written examination. The training courses for DGSAs are run by independent providers and the trade associations for each mode of transport, and vary in length from two to five days, depending on the mode of transport covered.

Minimising risks during transportation

Whichever mode of transportation is being used, there are a number of risks that could occur in transit, including damage, theft, chemical burns, fire, explosions, as well as many more. Consider the following before you make your journey:

  • Make sure large or heavy loads are protected properly and in the appropriate manner
  • Very important: ensure weight is evenly distributed.
  • You may need goods-in-transit or marine insurance to protect goods being transported, which may be paid for by the buyer or seller of goods.
  • Put suitable warning signs on vehicles to indicate wide, long or hazardous loads.
  • Take the appropriate security measures. For example, high-value goods should be tracked using a vehicle-tracking system.

Posted by Adi Oluwakemi,

08060874724, 07084594001

Best Practice for Identifying Leaks in GC and GC/MS Systems (Part 1)  

Leak symptoms

GC gas leaks fall into two distinct categories: large leaks that prevent the instrument from functioning and smaller leaks that allow the system to operate, but negatively impact chromatography. Large leaks typically prevent a system from reaching a ready state, leading to an electronic pressure control (EPC) safety shutdown. These types of leaks can result from a column not being installed in the expected inlet, a column not being

connected to the expected detector, a broken column, broken or loose fittings, broken ferrules, cored septa, or tubing blockage, to call out a few possibilities. The cause of these symptoms typically can be rooted out quickly by visual inspection or review of the method settings.

Identifying smaller leaks that allow the system to continue to operate can be more involved. Symptoms of smaller leaks can include constant cycling of actual pressure readings (oscillations greater than 0.02 psi), poor retention time reproducibility, higher than typical background, higher than typical bleed (particularly at temperatures greater than 230 °C), baseline drift, higher than usual inlet activity, tailing peaks, the need for more frequent inlet maintenance, and poor area reproducibility.

Figure 1 shows the elution of US-EPA 8081 pesticides on an Agilent J&W DB-1701 phase before and after exposure to 1,000 μL/L oxygen in helium carrier gas. After just 10 injections, column bleed increased significantly and a shift to shorter peak retention times was apparent

  1. No O2 exposure

2. Increased bleed shift to shorter retention after O2 exposure

Figure 1. US-EPA 8081 pesticides before and after exposure to 1,000 μL/L oxygen in helium

Carrier Gas Considerations

High quality carrier and detector gases of known purity are essential for obtaining optimal results in gas-phase analysis. Agilent specifies carrier and detector gas purity of at least 99.9995% (5.5 nines). Zero-grade air is recommended for flame detectors . Inline indicating gas traps are highly recommended to remove hydrocarbon, moisture, and oxygen. Gas certification testing and product descriptions vary by supplier and so obtaining a certificate of analysis (COA) for the gases in use is essential to understand gas quality. On the COA, key items to look for include tests conducted, specification for contaminants, and indications of whether testing was done on individual (preferred) or representative cylinders from a batch

Ferrule Selection

Selecting an appropriate ferrule for the column tubing size and particular fitting being used are critical for minimizing potential leaks and keeping the flow path free of contamination. Agilent J&W columns require one size ferrule for 0.1 to 0.25 mm id columns, while 0.32 mm and 0.53 mm id columns each require ferrules with larger diameter holes to accommodate the wider outside diameter of these columns. Ferrule material choice is also important for achieving the desired results for specific applications. Graphite ferrules are a popular choice for general-purpose and high-temperature applications (above 350 °C) but, typically, are not as contaminant-free as polyimide/graphite or metal ferrules. Further, graphite is a porous material and slightly permeable to gases, creating a very small continuous leak. Graphite also has a tendency to flake off, becoming a source of contamination. Pure polyimide ferrules are recommended for use outside of heated temperature zones only as they shrink dramatically with exposure to heat cycling.

Polyimide/graphite ferrules are a good choice for GC/MS and trace-level analysis, but they also have a tendency to shrink with repeated heat cycles, forcing the operator to snug the fitting repeatedly to avoid leaks. Ferrule shrinkage results in a tendency for analysts to over tighten fittings using polyimide/graphite ferrules.

Flexible metal ferrules are recommended for use with Capillary Flow Technology (CFT) devices as they are specifically designed for the fittings in these devices . Analysts are finding flexible metal ferrules an attractive alternative to other ferrules for standard column connection, such as the split/splitless inlet.

Table 1 identifies some common benefits offered by various capillary column ferrules

Table 1. Ferrule material selection attributes

Avoid Over Tightening

Be aware that over tightening can break the column or permanently damage fittings and actually produce leaks. The Agilent UltiMetal Plus Flexible Metal ferrule was designed to reduce column breakage by ompressing around the column. With a deactivated surface, these stainless-steel ferrules provide a robust and inert leak-free connection. Over tightening of Swagelok, SilTite or UltiMetal Plus Flexible Metal ferrules can damage fitting threads, making it impossible to obtain a seal, and resulting in costly instrument repairs. Carefully read and follow manufacturer’s instructions on fitting installation and use to avoid chronic leaks from

damaged fittings. Proper installation of graphite, polyimide/graphite, inlet seals, O-rings, and septa is also critical to maintaining leak free connections. Just tight enough (JTE) is the goal for proper installation of these somewhat pliable components. If tight is good, tighter is not better (TNB), as these pliable materials and can easily be crushed beyond their design specifications, causing them to leak sooner and more often. In extreme cases with repeated over tightening of brass mass spec transfer-line nuts, the nuts themselves can crack and potentially cause permanent damage to the mass transfer line. The Agilent septum nut has a C-shaped clip at the top that should not be turned more than 3/4 of a turn past where it begins turning with the nut assembly when a septum is being installed. Over tightening of the septum nut will cause premature septum coring with repeated injections, which in turn causes the septum to leak during a run. The septum nut is another fitting that needs to be JTE.



Best Practice for Identifying Leaks in GC and GCMS:  Technical Note, Agilent Technologies



Written by Muyiwa Adebola,

07084594001, 07084594004

Sample Preparation Techniques Used for Gas Chromatography

By Robert L. Grob, Ph.D., Emeritus Professor of Analytical Chemistry, Villanova University

Most scientists when faced with the analysis of a sample give little or no attention to how the sample was obtained, i.e., the sampling process. This is not the topic of discussion for this editorial. Suffice it to say one should realize that the results of the analysis of a sample can only be as reliable as the sample is representative. One must remember to obtain reliable analytical data three components are always involved:

1) the system (a representative sample), which consists of the analytes of interest and the matrix (the part not requiring analysis but which can interfere with the instrumentation),

2) a measuring instrument, and

3) the analyst or observer (a human being!). Well over 50% of the analysis time is spent on sample preparation and, since you have a human being as part of this system, sample preparation is the most error-prone and labour-intensive task in the analytical laboratory. For this discussion we will assume ALL samples are homogeneous and representative! If the final measurement of analyte concentration is by GC, one should strive to circumvent complete sample matrices; e.g., non-volatile components and interfering analytes.

One criterion for an analyte is that it must have a vapor pressure of 0.1 Torr at operating conditions in gas chromatography (GC). Thus, it must be able to be vaporized in the system inlet. The sample can be a gas, liquid or in some cases a solid (thermally stable or capable of producing a definite pyrolysis pattern). Most sample preparation techniques for GC are based on variations of extraction theory whereby the analytical chemist may change solvent, temperature, pressure, phases or volumes. Thus, an understanding or comprehension of liquid-vapor, liquid-liquid and liquid-solid equilibria are assumed.

IMPORTANT NOTE: Before you attempt the gas chromatographic analysis of an unknown sample obtain as much information about the sample as possible. Randomly injecting an unknown sample into a gas chromatograph does not reveal complete analytical data about the sample!

Having three states of matter (gas, liquid & solid), we have a series of sample preparation techniques available.

Gas-Liquid or Gas-Solid Equilibria

The main sample preparation techniques which can be classified by these equilibria are:

Static headspace technique: analytes of interest (volatiles) are equilibrated in a closed vial at a specified temperature & pressure. A gas-tight syringe is used to transfer the headspace sample into the gas chromatographic injection port.

Dynamic headspace technique: analytes of interest are swept or purged onto an adsorbent and then thermally desorbed into the gas chromatograph.

Solid-phase extraction (SPE): may be used to concentrate analytes from gaseous or liquid samples and often is used to clean up and concentrate liquid extracts. The adsorbed analytes can be eluted with a solvent or thermally desorbed.

Solid-phase microextraction (SPME): may be used for both gaseous and liquid samples. A fused-silica polymer coated fiber (e.g., with polydimethylsiloxane) is exposed to the stirred sample. The shielded-fiber is then inserted into the injection port of the gas chromatograph.

Distillations: these are predominately macro scale techniques and are rarely employed as sample preparation techniques for GC.

Liquid-Liquid or Liquid-Solid Equilibria

The technique of liquid-liquid extraction has lost appeal to the analytical chemist because of

(1) the time needed to reach equilibrium, and

(2) the volume of solvent needed for quantitative recovery of analytes (the environmental restrictions on waste disposal of used solvents).

Quantitative extraction of organic species from aqueous systems requires that the organic moiety

(1) is non-polar,

(2) does not dissociate in the aqueous phase, and

(3) does not dimerize or polymerize in the organic phase.

Thus, liquid-liquid extractions have limitations as sample preparation techniques for GC UNLESS the equilibrium between the two phases exhibits a large numerical partition coefficient. If this is the case, one may resort to micro liquid-liquid extractions, where the concentration factor is >1200 times that for the macro-technique [J. Chromatogr. 106,299 (1975) and J. Chromatogr. 177,135 (1979)]. Thus, classical liquid-liquid extractions have been replaced by modern & efficient techniques and are more prevalent in organic synthesis laboratories or for the separations of metal complexes, metal chelates, and/or ion-pairing reagents.

A number of classical liquid-solid equilibria techniques are available (e.g., ion exchange or Soxhlet extraction) but only Soxhlet extractions have application in sample preparation prior to GC. This technique is not commonly used because:

(a) a large volume of solvent is needed for the sample extraction,

(b) an evaporation step is required to concentrate the sample,

(c) lack of thermal stability and volatility of some sample analytes, and

(d) interference from contaminants in the extraction thimbles (requires a blank extraction prior to sample extraction).

In the past several years, newer techniques have become available. Accelerated solvent extraction (ASE), sometimes referred to as pressurized liquid extraction (PLE) or pressurized fluid extraction (PFE), may be used for solid and semi-solid samples. Elevated temperatures and pressures used in these techniques cause hydrogen bonds and dipole interactions to be reduced, and surface wetting is increased. Water may be used as the solvent if it is below its critical point. Then, it is known as subcritical water extraction (SWE); which makes it similar to SFE.

A similar sample preparation technique is microwave-assisted extraction (MAE); sometimes referred to as microwave-assisted solvent extraction (MWE). The pressure generated is ca. a few hundred psi; however, the extraction container must be microwave transparent (e.g., PTFE or quartz). The solvent used may be microwave absorbing or non-microwave absorbing. In place of microwaves, ultrasonic vibrations may be used to assure good contact between sample and solvent. This is a fast technique but efficiency is not as high as with other techniques. Low concentrations of analytes in samples require multiple extractions. This sample preparation technique is referred to as ultrasonic extraction (USE). A technique which became very popular during the 1980s is supercritical fluid extraction (SFE). Supercritical fluids (SFs) are dense gases above their critical temperature & pressure. Thus, SFs possess properties which resemble both liquids and gases. Analytes are more soluble in SFs when they are in their liquid state; thus, analyte melting points and solubility in the SF are important properties to consider. SFEs are fast and very efficient.

Another group of sample preparation techniques are solid-phase extraction (SPE), solid-phase microextraction (SPME) and stir bar sorptive extraction (SBSE). SPE is a technique (invented in the late 1970s) referring to a non-equilibrium exhaustive removal of analytes (semi-volatiles and non-volatiles) from a liquid sample by retention on a solid phase (sorbent) and then subsequent removal of selected analytes by solvent elution. Particulate matter in the sample can interfere with the analysis. Thus, particulate matter may sorb some analytes of interest and cause low analytical recoveries.

NOTE: Remove particulates, by filtration, prior to SPE analysis! The efficient use of this technique requires optimization of the sorption and desorption processes.

An extension of SPE, known as solid-phase microextraction (SPME), came about in 1989; the technique utilizes a liquid or solid stationary phase on a fiber, tube, vessel walls, suspended solids, stirrer, or disk/membrane. The technique may be applied to volatiles, semi-volatiles, and non-volatiles and is an equilibrium technique.

An excellent treatment of sample preparation techniques may be found in Chapters 11 and 15 of Modern Practice of Gas Chromatography, R.L. Grob and E.F. Barry (Eds.), John Wiley & Sons, Hoboken, NJ, 4th ed., 2004.

Akinbuli Opeyemi,



Taking Care of Laboratory Chemicals: Part 2

 Taking Care of Laboratory Chemicals: Part 2

Procedures to keep or improve the quality of chemicals

Part 1 of this paper showed that a packed chemical has sufficient time to reach thermodynamic equilibrium with the inner atmosphere, which necessarily includes the vapor pressure of the substance or its decomposition products and generally atmospheric gases. During the service time, it is critical to obtain and keep the atmosphere internal to the container as uninfluential as possible. Laboratory air at room temperature and pressure contains the very active compounds O2 (≈150 Torr, 240 mg per liter), H2O (10‒20 Torr, 10‒20 mg per liter) and CO2 (≈0.3 Torr, 0.7 mg per liter) so that it is unsuitable for preserving the quality of chemicals. Thus its presence and ingress into the container needs to be avoided as much as possible. This can be achieved by a suitable choice of container (inclusive of its multiusable closure) and its internal atmosphere.


The container should be sufficiently small (suitable for 10‒20 withdrawals) to allow easy flushing with inert gas, as detailed below. As a rule, for liquids, we use 25- or 50-mL borosilicate glass bottles for reagents and solvents, and 5-mL containers for spectroscopic solvents, all with a screw cap. For solids, commercial wide-mouth containers of 50 mL or less are used. The cap is the most problematic component because, besides possessing long-term stability to the contents, it should be as gas tight as possible to avoid exchange of the internal atmosphere with the laboratory atmosphere. It should also be as small as possible and checked periodically for gas-tightness.

Figure 1 ‒ Discouraged operation.

For reactive substances we use a solid “inverted hat” of polytetrafluoroethylene compressing an O-ring (in crush seal mode) of material suitable for the particular contents (i.e., Buna-N, viton, neoprene, ethylene propylene) on the rim of the glass neck by means of a screw cap. If the chemical was synthesized in the laboratory, rather than purchased, find which materials are suitable for its container and relative cap. For air-sensitive, even pyrophoric, chemicals, special bottles4 (see Figure 1) can avoid the use of a glove box or glove bag.

Internal atmosphere

There are two kinds of suitable atmospheres internal to the container, henceforth designated as “inert” and “equilibrium” atmospheres.

Inert atmospheres consist of a gas of very low reactivity (inert gas) such as Ar or N2 plus the vapors of the sample at their equilibrium partial pressure, and are suitable for most stable chemicals.

Liquids should be removed from the bottle while standing vertically by means of a syringe connected to an extraclean pipet or needle (Figure 2). Solids should be removed with a 90° curved spatula or spoon while keeping the container vertical. When finished, fill the container with the inert gas (Figure 3), keeping the cap positioned to allow rapid closure of the container when enough inert gas has been introduced. Argon is generally the most convenient gas (Figure 4) because it is unreactive even in the few cases in which nitrogen can react, and is heavier than air so that it accumulates from the bottom of the container, displacing other gases. Nitrogen is less expensive and becomes necessary if the container will be immersed in liquid nitrogen (LN2), where argon condenses to a liquid that gives over 800 times its volume as a gas at room temperature and pressure.

Figure 2 ‒ Storing or taking out a precise quantity of a pyrophoric liquid (Al(Et)3 25% in toluene) from the special container considered in Ref. 18 in Part 1.
Figure 3 ‒ Removing a measured volume of a solvent.

Figure 4 ‒ Filling to overflow with argon.

Since adsorption and absorption are invertible or reversing processes, repeated fillings of inert gas progressively displace dissolved or adsorbed oxygen, carbon dioxide and water, improving the quality of the compound or sample if it was previously stored under a lab atmosphere. If the substance has non-negligible vapor pressure, within a few minutes from closure of the cap its partial pressure reaches its equilibrium value, which adds to that of the injected inert gas at atmospheric pressure. Thus, in the container the equilibrium internal pressure will be higher than that of the external atmosphere, hindering leaks from there.

A plastic syringe of suitable volume and needle length (plugged when not in use) is a very convenient method of storage and source of the quantities of inert gas required to fill or flush the containers after partial removal of the contents. This simple procedure substantially increases, sometimes indefinitely (i.e., no reactant, no reaction) the service life of many chemicals and deters the degradation of samples.

The partial withdrawal of the chemical is completed by marking on the label the date and identity of the gas injected into the container.

Equilibrium atmospheres are appropriate for the compounds that undergo decompositions at laboratory temperature and pressure, where at least one of the reaction products is a gas or vapor. This case comprises most solvates (e.g., Na2SO4•10 H2O, SnCl4 • tetrahydrofurane), NaHCO3,  NH2COONH4,  H2SO4, HNO3 and many others. The same applies to most of the solutions of titrimetry. In such cases, the initial composition of the chemical is changed minimally if the internal atmosphere has an equilibrium composition, which is modified slightly by keeping the time of aperture of the cap as short as possible and the container vertical.

The post-use phase can be minimized by purchasing the quantity of chemical needed for the project or using dedicated secondary containers of that volume. Otherwise the residue can be checked, possibly purified or disposed of.


Flushing with inert gas after partial removal of contents of a bottle will force out part of the internal atmosphere containing the vapors of the substance. Unless its vapor pressure is negligible, as is the case with KCl, for instance, this operation should be carried out under a fumehood.


Use of the above procedures resulted in a drastic reduction in the dispersion and an increase in the reproducibility of data characterizing adducts in solution, keeping solvents and reagents free from detectable deterioration at their fifth-year check. The procedure is even applicable to substances that are particularly sensitive to oxygen, like chloroform and diisopropyl ether, or sensitive to water, like heavy water and CH3OD. Bright spheres of recovered sodium when covered with kerosene and flushing the container with argon after each withdrawal were shiny at the same control time.


Since all condensed phase chemicals must contain characteristic mass fractions and possibly reaction products of the components of the atmosphere internal to the container, choosing and keeping such an atmosphere as uninfluential as possible avoids the adsorption, absorption and formation of unwanted products that deteriorate the sample. The simple procedures indicated to achieve this significantly reduce or avoid the degradation of samples, extending their useful life. The consequent savings of time and other resources make up for the brief additional time (about 60 seconds per withdrawal) required to follow the reported recommendations.


Posted by Oluwakemi Adi,



With recent increases in helium pricing and an increasing number of regulated methods allowing the use of hydrogen as a carrier gas for GC, more labs are looking to switch to alternative carrier gases. This step by step guide will provide you with the necessary information to convert your GC’s carrier gas supply from helium to hydrogen. Hydrogen produced by a gas generator offers a safe, consistent and reliable source of gas as well as being a more convenient and greener source of hydrogen than cylinders.

Once you are satisfied that your method allows for conversion from helium to an alternative carrier gas, follow the steps in this guide to quickly get up and running with an alternative carrier gas while avoiding some of the common pitfalls of switching carrier gas.

Step1. Review all current method information and instrument conditions

  1. Review all methods that are run on the GC in the software and make hard copies.
  2. Leak-check the GC to ensure that the flow rates in the method will be achieved when running your analysis.
  3. Measure all flow rates indicated in your method (septum purge flow, split flow, carrier gas, and detector gases (if applicable), to ensure that the GC is meeting all set points correctly.
  4. Keep a note of the linear velocity of your carrier gas.
  5. Run a sample and monitor the system to ensure that flows are stable. If any flows change, make a note of these.
  6. Keep a record of a good chromatogram to compare with results after changing carrier gas.


Reference Chromatogram

Step 2. Perform routine GC maintenance before switching carrier gas

  1. Change the inlet septum. Use a low bleed septum that is recommended for your GC system.
  2. Change the liner. A tapered liner can improve results with hydrogen carrier gas for GC analysis. If you are not using a tapered liner, it may be advisable to purchase some tapered liners before setting up the system.
  3. If you are using gas purifiers, it may be advisable to switch these for new ones, depending on how old they are.
  4. If you are using a GC-MS and the ion source requires cleaning, this would be a good time to carry this out. Hydrogen carrier gas should maintain the condition of the ion source for longer than helium, so once you have switched carrier gas, you should find that ion source cleaning is required less regularly than before.
  5. It is possible to use the same column, provided it is still in good condition. Details are given below regarding conditioning a new column with hydrogen carrier gas.

Step 3. Install new tubing

  1. Tubing that was used to supply helium should be discarded. If you use the same tubing to supply hydrogen, you will see high background and it will take longer to optimize your analysis. If you are switching to nitrogen in place of helium for make-up gas, it is also advisable to change this tubing.
  2. Within 3m (10 feet) of the application, 1/8” laboratory grade copper or stainless steel tubing should be used. If you are piping gas from a distance greater than 3m, it may be necessary to use ¼” tubing and reduce to 1/8” at each GC. If you are unsure, Peak Scientific can assist you regarding your setup requirements.
  3. If you are supplying a GC-MS, you will need to connect just 1 line between the Hydrogen generator and the GC.
  4. All fittings should be compression fittings. DO NOT USE SEALING AGENTS or WELD CONNECTIONS. These can introduce volatile organic compounds (VOCs) into the gas stream and contaminate your system.

If you are connecting a GC-FID, you can supply both the carrier gas and fuel gas supply from a single generator. To connect to both gas inlets, prepare a T connection between the carrier and fuel inlets with a single supply line running from the generator. If you are unsure, please contact Peak Scientific for advice on how to connect the generator to your application.

Step 4. Gas supply to GC

The gas generators being used to supply your GC should be installed according to site preparation advice from the manufacturer.

Ensure that the connections into the GC are leak-free using an electronic leak detector – DO NOT USE LIQUID LEAK DETECTORS since these can cause contamination of your gas lines.

Detector gas supply:

  1. If your detector requires hydrogen, start the generator and ensure that all connections between the generator and GC are leak-free.
  2. If your detector requires nitrogen make-up gas, check that this line is also leak-free by checking before switching on the GC.
  3. If your detector requires an air supply, check that this line is also leak-free by checking before switching on the GC.
  4. Switch on the GC, making sure that the inlet gas flows are off and check that the detector signal stabilizes.

Carrier gas supply

  1. If you are using the column that was already installed, switch on the inlet pressure to allow carrier gas to flow through the column.
  2. If you are installing a new column, ensure that the column is correctly installed into the GC inlet. If the column requires conditioning, DO NOT CONNECT IT to the detector. Install the column into the inlet, following the guidelines from your GC manufacturer, ensuring that the connection is leak-tight.
  3. To condition the column using hydrogen carrier gas for GC-MS, carefully position the detector end of the column outside the GC oven and very carefully close the oven door, taking care not to break the column. You can see an example of this below:
  4. Start the hydrogen flow through the capillary column to purge oxygen and other impurities from the column.
  5. Check recommended heating programs and temperatures for the column which you have installed.
  6. Once the column is conditioned, switch on the detector, connect the column, and allow at least 1hr stabilization time.

System checkout

  1. Once the column is conditioned, connect to the detector and allow at least 1h stabilization time.
  2. Check that all method set points are met by the system and are stable.
  3. Run a blank sample to make sure that the detector baseline is stable.
  4. Adjust the linear velocity of the carrier gas to match the value when using helium carrier gas. Running hydrogen at the same linear velocity as helium, with the same oven temperature program should give you almost identical results. Compare the last chromatogram using helium with the results with hydrogen. The peaks should elute at the same time, but may have a slightly different shape.
  5. Compare the reference chromatogram obtained using helium carrier gas with the new chromatogram to ensure that all peaks have eluted and that their retention times are consistent. If there is plenty of peak separation, you can carry out optimization of the method to increase the linear velocity of the carrier gas and shorten run times. You may be able to halve the analysis time in some methods.
  6. Ensure that you can identify all peaks in your mixture. Run calibration standards to revalidate the method.

New Chromatogram


For enquiry and further information on this product, kindly contact us at Applied Analytical systems Ltd.


written by Ayodeji Ogunlowo



Determining Whether Your Non-Digital Fever Thermometer has Mercury in it : Newer non-digital fever thermometers often use:

  • alcohol, or
  • A non-toxic compound that looks similar to mercury.

To know if you Thermometer has Mercury


  • Is the liquid in the thermometer any color other than silver? Then it is most likely alcohol.
  • Is it silver? Then it may be mercury or possibly a non-mercury substance.  NOTE: Mercury is a liquid metal with properties different from most substances.  Small droplets will combine into a larger sphere shape, which will roll on a flat surface and break back into smaller droplets if dropped or if pressure is applied. Care must be taken to avoid scattering the mercury or allowing it to roll to a hard-to-reach location.  A standard oral/rectal/baby mercury thermometer contains about 0.61 grams of mercury.

NOTE ABOUT THERMOMETERS WITH SILVER LIQUID: If there is a paper calibration strip inside of the thermometer that includes the words “mercury free”, then the liquid in the thermometer is not mercury. If you do NOT see the words “mercury free”, assume that the liquid is mercury.


 What NEVER to Do After a Mercury Spill

  1. Never use a vacuum cleaner to clean up mercury. The vacuum will put mercury into the air and increase exposure.
  2. Never use a broom to clean up mercury. It will break the mercury into smaller droplets and spread them.
  3. Never pour mercury down a drain. It may lodge in the plumbing and cause future problems during plumbing repairs. If discharged, it can cause pollution of the septic tank or sewage treatment plant.
  4. Never walk around if your shoes might be contaminated with mercury. Contaminated clothing can also spread mercury around.

Prepping for Cleanup of a Broken Mercury Thermometer

  • Have everyone else leave the area; don’t let anyone walk through the mercury on their way out. Make sure all pets are removed from the area. Open all windows and doors to the outside; shut all doors to other parts of the house.
  • Mercury can be cleaned up easily from the following surfaces: wood, linoleum, tile and any similarly smooth surfaces.
  • If a spill occurs on carpet, curtains, upholstery or other absorbent surfaces, these contaminated items should be thrown away in accordance with the disposal means outlined below. Only cut and remove the affected portion of the contaminated carpet for disposal.Items Needed to Clean Up a Small Mercury Spill:
  1. 4-5 zip locking plastic bags
  2. trash bags (2 to 6 mils thick)
  3. rubber, nitrile or latex gloves
  4. paper towels
  5. cardboard or squeegee
  6. eyedropper
  7. duct tape, or shaving cream and small paint brush
  8. flashlight or small task light
  9. optional: powdered sulfur
    • Do not worry if you don’t have this available.
    • The sulfur binds to the mercury and makes clean-up easier. It is sometimes found in the gardening departments at hardware stores, near the fertilizer, or with garden pesticides and fungicides.  Pharmacists may also have it.

Mercury Spill Cleanup Instructions

  1. Put on rubber, nitrile or latex gloves.
  2. If there are any broken pieces of glass or sharp objects, pick them up with care. Place all broken objects on a paper towel. Fold the paper towel and place in a zip locking bag. Secure the bag and label it as directed by your local health or fire department.
  3. Locate visible mercury beads. Use a squeegee or cardboard to gather mercury beads into small mercury balls. Use slow sweeping motions to keep mercury from becoming uncontrollable. Take a flashlight, hold it at a low angle close to the floor in a darkened room and look for additional glistening beads of mercury that may be sticking to the surface or in small cracked areas of the surface. Note: Mercury can move surprising distances on hard-flat surfaces, so be sure to inspect the entire room, including any cracks in the floor, when searching.
  4. Use the eyedropper to collect or draw up the mercury beads. Slowly and carefully squeeze mercury onto a damp paper towel. Alternatively, use two pieces of cardboard paper to roll the mercury beads onto the paper towel or into the bag. Place the paper towel in a zip locking bag and secure. Make sure to label the bag as directed by your local health or fire department.
  5. After you remove larger beads, put shaving cream on top of small paint brush and gently “dot” the affected area to pick up smaller hard-to-see beads. Alternatively, use sticky tape, such as duct tape, to pick up any remaining small glass fragments. (Peel the tape very slowly from the floor to keep the mercury beads stuck to the tape.) Place the paint brush or duct tape in a zip locking bag and secure. Make sure to label the bag as directed by your local health or fire department.
  6. OPTIONAL STEP: It is OPTIONAL to use commercially available powdered sulfur to absorb the beads that are too small to see. The sulfur does two things:
    • It makes the mercury easier to see since there may be a color change from yellow to brown, and
    • It binds the mercury so that it can be easily removed and suppresses the vapor of any missing mercury.

Where to get powdered sulfur? It is sometimes found in the gardening departments at hardware stores, near the fertilizer, or with garden pesticides and fungicides.  Pharmacists may also have it.​

Note: Powdered sulfur may stain fabrics a dark color. When using powdered sulfur, do not breathe in the powder as it can be moderately toxic. Additionally, users should read and understand product information before use.

  • Remember to keep the area well ventilated to the outside (i.e., windows open and fans in exterior windows running) for at least 24 hours after your successful cleanup. If sickness occurs, seek medical attention immediately.


Written by Gabriel Oweh,


Maintenance Planning for Laboratory Equipment

Troubleshooting vs Maintenance


  • Logical, systematic search for the source of a problem
  • Helps to identify the cause(s) of a problem with a view
  • to correcting it
  • Works usually by the process of elimination


  • Activities carried out in order to preserve the operational status and life of an asset (equipment)
  • Does not necessarily extend the life of an asset; but lack of it can reduce the asset’s life
  • Basically two types: Preventive and Corrective Maintenance
  • Preventive Maintenance: Activities that help to detect and prevent failures before they occur or before they develop into major defects.
  • Corrective Maintenance: carried out to correct a fault so that a failed equipment can be restored to normal operational status

Maintenance Planning

  • Starts before instrument purchase
  • Critical factor in the choice of instrument model to be purchased, the instrument configuration and the instrument vendor
  • Overall goal is ease of maintenance and low cost of
  • ownership without compromising the quality of data
  • required from the instrument

Maintenance Planning: What to consider Before Instrument Purchase

  • Instrument type, model, and configuration
  • Stage of instrument model in manufacturer’s support’s life span
  • Availability of adequate and responsive local technical support
  • Ease of access to critical instrument parts and consumables
  • Local availability/ease of access to high quality reagents, gases etc mandatory for instrument’s operation
  • User Training : local or overseas
  • Environmental Conditions: Power, temperature, humidity, bench availability, location in the lab

Maintenance Planning: During Installation & Commissioning

  • Proper instrument installation
  • Installation Qualification
  • System Suitability Testing
  • User’s Familiarization/Training
  • Appropriate documentation: Manuals, reports, lists, etc

Maintenance Planning: After Installation

  • Develop SOP
  • Develop list of critical spares and consumables and stocking programme
  • Develop and setup an Instruments Replacement Schedule
  • Develop a planned maintenance programme
  • Implement, Review, Take remedial actions, Implement.

Planned Maintenance    

  • Also called Preventive Maintenance or Scheduled Maintenance
  • It is Pre-planned and scheduled
  • Involves scheduled service visits by competent service agent, but may also include periodical checks  and care by User
  • Can be date-based or hours-based
  • Allows for easier planning of maintenance
  • Allows for easier ordering and inventory of spares
  • Allows for easy budgeting and costs management
  • Allows for an essentially trouble-free instrument life

Impact of Environmental Conditions and Other Factors

Environmental Conditions

  • Basically temperature, humidity and light
  • Comply with Manufacturers recommended environmental conditions
  • Temperature affects instrument stability; high temperature may damage some instrument components and PCBs
  • High humidity can also damage instrument components and PCBs
  • Use of air-conditioners and/or dehumidifiers may be necessary.
  • Light may damage optical components in spectrophotometers


  • Instruments work within precise and specified gas pressure ranges
  • Use high quality and functional pressure regulators; preferably 2-stage regulators
  • High gas pressures can damage components such as valves and electronic flow regulators in instruments
  • Instruments also require highly pure gases devoid of moisture, hydrocarbons and other impurities —–VERY IMPORTANT
  • Impure gases can damage valves, electronic flow regulators, GC columns and block GC capillary gas lines
  • Use high quality gases and gas filters


  • Perhaps, the greatest singular cause of equipment malfunction in developing countries where power supply is irregular and inadequate
  • Voltage and frequency very important
  • Damage to instrument components can occur due to voltage and/or frequency fluctuation and intermittent power supply
  • Facility wiring and grounding also very important and critical
  • Use of good quality line conditioners, voltage stabilizers and UPS may be necessary


  • An untrained User is the greatest source of danger to an equipment, as well as to himself and the facility.
  • Users must be trained and qualified in the operation and basic user maintenance of an instrument before they begin to use
  • A freshly trained User must use an instrument under supervision until he has demonstrated adequate proficiency
  • Properly developed SOPs must be in place and must be understood and followed by all Users


(C) Muyiwa Adebola, 2017.


Written by Muyiwa Adebola,

07084594001, 07084594004

HPLC Mobile Phases – 10 bad habits to avoid

  1. Measuring the pH of the mobile phase after the organic has been added
    pH meters are calibrated to give the correct pH readback in aqueous solution – the buffers you verify this with are aqueous. If you measure the pH with the organic added, the pH will be different to that of measuring before organic addition.
    However, the most important point is to be consistent.If you do always measure pH after the organic is added, make sure you state this in the method so that everyone does it the same way. It won’t be 100% accurate, but at least it will be consistent. This is probably more important than having the exact pH.
  2. Not using a buffer
    Buffers are present to control pH and resist a change in pH. Many other parts of method (e.g. sample matrix, CO2 in air, source of water used for your mobile phase) can change the pH of the mobile phase causing shifts in retention, peak shape and peak response.
    Formic acid, TFA etc. are not buffers.
  3. Not using the buffer in its correct pH range
    Each buffer salt has a 2 pH unit wide range over which it provides the optimal pH stability. Outside this window the salt is ineffective at resisting change in pH.
    Either use your buffer within the correct range or pick a buffer whose range covers the pH you require.
  4. Adding buffer to organic
    Mixing aqueous buffer into the organic phase carries a high risk of the buffer being precipitated – in many cases so finely that it may not be obvious it has happened. ALWAYS add the organic to the aqueous phase, this greatly reduces the risk of buffer precipitation.
  5. Using the pump to mix gradients from 0%
    Modern pumps are very effective at mixing mobile phases and degassing online, however not everyone who ends up using your method has a high quality pump. Premix your A and B starting mix to a single solution that runs at 100% on line A. e.g. Prepare the starting mixture by mixing 950ml Aqueous with 50ml organic, then filter and degas. This reduces variability between HPLCs, reduces the risk of bubbles and precipitation in the system. Note however that 95:5 mixed on the pump will not give the same retention time as 95:5 premixed in the bottle – you normally need to add a few more percent organic when premixing.
  6. Not using the correct pH modifying acid or base for your buffer
    Only use the acid or base that forms the buffer salt you are using. E.g. sodium phosphate buffers should be adjusted with only phosphoric acid or sodium hydroxide.
  7. Not stating the full information of your buffer in the method e.g. weigh 5g of sodium phosphate into 1000ml of water
    The type of buffer (mono, di or tribasic) determines its pH buffering range.
    The required molarity is what determines the buffer strength. 5g or anhydrous sodium phosphate and 5g of monohydrate sodium phosphate will have different buffer strengths and will affect retention.
  8. Filling lines with organic without checking what was in there before
    If the previous method used buffer in line B and your method uses organic in line B there’s a good chance you will precipitate buffer in your pump tubing / pump head. I did it in my early days and it caused a lot of damage. If in doubt – flush it out (80:20 water : organic).
  9. Propping up bottles to get last drop out
    It’s 5 to 5 and you’ve barely got enough mobile phase to finish the run – it’ll be running on fumes by the last few samples. Apart from the risk or running your pump and column dry, mobile phases evaporate from the surface, so the mobile phase at the top of the bottle will have changed composition from the bulk. This portion from the top is exactly what will be running through the column if you use the last dregs in the bottle.
  10. Using sonication to degas mobile phase
    It’s great for making sure all your buffer salts have dissolved, but it’s the least effective method of degassing AND it quickly heats up the mobile phase causing the organic portion to evaporate. Save yourself problems later – take 5mins to vacuum filter your mobile phase – it degasses and filters in a single step.


LCGC Editors, March 8, 2017.

Posted by Opeyemi Akinbuli,,


Taking Care of Laboratory Chemicals: Part 1

All solid or liquid laboratory chemicals necessarily adsorb and possibly absorb and react with the components of the atmosphere inside the container, which is exchanged for the external atmosphere upon any withdrawal of the product, unless suitable procedures are adopted. Since interactions of the chemicals with atmospheric components generally degrade their quality, this paper explains how to minimize this loss and preserve quality. Part 1 considers the theoretical aspects of the topic, and Part 2 will examine its practical facets.


A chemical (short for “chemical compound,” “chemical substance” or “pure substance) is characterized by composition and propertiesoften available from the literature that do not change with time. However, this is the concept, or virtual, or symbolic chemical, represented by a single formula (generally conventional) that allows the symbolic handling of the chemical, like writing possible chemical reactions and carrying out the relative zero-order stoichiometric calculations.

Image result for lab chemicals

The real chemical product (i.e., chemical or product) is different because 1) its composition, generally shown on the label of the container, only approximates that of its conventional formula, since it necessarily contains more or less small fractions of identified (such information is labeled “chemical speciation”) or unidentified diverse compounds; 2) it has time-dependent properties due to the fact that it is subjected to several time-dependent interactions, both of the field and contact type. Even the most important properties—elemental and molecular composition—may change from those shown on the long-lasting container label, which thus can potentially provide misleading indications. Worse, composition changes, if unnoticed, may be dangerous, and can result in errors and increased dispersion of data from related measurements.

A chemical is designated as “degraded” when the values of the properties of interest differ from those acceptable for the programmed use. In many cases, the concentration of particular substances must be below critical, very low, limits, attainable in solids by adsorption (or physisorption) only.

The successful use of a chemical requires knowledge of its current composition to establish whether it is compatible with the intended operation. To avoid the time-consuming and potentially useless analysis of each chemical prior to each use, it is possible to circumvent or adjust, sometimes drastically, some of the interactions listed above, and update the label to show reliable information.

Laboratory chemicals

The time dependence of the composition of ordinary, liquid or solid laboratory chemicals, packed in a container with impermeable walls and reversible closure, is taken into account by defining for each a characteristic life cycle that can span several orders of magnitude, from days to indefinite. This consists of 1) a storage time, running from the purchase or preparation to the first opening of the container to use the chemical; 2) a service time, from first opening of the container to end of use of the contents and 3) postuse time.

Quality preservation in the former interval rests mainly on the expertise of the vendor, sometimes adjustable on request, for instance, of packing under inert gas. The normally limited usefulness of chemicals in time led to their classification in terms of shelf life, which (under appropriate conditions) can be excellent (>5 years), good (3–5 years), fair (1–3 years), poor (<1 year) and the consequent expiration date.13

During the service period, parts of the contents are taken out several times, using procedures that are dependent on the sensitivity of the product to the atmosphere. Here we concentrate on the common handling of the product in the atmosphere, referring to published information for the handling of the most sensitive, frequently very dangerous, products.

Presently an enduring protective medium that permits (trained) handling in the atmosphere is provided by vendors only in the cases of substances that are very sensitive to atmospheric components. Examples are Li, Na, K (kept under mineral oil) and yellow P (kept under water). To reduce the introduction of atmospheric moisture, but not oxygen, containers may be kept in dessiccators. Recently a proprietary packing claimed similar results. The concentration of water is kept low by the addition of insoluble dessiccants such as an appropriate molecular sieve sometimes enclosed in a gas-permeable container attached to the cap, known as a water trap.

The effects of atmospheric oxygen are generally kept to a minimum by the addition of stabilizers (hydroquinone, H3PO2, 2,6-di-t-butyl-4 methyl phenol or BTU). Contamination by atmospheric components is prevented in closed, dedicated systems for high-purity liquids. However, the only published device and procedure usable for opening and closing the container of liquids in the atmosphere that is very sensitive to atmospheric components. For normal chemicals, we found that simpler practices, indicated below, meet most needs, provided adequate attention is given to the cap and the volume of the containers.

The “postuse time” basically consists of checking and/or purifying for a new storage period, or disposing of the remaining product. In the laboratory, this phase can easily be minimized.

Analysis and model of the system during the service stage

During the service stage, most of the time the substances rest on the lab shelf (idle time, 1 month‒5 years) and their container is repeatedly but briefly opened to withdraw part of the contents (working time, e.g., 20 times for 30 seconds each).

In the cumulative idle time, the chemical is enclosed in a container provided with a temporary gas-tight port with impermeable, rigid and thermally conducting walls and subjected to both contact and field interactions. The contact interactions take place with stabilizers, inner atmosphere, container walls, inlet/outlet port and cosmic rays. The field interactions occur with gravity, electric and magnetic fields from the environment or the chemical itself, and from the scalar temperature and pressure fields from the surrounding atmosphere. With the exception of thermostated spaces (e.g., within refrigerators) lab temperature and pressure change during the day/night and season sequences, providing a continuous drive toward cyclic equilibrium conditions.

In addition to the easily avoidable photochemical effects of UV-VIS radiation and the unavoidable gravitational setting, the relevant processes involving the chemical are:

  • Adsorption (involving mainly surfaces, inclusive of pores)
  • Absorption (involving the bulk of the material, controlled by diffusion)
  • Mixing
  • When possible, reactions of the substance with the components of the atmosphere (or gas phase) internal to the container.

Most of these processes appear to have a characteristic time that is short with respect to the idle time. Thus, the approximate description of the product as a thermodynamic closed system consisting of a chemical and a gas phase internal to the container should be a useful guide.

Any chemical in a condensed phase contains as adsorbates, solutes or reaction products the components of the internal atmosphere, because their initially zero concentration in the substance implies (in the usual absence of impermeable or semipermeable boundaries) a very high propensity (initial ∆mix – ∞) to reach a finite concentration.

In the working phases, the normally closed and (assumed) gas-tight closure of the container (its most problematic part; see next section) is temporarily opened to remove a portion of the chemical. During such operation the system becomes an open system, exchanging intentionally part of the chemical and, unintentionally, by pressure difference, gravity and diffusion, more or less of the internal atmosphere with the external atmosphere, which contains the necessarily soluble and reactive components oxygen, water and carbon dioxide. If the chemical is usually removed by pouring, each use will replace the entire (heavier or lighter) internal atmosphere plus the volume of the chemical removed, with the laboratory atmosphere.

Old, leaky caps and especially pouring are the main causes of emission into the lab atmosphere, leading to alteration of solid or liquid chemicals. The pervasive presence of pouring rings and dripless pour lips on the bottlenecks of the containers is detrimental. The oxygen entering at each withdrawal produces aldehydes or ketones from alcohols, explosive peroxides from ketones, ethers and olefines; phosgene from chloroform and oxides on the surface of metals. Water reacts with many compounds, inorganic—such as hydrides: LiH, NaH; oxides: Na2O, K2O, P4O10; carbides: CaC2, Al4C3; sulfides: Al2S3, P2S3; halides: AlCl3, SnCl4, TiCl4); AlP; organometallics such as carbonyls and organic substances such as anhydrides, acyl halides, orthoesters and esters (with the evolution of CO2 in the case of di-tert-butyldicarbonate), and must be substantially absent in many chemical procedures. Carbon dioxide, especially in the presence of water vapor, reacts with most basic substances giving various carbonates. Even very stable compounds like squaric acid show surface discoloration unless the atmosphere of the container is made inert; “activated” zinc deactivates in contact with air. In thermodynamic terms, such openings introduce uncontrolled composition variables, sometimes called chemical noise.

A recent paper analyzes in detail the changes of phase diagrams of pure substances in the presence of air, but considers air as a single component rather than a mixture of variable composition, and also inert, that is, insoluble and unreactive toward the substance of interest. Such results support the present goals, but are not useful to their attainment.


  1. IUPAC’s Gold Book (1997). Online corrected version 2006–; accessed May 30, 2014.
  2. Meaning “relevant properties” and referring to given environmental conditions. Relevant properties are those needed for the selective specification of thermodynamics systems opposed to the exhaustive specification of mechanical systems). Density is a relevant property; the distance from the moon is not.
  3. Most chemicals are ensembles of several microscopic entities, some of which differ from the analytical formula. For instance, liquid acetic acid contains molecules CH3COOH (CH3COOH)2, ions CH3COO and CH3COOH2+in concentrations dependent on temperature.
  4. ; accessed 11.06.2014.
  5. Current literature on “time sensitive chemicals” seems to neglect the effects of time on reactions and measurements, limiting to the effects on safety; see, for example; accessed 27.01.2015.
  6. As acquired from research on adducts in solution, see, for instance, Lunelli, B.; Francesconi, R. et al. Faraday Trans., 199793, 2527‒32.
  7. The most common is water, as evident, for example, from the commercial availability of anhydrous-grade solvents.
  8. Even the moisture adsorbed on borosilicate glass must be eliminated when using air-sensitive chemicals, see, for example, Aldrich Technical Bulletin AL-134 (Handling Air-Sensitive Reagents).
  9. Gases do not interact with the atmosphere internal to the container because they occupy all the available volume.

Posted by Oluwakemi Adi,


11 Questions to Ask Before Installing a Remote Monitoring System for Medical Refrigerators and Freezers

To maintain the quality of vaccines, pharmaceuticals, blood and tissue samples, research materials, and other medical inventory stored in refrigerators and freezers, personnel must frequently monitor and record the temperature inside each cold storage unit. Installing a remote monitoring system with temperature sensors can save time, money, and anxiety. Systems that use cloud-based technology display the real-time status of all monitored conditions, on a mobile device, and send alerts when temperatures move outside the safe range (see Figure 1).

Figure 1 – Cloud-based monitoring system in protective enclosure.

Below are answers to questions to ask before installing a cloud-based monitoring system:

1.   What is required to use a remote monitoring system?

Most remote monitoring systems require an internet or WiFi connection and access to an electrical outlet. Programming is done through a website, so it’s easiest to use a computer for the initial setup. If you don’t have an internet connection at your location, you’ll want to choose a cellular system. Make sure there’s sufficient signal strength at your site, and check the signal quality in the area before purchasing a cellular device.

2.   How do we determine what kind of monitoring system and sensors we need?

A reputable manufacturer will have a well-trained support team that can assess your needs even without a site visit to determine which products are best for your application. The monitoring system representative will determine the type of system that would best serve your operation, the number of base units you’ll need, and the types of sensors required. The number of sensors a base unit can monitor varies. Make sure to evaluate your needs and select one that can accommodate your present situation and future growth. The representative should also be able to provide tips on the placement of the sensors you’re purchasing.

If you feel you need the rep to check out your refrigerators, freezers, and work space, many companies can set up a video conference or Face Time chat to substitute for being on site. Note that there should not be a cost for a demo, consultation, or assistance throughout the sales process. Be sure to ask if there are any fees or licenses to keep using the monitoring equipment after you purchase it.

3.   Are sensors included with a monitoring system?

In most cases, sensors are sold separately. The sensors you select depend on the conditions you want to monitor, how many you can connect to your base unit, and your specific application. For example, cloud-based monitoring systems and sensors are a great help to facilities that must comply with CDC cold storage requirements, such as those participating in the Vaccines for Children Program. That is because they include CDC-compliant features such as temperature displays adhered to the outside of the refrigerator to clearly show the current interior temperature, NIST-calibrated temperature probes buffered in a bottle filled with either glycol or glass beads, and data-logging capability.

Temperature sensors pre-installed in a bottle filled with glycol or glass beads are ideal for medical cold storage units (see Figure 2). These bottles keep the sensors at the same temperature as the liquids inside the refrigerator or freezer. They also prevent false high-temperature alarms when a door is opened or the freezer goes into a defrost cycle.

Figure 2 – Temperature sensor display with glass bead bottle.

4.   Do monitoring systems only work with the manufacturer’s sensors?

Not necessarily. For example, certain monitoring units can connect with most 4–20 mA sensors and transmitters regardless of the brand. When selecting sensors, you might have a choice between ones that are designed by the manufacturer to work specifically with the monitoring system or universal components made by a third party. If the components are not made by the system manufacturer, you’ll want to find out if they have been tested with the monitor you are choosing and if you need to work with another vendor to purchase the parts.

5.   Is the monitoring system easy to set up, or do we need to hire an electrician?

Many monitoring systems are quick and easy to install, and users can often set them up without hiring an outside expert. Look for one that has only a few simple physical installation steps, for example, 1) mount the device to the wall or somewhere secure, 2) plug it into an electrical outlet and an internet connection, and 3) connect the sensors.

The sensors are connected to the base unit’s terminal strip with wire, which is included with many sensors. The range of many wired sensors can be extended up to 2000 feet away from the base unit by adding wire that can be easily purchased at any home store. It’s a good idea to hire an electrician if you need to run wires through walls or ceilings.

Usually, once you plug in the device and connect the sensors, you create an account on the manufacturer’s designated website and begin using the device. There should be no fee to create an account and use the site.

If the manufacturer does not offer installation services, ask if they can recommend a local representative in your area who can set up your system. If not, make sure they provide free technical support via the phone or e-mail to walk you through the installation and answer any questions you might have about programming and daily usage.

6.   Is there a monthly fee to access all of the functionality of a monitoring device?

Many web- or cloud-based systems provide free functionality, with some limitations. You might have to purchase a premium subscription to unlock features such as text messaging, phone call alerts, and unlimited data-logging access.

7.   Should we get a system that is wired or wireless? Will we need to have a phone line, cable, internet, or something else?

Wireless can mean two different things as it relates to monitoring: how the system communicates its data to the outside world and how the sensors communicate with the system. The most popular systems require an internet or WiFi connection, but if that’s not an option, cellular- and phone-based systems are available.

A hard-wired monitoring system connects the sensors to the base device with wires. A wireless system uses built-in radio transmitters to communicate with the base unit. Some monitoring systems can accommodate a combination of hard-wired and wireless sensors.

8.   Can one system monitor several sensor inputs around the clock?

Once the monitoring system is installed and programmed, it will constantly read the information from the sensors 24/7. Cloud-based systems have data-logging capabilities and store limitless amounts of information that you can view from any internet-connected device via a website or app.

If the system detects any sensor readings outside the preset range, it will immediately send an alert to the people on your contact list. If you don’t want all your personnel to receive notifications at the same time, some devices can be programmed to send alerts in a tiered fashion or on a schedule. Multiple communications methods like phone, e-mail, and text provide extra assurance that you’ll get the alert. It’s a good idea to check the number of people the system can reach and if the system automatically cycles through the contact list until someone responds. Some systems allow for flexible scheduling so that off-duty personnel don’t receive alerts.

9.   How does the monitoring system supplier or manufacturer respond to emergencies? When an alarm is triggered, do they call our staff, police, fire department, and emergency medical professionals?

In most cases, the environmental monitoring system supplier is not a call center. They manufacture devices that constantly monitor conditions like temperature, power failures, and intrusion. You can program the system to notify the appropriate personnel when sensor readings fall out of the preset range or when the power goes out. It is up to your staff to take action at that point. These systems are not intended for life safety applications.

10.  Do monitoring systems have a back-up power system that will ensure the alarming function still works if the power goes out or if someone disconnects the power?

The safest choice is a cloud-based system that comes with a built-in battery backup that will last for hours in the event of a power failure. Cloud-based units constantly communicate a signal to the cloud to validate its online status. If the communication link is interrupted—for example, by a power outage or an employee accidentally switching off the unit—the system generates an alert indicating that the internet connection is lost or that there is a cellular communications problem. Users are notified about the disruption through phone, text, or e-mail. All data collected during this time will be stored in the device and will be uploaded to the cloud when the internet connection is restored.

If you opt for a cloud-based monitoring system, make sure the infrastructure used to create the cloud platform is monitored 24/7 by the manufacturer’s team. Ask if they have multiple backups across the country to ensure the system is never down.

11.  What are the costs associated with repairs to the system?

Purchase your system from a reputable manufacturer that provides a warranty and offers full repair services in the event the product stops working as it should. Also, research to make sure their tech support team is knowledgeable and willing to walk you through any questions you have about your monitoring system. Often, support specialists can diagnose and correct unit setup and programming issues over the phone.

It helps to record your observations regarding the problem so that the tech team can look for trends and circumstances concerning the issue and better diagnose the problem. Ideally, the manufacturer can provide loaner units if the problem requires mailing the device to their facility for repair.



Written by Oluwakemi Adi,,


Practical Guidelines in the Care and Maintenance of Capillary GC Columns

Many gas chromatographers rely on their gas chromatography (GC) system to function properly under extreme and demanding conditions that put their system and column under heavy stresses. The column, a simple fused-silica open-tube capillary, is often held responsible when poor chromatography arises, and yet it never is praised for the hard work that it does on a daily basis. These columns have fallen victim to the high-throughput analysis that is common in today’s laboratories. Often, little regard is given to what is injected onto the column. When analysts change columns or perform routine maintenance on a system, the columns are sometimes mishandled. Many chromatographers believe that columns have become indestructible, and treat them as such. While technological advances have been made in column manufacturing, care still should taken when handling or using a capillary column.


Capillary column                                          Packed column

Even if your GC instrument is used in a relatively “light duty” situations (for example, very clean sample matrices, few samples run per day) there are things that you can do to ensure that the column and the GC system as a whole perform at an optimum. We will discuss some precautions that will help prolong the life of capillary columns, thus, minimizing downtime and increasing productivity.

Column Breakage

Many chromatographers have one of two perspectives when it comes to capillary GC columns. Some think that they are nearly indestructible and that a 0.53-mm i.d. column can handle a bending radius of a finger. Others feel that the capillary column should be treated as if it were a fine piece of crystal. The correct perspective is somewhere in between. The reality is that the column is very durable and robust but still needs to be handled with care.

The capillary column is a fused-silica “pure quartz” open tube that is coated on the outside with a polyimide polymer and on the inside with a liquid or solid stationary phase. The capillary tube, being constructed of quartz, is actually very strong on its own. The polyimide coating provides the outer surface with a layer of protection against abrasions that could scar the tube and eventually lead to column breakage. It also protects the column from any surface flaws that if exposed to the elements in the oven (heat and moisture) would grow at an accelerated rate and lead to a failure (breakage). As the diameter of the column cage winding decreases, the stresses on the fused-silica tube increase. This observation is equally true for an increase of the inside diameter of the capillary tubing. The minimum bending or cage radius increases with an increase in column diameter. Some chromatographers try to determine how well a column will perform by bending the column to an extremely small radius, thus, testing its brittleness. If the column breaks at a larger bending diameter than what they are used to, the column is assumed to be “bad.” How tightly you can bend a column has no bearing on how well it will perform chromatographically. The performance of the column depends upon the stationary phase inside the column, not the strength of the capillary itself.

The protective coating on the column helps it withstand breakage from its environment. If the integrity of this coating is compromised, so is the strength of the column. When you trim a column, you score the polyimide coating and slightly nick the quartz tube, creating an imperfection, at which point the column breaks freely. The column hangers inside of a GC oven help keep the column out of contact with any surfaces that could potentially scar the polyimide coating and expose the quartz column. As the GC oven goes through heating and cooling cycles, the column vibrates a little, and over time, this exposed area could lead to a break.

A broken column is not necessarily a fatal event. If the break occurs within a meter or two of one of the ends, discarding that short portion of the column, trimming it to make sure that the end of the capillary is perfectly flat, and reinstalling it will cause minimal differences in performance, if any at all. Adjusting your data station for the difference in column length will help retain the appropriate flow parameters for the analysis, and resolution will not change noticeably. If a break occurs in the middle of the column, rather than discarding a column that might still be good, try using a column union to rejoin the pieces. A union such as a press-fit connector can be used to join the two sides of the column together quickly and inexpensively. Again, because the break probably will not be clean, trim the ends of both sections of the capillary column before rejoining. There are other column connectors available if you are wary of the press-fit option. The number of unions down the length of a capillary column should be limited to two or three. The larger the number of unions, the more dead volume there is, which can lead to peak tailing. A union will be a less expensive alternative to replacing the entire column and your chromatography generally will not suffer as much as you might think.

The easiest way to avoid column breakage is to use good common sense and treat your column with care. Take care when installing or removing a column, and try not to scrape it with or against any object that could potentially weaken the polyimide coating. When storing a column, seal the ends using an old septum and place it back in its original box instead of putting it unprotected in a drawer or on a shelf where it can come into contact with sharp objects. Using care and common sense will help keep the column in good shape for a longer period of time.

Stationary Phase Damage

The root cause of stationary phase damage is often difficult to determine because the symptoms usually are similar. The symptoms of stationary phase damage or degradation often are indicated by excessive column bleed, a higher degree of column activity, a loss of efficiency, or a combination of these effects. The three main causes of stationary phase failure are prolonged exposure to oxygen, sample contamination, and exceeding the upper temperature limit of the column.

Prolonged oxygen exposure: stationary phase damage due to prolonged exposure to oxygen is the most destructive and the most common mode of column failure (1). Oxygen exposure primarily is due to the presence of a leak in the system. Leaks usually occur at the inlet and are caused by a loose septum nut or a bad septum. Sometimes leaks can be found in the tubing and connections after the gas traps have been replaced. Stationary phase damage due to oxygen in the column begins just above room temperature and the severity increases with increasing temperatures. Normal injections of small volumes of air or samples that contain air are not of concern, but prolonged exposure of oxygen is especially detrimental to a column.

One of the early signs of stationary phase damage will be an increase in the baseline signal at low temperatures. Normal column bleed occurs at temperatures 30–40 °C below the upper temperature limit of the column. Each stationary phase has its own upper temperature range. Not surprisingly, these temperature limits are related directly to the overall stability of the stationary phase. The higher the temperature limit, the more stable the phase. The amount of bleed produced by a given column is related directly to the type of stationary phase as well as the amount of phase present. Therefore, for a given stationary phase, the bleed will be higher for thicker films than for thinner films. The same also holds true for an increase in polarity or column diameter and length. Column bleed is an equilibrium process, and as such, produces a rising baseline at elevated temperatures at which the stationary phase becomes less stable. Bleed produces neither discrete peaks nor an elevated baseline at low temperatures. Once you have introduced oxidative damage to the column, this “normal” baseline rise occurs at much lower temperatures, or might even be seen as an increased resting baseline under isothermal conditions.

There are several ways to limit the amount of oxygen exposure to a GC system. The first is to make sure that you use a high grade of carrier gas. However, using the highest grade of gas from a supplier is never a guarantee that no oxygen is present in the cylinder. The next line of defense is to use the appropriate gas traps or purification system and to make sure that these are maintained or replaced on a regular basis. Initially, a large capacity trap followed by a smaller indicating oxygen trap ensures that oxygen is scrubbed from the carrier gas. An indicating trap that changes color as its trapping efficiency decreases will notify you visually when your large capacity trap needs to be replaced before oxygen damage can begin to occur in your column. After you are sure that your gas supply is oxygen free, a leak test on the column installation is always a good idea. Snoop (Swagelok, Solon, Ohio), a commonly used leak detector, is never recommended to use for a leak test around a column. Because it is essentially a soap solution, components of that solution are semivolatile to nonvolatile and can cause problems within your column or instrument if it happens to get inside the flow stream. A 50:50 mixture of methanol or isopropanol and water is generally a safer solution used for leak testing around a column. The solution can be dispensed with a squeeze bottle. An alternative device to use is a leak detector available from GC accessory suppliers. These probes are very sensitive to trace amounts of carrier gas, feed gases for detectors such as the flame ionization detector, or make up gases.

One of the best ways to be certain of a leak-free system is to inject an unretained compound. This is a compound that has no interaction with the stationary phase and, therefore, should be eluted as a very symmetrical peak. Any deviation in peak shape, such as tailing, is indicative of a poor column installation or a flow-path obstruction. Butane is an easily obtained inert compound, especially good for the flame ionization detector. All one has to have on hand is a disposable lighter.

Sample-related degradation: A second contributor to column stationary phase degradation is sample related. Column technology has progressed through the years, and the stability of the stationary phases has increased during this time. Most stationary phases today are bonded and cross-linked. This characteristic increases the robustness of a column and provides significantly more resistance towards unfavorable injected solutes and solvents. A bonded and cross-linked column will resist damage to its stationary phase by water or organic solvents. Even with prolonged residence times of these solvents, the stationary phase will remain unharmed.

However, there are still certain solutes that should be avoided if at all possible. Samples to avoid are those that contain inorganic acids and bases such as KOH, NaOH, H2SO4, HNO3, and HCl to name a few. These are quite damaging to the stationary phase, even when neutralized. Occasionally, there might be small amounts of residual base or acid left in the sample. Of these, HCl will be the least damaging to a column under certain conditions. Because HCl is a gas at room temperature, it will have little to no residence time on the stationary phase itself, unless water is present.

If water is present, the HCl will travel with the water through the column and will likely have an extended residence time in the stationary phase and begin to degrade it. If water is present during an isothermal run, temperatures above 100 °C will prevent the water from condensing on the stationary phase, and, as such, also will minimize any residence time that the HCl will have. If you are using a temperature-programmed run that starts at temperatures below 100 °C and HCl-acidified water is present, the front end of the column will be in the harshest environment. If damage occurs, the front end (1–2 m) can be trimmed to remove the affected area and hopefully restore the chromatography.

As with oxygen damage, the symptoms of stationary phase damage initiated by chemicals are loss of resolution, peak broadening, peak tailing, and a shift in retention time, to list a few. Damage by acids and bases not only affects the column, but they can cause interferences within the inlet as well. The solutions can “salt out” in the liner and react with samples before they even reach the column resulting in some of the same characteristics of stationary phase damage. This interaction will be particularly noticeable with acidic and basic compounds. Changing the liner and trimming the column by 1 m or so can eliminate the problems.

Exceeding upper temperature limit: A third facilitator to column phase degradation is a problem that is often overlooked. Exceeding the posted upper temperature limit for a column or maintaining a column at the programmed temperature limit for prolonged periods of time can speed up the degradation of the phase. Stationary phase bleed begins at 30–40 °C below the upper temperature of the column. The lower of the two is called the isothermal temperature limit, and the other is the programmed temperature limit (usually in parentheses or depicted to the right of the isothermal temperature limit). The latter is the temperature that the column can be exposed to for a short period of time (~15 min or so) that can occur during a temperature program.

Sometimes when a new column is installed, or after maintenance with a reinstallation of the column, the carrier gas is left off accidentally, or a different inlet is used on a dual-inlet system. Without the flow of a carrier gas, oxygen will not be purged from the column. As was described in the previous section, oxygen in the presence of elevated temperatures will cause stationary phase damage. Surprisingly, this is a common problem associated with elevated column temperature. The second most common failure due to higher column temperatures occurs when two columns with different stationary phases or with phases of different polarity are installed in the same GC oven. Typically, the higher the polarity of a column, the lower the stability of the stationary phase and the lower its upper temperature limits. One column can have an upper temperature limit of 260 °C, and the other of 325 °C. Upon conditioning, an analyst inadvertently could raise the temperature of the oven to 325 °C and damage the lower temperature column. Typical signs of thermal damage to a column will be similar to those of oxidative or chemical damage including poor peak shape, high baseline, or excessive column bleed.

Column technology has changed over time, and column manufacturers have produced more stable polar stationary phases that minimize some of the dual column setup temperature mismatches. However, it is always a good practice to set the maximum column temperature in your method to the isothermal limit of the column. When two columns are installed in the GC system, always set all methods to the lowest isothermal temperature limit of the two columns.

Column Contamination

Contamination exists in even the cleanest samples and routinely is found in standards and even high-grade solvents. In many cases, there are more sample components that do not go through the column. Residuals exist to one degree or another in everything that is injected. Residuals are not always apparent. One simple way to check for residuals is to place 5–10 μL of the sample or standard onto a clean watch glass or microscope slide and place it on or near the hot injection port. After the solvent has evaporated, whatever is left behind on the glass is what will likely remain in the inlet or at the head of the column.

The two main classes of column contamination are nonvolatile and semivolatile. Nonvolatile contaminants are those that essentially are trapped either in the injector liner or near the front of the column and do not migrate under “normal” GC operating conditions. Semivolatile contaminates are more notorious and are those contaminants that migrate very slowly through the column and eventually are eluted. These are often the “big blobs” that are eluted unexpectedly and randomly from the column. However, such baseline upsets also might be due to carryover from poorly optimized injection conditions. These extraneous peaks or baseline upsets might have been injected as part of a previous sample several hours or even days before. One way to identify semivolatile contamination is to examine the width of the offending peak compared with the peaks of interest. Very seldom will a very broad peak occur during the elution of a series of narrow peaks (see Figure 4). Besides the fact that the peak is not supposed to be there, if the so-called “bonus peak” is significantly wider than the peaks around it, is an indicator that the material has been around for a while.

Contamination can mimic almost every type of chromatographic problem. Symptoms include ghost peaks, high bleed, sample decomposition, peak tailing or fronting, and even changes in selectivity. Although we usually think of contamination as coming from samples, it also can come from several different sources namely split-vent traps, septa, ferrules, carrier gas, gas traps, sample vials, or syringes.

The best way to eliminate contamination is to attempt to remove it before injection by employing clean-up steps during sample preparation. These steps can be as simple as passing the sample through a filter, performing sample extraction, or the use of solid-phase extraction cartridges. Indeed, we find that the most fortunate chromatographers are those performing volatiles analysis by introducing their samples via purge and trap or headspace systems, or by volatile analyte desorption from a suitable sorbent medium (for example, solid phase microextraction, polydimethylsiloxane-coated stir bar, and so forth). Columns used for these types of “clean” applications can last for years. These analyses lend themselves to longer column life because only the volatile materials are allowed onto the column, leaving any residues behind with the sample matrix. Even so, these ancillary injection techniques can be prone to gas leaks from an overused septum or worn valves and transfer line connectors, so attention must be paid to maintaining a leak-free gas system.

In the event that residues cannot be removed before injection, there are other techniques that can be employed. The first such method is to use glass wool placed at the lower end of the inlet liner. The glass wool will help to trap any nonvolatile material before the column. Another method is to use a guard column or retention gap, which is simply a 3–5 m piece of bare fused-silica capillary that is attached to the front of the column or is built-in as part of the front of the column. Any semivolatiles or nonvolatiles that make it past the glass wool will deposit on this section of column. When it comes time for inlet maintenance, a section of the guard column is removed, thus, extending the overall life of the analytical column.

Many GC problems are inlet-related or more accurately described as “front-end” related. As part of a normal inlet maintenance procedure, many times, only the liner is changed while the front end of the column is ignored. Presumably the liner is being changed because of known contamination problems. Because of the close proximity of the head of the column to the inlet liner, it is a good practice to always trim 10–20 cm of the column in conjunction with changing the inlet liner. For electronic pneumatic controlled GC systems, after trimming the front end of the GC column, a commonly overlooked step is to update the length of the column in the software. This will keep your column flows consistent because the carrier gas flow provided by the instrument relies on this dimensional information. Informing the GC software of this change will keep your retention times from shifting slightly after the column is trimmed.

After you have determined that there is a contamination problem, the simplest remedy is to trim a portion of the front of the column. Depending upon the severity of the problem, as much as 1 m or more might need to be removed. Normally, on a column longer than 20 m, this operation will not have a major impact on resolution. However, if trimming the column does not restore the performance, then the contamination is not localized to the front of the column. The contamination can reside in other parts of the inlet such as the liner or inlet body. The contamination can exist throughout the entire length of the column. When this form of contamination occurs, many analysts immediately bake-out the column by heating it to its upper temperature limit for a period of time. A prolonged bake-out is tempting because of its simplicity; however, it is the least effective way to remove contaminates and can cause more problems than it solves. Baking out the column can cause contaminants to irreversibly polymerize or caramelize on the stationary phase, thus, permanently damaging the column.

A better solution is to rinse the column with solvent. Although the rinse procedure is more involved than a simple bake-out, the procedure is still relatively simple and definitely more effective. Solvent rinsing can be performed on any bonded and cross-linked phase only. Nonbonded phases cannot be rinsed. If there is any question as to whether your column can be rinsed, contact the manufacturer to be sure. Rinse kits are available for purchase or can be homemade (see Figure 5). Solvents with varying polarities should be used so as to remove the widest range of contaminants. Rinse in the order of most polar to least polar. Each successive solvent must be soluble in the previous one. The injection solvent also should be included when possible. A good general-purpose solvent list might be methanol, methylene chloride, and hexane. If aqueous-based samples have been injected or salts are suspected as contaminates, include water as one of the rinse solvents. Try to avoid any high boiling solvents. Anywhere from 5 to 10 mL of each solvent should be used, performing the rinse from the detector end to the injector end. This reversing of the column is recommended strongly, because most contaminants are likely in the front part of the column and the removal path is shorter for solubilized contaminants.

Be sure to “dry” the column phase thoroughly by purging with nitrogen for ~30 min after rinsing before placing the column back in the GC oven. Install the column in the injector side only and pass carrier gas through the column for 10–20 min, then install the column on the detector side and check for leaks. Start the oven program at 40 °C and ramp the temperature at 2–3 °C/min to the isothermal maximum. Hold this temperature until the baseline stabilizes, which should take only 30–60 min. All of these steps are meant to ensure that no residual rinsing solvent is left behind in the stationary phase. If solvent remains and the column is placed in the oven and heated too quickly, the rapid vapor expansion could cause damage to the internal polymeric layer.



While capillary columns are not as fragile as a piece of crystal, they also are not indestructible. Care should be taken to ensure the robustness and productivity that they can provide. Maintenance requires some periodic downtime. Minimizing this downtime is pertinent to the high sample throughput labs of today. Maintenance is always less time consuming than repair primarily because maintenance is a scheduled event that when properly implemented, will always be less timely than unexpected column, injection-port, or detector failures caused by neglect. Topics that we discussed earlier, such as handling the column more gently, taking an extra moment to cleanup a sample, or taking an extra second to double check the instrument method parameters before starting the system, is critical to extending the life of a column. It is highly recommended that more frequent replacement of inlet liners will help to cut down on column maintenance problems. Those extra steps taken to maintain the health and performance of your column will pay dividends in production efficiency and analysis reproducibility.


(1) E.J. Guthrie and J.J. Harland, LCGC 13(6), 446–455 (1995).

(2) R.E. Majors, LCGC 16(11), 982–991 (1998).

(3) D. Rood, A Practical Guide to the Care, Maintenance, and Troubleshooting of Capillary Gas Chromatographic Systems (Huthig Buch Verlag GmbH, Heidelbe

Written by Aduroja Opeyemi



What are total petroleum hydrocarbons (TPH)?

Total petroleum hydrocarbons (TPH) is a term used to describe a large family of several hundred chemical compounds that originally come from crude oil. Crude oil is used to make petroleum products, which can contaminate the environment. Because there are so many different chemicals in crude oil and in other petroleum products, it is not practical to measure each one separately. However, it is useful to measure the total amount of TPH at a site.

TPH is a mixture of chemicals, but they are all made mainly from hydrogen and carbon, called hydrocarbons. Scientists divide TPH into groups of petroleum hydrocarbons that act alike in soil or water. These groups are called petroleum hydrocarbon fractions. Each fraction contains many individual chemicals.

Some chemicals that may be found in TPH are hexane, jet fuels, mineral oils, benzene, toluene, xylenes, naphthalene, and fluorene, as well as other petroleum products and gasoline components. However, it is likely that samples of TPH will contain only some, or a mixture, of these chemicals.

What happens to total petroleum hydrocarbons (TPH) when they enter the environment?

  • TPH may enter the environment through accidents, from industrial releases, or as by-products from commercial or private uses.
  • TPH may be released directly into water through spills or leaks.
  • Some TPH fractions will float on the water and form surface films.
  • Other TPH fractions will sink to the bottom sediments.
  • Bacteria and microorganisms in the water may break down some of the TPH fractions.
  • Some TPH fractions will move into the soil where they may stay for a long time.

How might I be exposed to total petroleum hydrocarbons (TPH)?

  • Everyone is exposed to TPH from many sources.
  • Breathing air at gasoline stations, using chemicals at home or work, or using certain pesticides.
  • Drinking water contaminated with TPH.
  • Working in occupations that use petroleum products.
  • Living in an area near a spill or leak of petroleum products.
  • Touching soil contaminated with TPH.

How can total petroleum hydrocarbons (TPH) affect my health?

  • Some of the TPH compounds can affect your central nervous system. One compound can cause headaches and dizziness at high levels in the air. Another compound can cause a nerve disorder called “peripheral neuropathy,” consisting of numbness in the feet and legs. Other TPH compounds can cause effects on the blood, immune system, lungs, skin, and eyes.
  • Animal studies have shown effects on the lungs, central nervous system, liver, and kidney from exposure to TPH compounds. Some TPH compounds have also been shown to affect reproduction and the developing foetus in animals.

    How likely are total petroleum hydrocarbons (TPH) to cause cancer?

  • The International Agency for Research on Cancer (IARC) has determined that one TPH compound (benzene) is carcinogenic to humans. IARC has determined that other TPH compounds (benzo[a]pyrene and gasoline) are probably and possibly carcinogenic to humans. Most of the other TPH compounds are considered not to be classifiable by IARC.
    Is there a medical test to show whether I’ve been exposed to total petroleum hydrocarbons (TPH)?
  • There is no medical test that shows if you have been exposed to TPH. However, there are methods to determine if you have been exposed to some TPH compounds. Exposure to kerosene can be determined by its smell on the breath or clothing. Benzene can be measured in exhaled air and a breakdown product of benzene can be measured in urine. Other TPH compounds can be measured in blood, urine, breath, and some body tissues.

Total Petroleum Hydrocarbon analysis

Modern science includes a wide array of testing methods for thorough Total Petroleum Hydrocarbon analysis. Some of the most commonly used methods include:

  • Gravimetric – A sample is extracted using an organic solvent and then evaporated to leave the grease residue behind. However, this method only measures heavy hydrocarbon ranges. It does not provide detailed information as to the carbon range.
  • Immunoassay – This is a relatively new biochemical test used in field measurements of the concentration of total petroleum hydrocarbons in a solution. However, this method does not give information regarding the carbon range and results are prone to interference.
  • Infrared Spectroscopy – A sample is extracted using an “IR transparent” solvent and the transmittance or absorbance is measured in an Infrared Spectrophotometer. The method is prone to interferences including solubility issues and does not give information as to carbon range
  • Gas Chromatography – is perhaps the most commonly used TPH analysis method. Samples are extracted using methods such as tumbling, sonication, or microwave extraction. Extracts are then analyzed using gas chromatography. TPH compounds are detected in the order of boiling point and quantified by comparison to standards. The results are expressed in relation to hydrocarbon ranges:
  • C6-C9
  •  C10-C15
  • C16-C28

Source:   Agency for Toxic Substances and Disease Registry ASTDR

Adebola Muyiwa,,

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Fermentor (Bioreactor): Design and Its Functions

Meaning of Fermentor:

A fermentor (bioreactor) is a closed vessel with adequate arrangement for aeration, agitation, temperature and pH control, and drain or overflow vent to remove the waste biomass of cultured microorganisms along-with their products.

Figure 38.8 : A fermentor (bioreactor)

A fermentor is used for commercial production in fermentation industries and is a device in which a substrate of low value is utilized by living cells or enzymes to generate a product of higher value. Fermentors are extensively used for food processing, fermentation, waste treatment, etc.

Figure 38.9 : A fermentor

Design of Fermentors:

All bioreactors deal with heterogeneous systems dealing with two or more phases, e.g., liquid, gas, solid. Therefore, optimal conditions for fermentation necessitate efficient transfer of mass, heat and momentum from one phase to the other. Chemical engineering principles are employed for design and operation of bioreactors.

A bioreactor should provide for the following:

(i) Agitation (for mixing of cells and medium),

(ii) Aeration (aerobic fermentors); for O2 supply,

(iii) Regulation of factors like temperature, pH, pressure, aeration, nutrient feeding, liquid level etc.,

(iv) Sterilization and maintenance of sterility, and

(v) Withdrawal of cells/medium (for continuous fermentors).

Modern fermentors are usually integrated with computers for efficient process monitoring, data acquisition, etc.

Figure 39.0 : A Modern fermentor

Generally, 20-25% of fermentor volume is left unfilled with medium as “head space” to allow for splashing, foaming and aeration. The fermentor design varies greatly depending on the type and the fermentation for which it is used. Bioreactors are so designed that they provide the best possible growth and biosynthesis for industrially important cultures and allow ease of manipulation for all operations.

Size of Fermentors:

The size of fermentors ranges from 1-2 litre laboratory fementors to 5,00,000 litre or, occasionally, even more, fermentors of upto 1.2 million litres have been used. The size of the fermentor used depends on the process and how it is operated. A summary of fermentor or size of fermentor (litres) Industrial product sizes for some common microbial fermentations is given in Table 39.6.

Fermentor sizes for various microbial fermentations

Construction of Fermentors:

Industrial fermentors can be divided into two major classes, anaerobic and aerobic. Anaerobic fermentors require little special equipment except for removal of heat generated during the fermentation process, whereas aerobic fermentors require much more elaborate equipment to ensure that mixing and adequate aeration are achieved.

Since most industrial fermentation process are aerobic, the construction of a typical aerobic fermentor (Fig. 39.1) is the following:

An industrial aerobic fermentor

1. Cooling Jacket:

Large-scale industrial fermentors are almost always constructed of stainless steel. A fermentor is a large cylinder closed at the top and the bottom and various pipes and valves are fitted into it. The fermentor is fitted externally with a cooling jacket through which steam (for sterilization) or cooling water (for cooling) is run.

Cooling jacket is necessary because sterilization of the nutrient medium and removal of the heat generated are obligatory for successful completion of the fermentation in the fermentor. For very large fermentors, insufficient heat transfer takes place through the jacket and therefore, internal coils are provided through which either steam or cooling water is run.

2. Aeration System:

Aeration system is one of the most critical part of a fermentor. In a fermentor with a high microbial population density, there is a tremendous oxygen demand by the culture, but oxygen being poorly soluble in water hardly transfers rapidly throughout the growth medium.

It is necessary, therefore, that elaborate precautions are taken using a good aeration system to ensure proper aeration an oxygen availability throughout the culture. However, two separate aeration devices are used to ensure proper aeration in fermentor. These devices are sparger and impeller.

The sparger is typically just a series of holes in a metal ring or a nozzle through which filter-sterilized air (or oxygen-enriched air) passes into the fermentor under high pressure. The air enters the fermentor as a series of tiny bubbles from which the oxygen passes by diffusion into the liquid culture medium.

The impeller (also called agitator) is an agitating device necessary for stirring of the fermenter.

The stirring accomplishes two things:

(i) It mixes the gas bubbles through the liquid culture medium and

(ii) It mixes the microbial cells through the liquid culture medium. In this way, the stirring ensures uniform access of microbial cells to the nutrients.

The size and position of the impeller in the fermentor depends upon the size of the fermentor. In tall fermentors, more than one impeller is needed if adequate aeration and agitation is to be obtained. Ideally, the impeller should be 1/3 of the fermentors diameter fitted above the base of the fermentor. The number of impeller may vary from size to size to the fermentor.

3. Baffles:

The baffles are normally incorporated into fermentors of all sizes to prevent a vortex and to improve aeration efficiency. They are metal strips roughly one-tenth of the fermentors diameter and attached radially to the walls.

4. Controlling Devices for Environmental Factors:

In any microbial fermentation, it is necessary not only to measure growth and product formation but also to control the process by altering environmental parameters as the process proceeds. For this purpose, various devices are used in a fermentor. Environmental factors that are frequently controlled includes temperature, oxygen concentration, pH, cells mass, levels of key nutrients, and product concentration.

Use of Computer in Fermentor:

Computer technology has produced a remarkable impact in fermentation work in recent years and the computers are used to model fermentation processes in industrial fermentors. Integration of computers into fermentation systems is based on the computers capacity for process monitoring, data acquisition, data storage, and error-detection.

Some typical, on-line data analysis functions include the acquisition measurements, verification of data, filtering, unit conversion, calculations of indirect measurements, differential integration calculations of estimated variables, data reduction, tabulation of results, graphical presentation of results, process stimulation and storage of data.

Types of Fermentor:

The fermentor (bioreactor) types used extensively in industries are the stirred tank fermentor, airlift fermentor, and bubble column fermentor.

(i) Stirred Tank Fermentor:

Stirred tank fermentors consists of a cylindrical vessel with a motor driven central saft that supports one or more impellers.

(ii) Airlift Fermentor:

In airlift fermentor (39.2) , the liquid culture volume of the vessel is divided into two interconnected zones by means of a baffle or draft tube. Only one of the two zones is sparged with air or other gas and this sparged zone is known as the riser.

The other zone that receives no gas is called down-comer. The bulk density of the gas-liquid dispersion in the gas-sparged riser tends to be lower than the bulk density in the down-comer, consequently the dispersion flows up in the riser zone and down-flow occurs in the down-comer.

Airlift fermentor

Airlift fermentors are highly energy-efficient and are often used in large-scale manufacture of biopharmaceutical proteins obtained from fragile animal cells. Heat and mass transfer capabilities of airlift reactors are at least as good as those of other systems, and airlift reactors are more effective in suspending solids than are bubble column fermentors.

All performance characteristics of airlift -fermentor are related ultimately to the gas injection rate and the resulting rate of liquid circulation. Usually, the rate of liquid circulation increases with the square root of the height of the airlift device.

Because the liquid circulation is driven by the gas hold-up difference between the riser and the down-comer, circulation is enhanced if there is little or no gas in the down-comer. All the gas in the down-comer comes from being entrained in with the liquid as it flows into the down-comer from the riser near the top of the reactor.

(iii) Bubble Column Fermentor:

A bubble column fermentor (Fig. 39.3) is usually cylindrical with an aspect (height-to-diameter) ratio of 4-6. Gas is sparged at the base of the column through perforated pipes, perforated plates, or sintered glass or metal micro-porous spargers.

O2transfer, mixing and other performance factors are influenced mainly by the gas flow rate and the rheological properties of the fluid. Internal devices such as horizontal perforated plates, vertical baffles and corrugated sheet packing’s may be placed in the vessel to improve mass transfer and modify the basic design.

The column diameter does not affect its behaviour so long as the diameter exceeds 0.1 m. One exception is the axial mixing performance. For a given gas flow rate, the mixing improves with increasing vessel diameter. Mass and heat transfer and the prevailing shear rate increase as gas flow rate is increased.

A bubble column fermentor


Written by Oluwakemi Adi,,

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