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,

opeyemi@aasnig.com, www.aasnig.com


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. http://www.cmu.edu/homepage/environment/2011/fall/chapter_04.pdf ; 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 http://education.qld.gov.au/health/pdfs/healthsafety/time-sensitive-chemicals.pdf; 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.
  10. http://clark.cleanharbors.com/tt/sl.ashx?z=776bb3d6&dataid=1605.

Posted by Oluwakemi Adi

kemi@aasnig.com, www.aasnig.com


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,

kemi@aasnig.com, www.aasnig.com


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,

muyiwa@aasnig.com, www.aasnig.com

07084594004, 07084594001


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,

kemi@aasnig.com, www.aasnig.com

08060874724, 09086118123

10 Steps to Pipetting Like a Pro!

Pipettes are an essential laboratory tool used to dispense liquids. Whilst pipettes can be used
simply as a liquid transfer device where the actual volume of liquid dispensed is not important,
more often they are required to dispense accurate and precise volumes of solutions which are
likely to be critical to experimental procedures. There are numerous pipette manufacturers
and many pipette types that are designed for all applications, with volumes ranging from 0.1
μL to 100 mL or more. You can get repeater pipettes if you are dispensing lots of aliquots of the
sample solution and multi-channel pipettes which are great for dispensing samples into plates;
especially useful for high-throughput assays. In this guide we will highlight 10 important factors
that will help you improve your pipetting technique and ensure accurate, reproducible results!

1. Identifying your pipettes
If working to Good Laboratory Practice (GLP) or Good Clinical Practice (GCP) regulations then all of
your pipettes should have a unique identifier, typically this will include the area or assay they are used
for and a number. All pipette information should be held centrally and include a list of all pipettes in
circulation, the make and model, the serial number and the unique identifiers you have assigned.
All details of the pipette metrology, its service history, any faults and repairs should also be stored
here as a regulatory authority may wish to view this information during an inspection.

2. Calibrate your pipettes
Pipettes should be calibrated regularly in accordance with the guidelines you are working to.
For effective calibration, use their lowest and highest volumes, which will be stipulated by the
manufacturer. This calibration can be done with water weighings using a 4 place balance; for
volumes below 10 μL a 5 place balance is required, or it can be done using a radioactive solution.
Once the calibration measurements have been performed the accuracy and precision should be
calculated. Typically the accuracy should be within 3% of expected weight or radioactive counts
and the precision within 2.5%. Always check the laboratory SOP’s for exact acceptance criteria.
If a pipette fails metrology, there are a number of things that can be done. Firstly, check that you
are using the correct tip for the pipette and that it has been put on the pipette correctly. Ensure
that the tip is not damaged or leaking and that there are no air bubbles present when you pipette
your water/radioactive standard. If calibration still fails after these checks have been carried out,
get a second person to perform the calibration, to ensure no operator error. If the pipette still
fails, it can be taken apart (follow manufacturer’s instructions) and cleaned with water, or pipette
grease added to improve movement. If the pipette still fails calibration it is advisable to send it to
the manufacturer or service provider for repair

3. Which pipette is best for a given volume?
The accuracy of variable volume pipettes is greatest at the maximum volume and it is therefore
important that the correct pipette is selected for each application. It is common practice to
only use a volume range of 20–100% of the pipette’s max capacity, meaning you are never
using a pipette at the lowest volume that it can go down to (usually 10%) – this limits the risk
of inaccuracy associated with using a pipette at its lower limits. Ideally, you want to select a
pipette where the volume you’re pipetting is 50–100% of the pipette’s range.

4. Pipette tips
Make sure you select the correct pipette tip for your chosen pipette. Most pipette tips can be
supplied loose in bags or set up in racks for easier/more convenient use. Racked tips can be
supplied sterile and non-sterile and some will stipulate, for example, RNAse free for specific
applications. Pick the tips most suitable for you application – bagged tips are cheaper and
sterile cost more than non-sterile! Ensure that the pipette tip you use is compatible with the
make and model of the pipette you are using.
When using an air displacement pipette for samples, for samples that must remain sterile
(e.g. plasma and cell cultures), or that are sensitive to cross contamination (such as next
generations sequencing library preparation) you can use tips with filters built in to prevent
liquids aspirating into the pipette barrel. This prevents the pipette becoming contaminated
with your biological sample and transferring to other samples.
Ideally you want to change your pipette tips between samples to avoid contamination, however
if you are preparing something like a calibration line where you are going up in concentration
you can use the same tip, in this instance it’s advisable to prime the tip with each solution
as you move up the concentration range. If you are moving down concentration range or if
you are pipetting samples of unknown concentration then always change your tip between
samples to prevent any contamination.

5. Air displacement pipettes
On air displacement pipettes the plunger is depressed to both draw up and dispense liquid.
Operation consists of depressing the plunger to the first stop position whilst the pipette is
held in air. The pipette tip is then submerged in the liquid to be pipetted and the plunger
released slowly, drawing the liquid into the pipette tip. The liquid then be dispensed into
the receiving vessel by depressing the plunger to the first stop position and then the second
stop position (also called the ‘blow out’ position) to expel all of the liquid from the pipette
tip. The volume of air displaced is equivalent to the volume of liquid aspirated.
Positive displacement pipettes

6. Positive-displacement pipettes, work like a syringe. There is no air cushion between the
disposable piston and the sample. With no air cushion to expand or contract, the aspiration
force remains constant, unaffected by the physical properties of the sample. This allows
the positive-displacement operator to pipette very viscous or high density samples, such as
glycerol and blood.

7. Pipetting technique
Once you’ve selected the most appropriate pipette for your sample and ensured the correct
pipette tip is on, you’re good to go.
• Firstly prime/pre-wet the tip 2–3 times with the solution/sample you wish to transfer.
• Immerse the tip into the liquid you wish to pipette – this should be sufficiently below the
meniscus such that you don’t aspirate air but not so far that the liquid level comes above
the top of the pipette tip.
• Leave the tip in the liquid for a couple of seconds after aspiration to ensure the tip has
been filled completely.
• During aspiration, try to hold the pipette vertically in the liquid and in the centre of the
vessel rather than touching the sides. If any droplets of liquid are on the side of the pipette
tip these can be removed using the edge of the vessel or by wiping with a lint free cloth –
ensure you don’t wipe the opening of the tip as this could cause some loss of sample.
• When dispensing the liquid, hold the pipette at a slight tilt rather than vertical and dispense
against the side of the receiving vessel. Watch the sample leave the pipette tip and when you
remove the tip from the vessel ensure there are no droplets on the end of the tip – if there
are, touch the tip against the side of the receiving vessel again so that the droplet transfers.
• Try to aspirate and dispense at a slow and consistent speed between samples as this will be
more likely to give consistent and reproducible results.

8. Which pipette is best for my sample?
Regardless of the volume you require, the nature of the sample directly impacts precision
and accuracy. Air-displacement pipettes will be better for aqueous liquids whereas positive displacement
pipettes should be used for problem liquids.

9. Looking after your pipettes
Pipettes should be cleaned with a soft cloth and water if they get any sample on them. They
can also be sprayed with IMS or ethanol to clean them prior to use in a sterile environment
or prior to service. You must not use organic solvents to clean the internal parts of a pipette
as this can cause damage or corrosion. It’s best to store pipettes vertically when not in use;
on a pipette stand or carousel. If a pipette is dropped or taken apart it should always be
calibrated again before use.
If working in a lab where a variety of different procedures take place it is a good idea to have
separate sets of pipettes for the different applications. For example, having one set that is
solely used for radioactive work eliminates the risk of other pipettes and other areas of the
lab becoming contaminated with radioactivity. Having separate sets for PCR or cell culture
work also reduces the risk of cross-contamination or introducing any infections into your

10. Unexpected results
Sometimes even when we do everything correctly our samples don’t behave as we’d like
and we don’t get the results we are expecting. This could be due to the properties of the
compounds in the solutions we are pipetting, for example compounds can stick to the
pipette tips or the sample vessels. There are some things you can do to try and eliminate
this. With regards to vessels, you can trial different plastics (e.g. low bind materials), glass
and salinized glass. With pipette tips you can prime/wet-the tip multiple times to ensure that
the compound will stick during the prime thus resulting in the correct amount being pipetted
when transferring your sample. You could also use a low retention tip designed to repel the
sample you are pipetting thus preventing adhesion to the tip. Most manufacturers supply a
variant of these for their pipettes.




Written by Aduroja Opeyemi

opeyemi@aasnig.com, www.aasnig.com



The Effect of Air Leaks on the Performance of GC and GC/MS Systems


In gas chromatography, air leaks can cause a cascading series of deleterious effects on system components and chromatographic results. Establishing and maintaining leak-free connections in GC systems is a basic yet critical aspect of gas-phase analysis. Leak-free systems provide consistent, reliable data and can improve productivity by increasing intervals between required maintenance. At elevated temperatures, generally higher than 260 °C, polysiloxane-based GC columns bleed and lose stationary phase, depending on the substituent group linked to the polymer . In the presence of oxygen, bleed increases dramatically at elevated temperature. Increased column bleed in turn leads to a shift to shorter retention for peaks of interest and an early demise of the GC column.

Liner activity is also impacted by the presence of oxygen in the flow path. Oxygen can strip deactivation layers from glass liners, leaving more active sites onto which polar analytes can sorb. This leads to increased tailing, poor peak integration, and inaccurate results. The need for more frequent inlet maintenance, along with system downtime, is a direct consequence of leaks, especially for analyses of active analytes, such as chlorinated pesticides

In GC/MS, air leaks in the flow path produce high levels of noise, increased bleed, shorter filament lifetime, more frequent source cleaning, and reduced electron multiplier life times. Cumulative effects of oxygen-contaminated carrier are illustrated in this application note to underscore the need to establish and maintain leak-free systems to the fullest extent possible.

Materials and Methods

System 1 consisted of a dual-channel FID Agilent 7890A GC equipped with a dual-tower Agilent 7693 Automatic Liquid Sampler and two inert split/splitless inlets. An inline three-way valve was installed in the carrier line leading to the rear inlet. One leg of the valve was plumbed to a pure helium (99.9999%) source, and the other was plumbed to a helium cylinder containing 1,000 μL/L oxygen. This installation enabled switching back and forth between pure helium and helium doped with oxygen to evaluate how lasting any effects might be. The front inlet was plumbed to the same pure helium source as the pure helium leg of the three-way valve on the rear inlet. Inlet maintenance and testing sequences were run simultaneously to ensure as close to a one-to-one comparison as possible.

System 2 consisted of an Agilent 7890B GC and an Agilent 5977A Series GC/MSD System equipped with a single-tower 7693 Automatic Liquid Sampler and an inert split/splitless inlet. An inline three-way valve was installed in the carrier line leading to the front inlet. One leg of the valve was plumbed to a pure helium source and the other was plumbed to a helium cylinder containing 1,000 μL/L oxygen. This installation enabled switching back and forth between pure helium and helium doped with oxygen to evaluate how lasting the effects might be

GC-FID conditions

Column: Agilent J&W DB-1701, 20 m × 0.18 mm, 0.18 μm

Carrier: Helium (front) versus 1,000 μL/L O2 in helium (rear),

constant flow 1.36 mL/min at 125 °C

Oven: 125 °C (0.34 min) to 275 °C (7.3 °C/min, 10.1 min hold)

Inlet: Pulsed splitless, 45 psi 0.32 min, 1 μL at 250 °C, total flow

54.4 mL/min, 3 mL/min switched septum purge, gas saver off,

50 mL/min purge flow after 0.33 min

Sample: CLP pesticide mix 4 μg/mL or endrin/DDT 20 μg/mL

Inlet liner: Ultra Inert splitless single taper with wool (p/n 5190-2293)

Dual FIDs: 300 °C at 40 mL/min H

GC/MS with Agilent J&W DB-1701 conditions

Column: Agilent J&W DB-1701, 20 m × 0.18 mm, 0.18 μm

Carrier: Helium versus 1,000 μL/L O2 in helium, constant flow

1.36 mL/min at 125 °C

Oven: 125 °C (0.34 min) to 275 °C (7.3 °C/min, 10.1 min hold)

Inlet: Pulsed splitless, 45 psi 0.32 min, 0.5 μL at 250 °C, total flow

63.9 mL/min, 3 mL/min switched septum purge, gas saver off,

60 mL/min purge flow after 0.33 min

Sample: CLP pesticide mix 4 μg/mL or endrin/DDT 20 μg/mL or

semivolatile mx

Inlet liner: Ultra Inert splitless single taper with wool (p/n 5190-2293)

MSD temps: Transfer line 280 °C, source 300 °C, quad 180 °C

Mode: Full scan, 10 to 450 amu

Results and Discussion


A 7890A GC dual-channel FID, a 7890B GC, and a 5977 Series GC/MSD system were set up with a three-way valve in the carrier line to enable switching between pure helium, and helium doped with 1,000 μL/L oxygen to simulate an air leak. Oxygen in helium at 1,000 μL/L is representative of a 5% by volume air leak into each respective system. This approach was chosen to rapidly demonstrate the deleterious effects of having oxygen in the carrier gas. Organochlorine pesticides included in US EPA Method 8081 were chosen as a test case for the dual-GC-FID system. DB-1701 columns (14% (cyanopropyl-phenyl) methylpolysiloxane phase) were used to demonstrate the effects of having oxygen in the carrier, with respect to endrin/DDT breakdown, bleed profiles, and retention time stability.

Figure 1 shows a typical FID trace for US EPA 8081 pesticides on a 20 m × 0.18 mm, 0.18 μm Agilent J&W DB-1701 column. The nominal concentration of the pesticide mix in this chromatogram was 4 μg/mL.

Almost immediately, oxygen in the carrier gas was observed to have a subtle impact on the column bleed performance with temperature cycling on the DB-1701 stationary phase. There was an increase in bleed on the oxygen-exposed column as the oven temperature climbed to 275 °C. The effect persisted, even after purging with pure helium carrier, indicating permanent column damage.

Figure 2 is an overlay of blank injection FID chromatograms with and without

oxygen in the carrier.


A 7890B GC with a 5977 Series GC/MSD system was set up with a three-way valve in the carrier line to enable switching between pure helium, and 1,000 μL/L oxygen-doped helium to simulate an air leak. The three-way valve was switched back and forth between the oxygen-doped and pure helium carrier gas. At first, the oxygen-doped carrier was inline only during acquisition sequences. Gradually, exposure to oxygen-doped carrier gas was increased to overnight, then weekends, and finally for five days continuously until auto tune no longer functioned. The cumulative total of oxygen exposure for the  GC/MS was 15 days before the system would no longer tune. The highest EMV (electron multiplier voltage) obtained was 2,350 volts, at which time source cleaning and filament replacement were required to return the instrument to normal function with pure helium.

GC/MS testing was conducted on a DB-5ms column with a 1 μg/mL GC/MS semivolatile analyzer check-out mix. Here, the impact of oxygen in the carrier gas was readily apparent in that the high signal background all but wiped out the analyte signal. At a level of 1 μg/mL, this represents an alarming loss of sensitivity.

Figure 3 is an overlay of the total ion chromatograms for an injection of 1 μg/mL GC/MS semivolatile mix with oxygen in the carrier and without oxygen in the carrier.


Air leaks into GC and GC/MS systems have a dramatic and cumulative effect on system performance. Permanent column damage, retention time drift to shorter retention, and increased inlet activity are characteristic of oxygen exposure at elevated temperature in both GC and GC/MS systems. All of these effects were observed. Dramatic signal loss, high background noise and rapid increase in electron multiplier voltage were seen on the GC/MS system.

The conditions used in these experiments were chosen to simulate an air leak of approximately 5% air into the system, and to rapidly visualize the deleterious effects. Both GC column and liner lifetimes were shortened, requiring more frequent maintenance. Under these conditions, after 15 days of cumulative exposure to oxygen, electron multiplier voltage climbed to 2,350 volts and the filament failed, dictating a major service event in just over two weeks. Having to clean the source and replace a filament twice a month would most certainly have a dramatic impact on system productivity. All of these deleterious effects build a strong case for doing everything possible to keep air and the oxygen it contains out of GC and GC/MS systems.


Culled from: Impact of Air Leaks on the Productivity of GC and GC/MS Systems by Ken Lynam. Application Notes. Agilent Technologies Inc.

Written by Muyiwa Adebola,

www.aasnig.com, muyiwa@aasnig.com

07084594004, 07084594001

Migraine: More Than Just A Bad Headache

Migraine is a debilitating neurological disease that is often misunderstood by those who do not suffer symptoms, according to the Migraine Impact Report a new survey recently released by Eli Lilly and Company and conducted by Nielsen. The report showed that respondents without the disease underestimate the pain and duration of a typical migraine. Ninety one percent of respondents with migraine further agreed that those who do not experience migraine pain do not have a complete understanding of the severity of the disease.

“As a practicing neurologist, I saw firsthand both the burden faced by people living with migraine, and the feeling that people must ‘power through’ their lives because those around them may not understand just how all-encompassing migraine can be,” says Dr. Sheena Aurora, a medical fellow at Lilly.

Migraine is characterized by recurrent episodes of severe headache, and is often accompanied by other symptoms including nausea, vomiting, changes in vision and sensitivity to light and sound. The pain can be so severe that most respondents to the survey on average rated the worst migraine pain as higher than both the pain associated with kidney stones and broken bones. More than 36 million Americans have migraine, with three times more women affected by migraine compared to men.

Migraine More Than Just A Headache infographic

Graphic courtesy of Eli Lilly and Nielsen

The survey found that the physical, social and economic impacts of migraine can be frequent and far-reaching. Respondents diagnosed with migraine on average experienced just 15.4 completely pain-free days over the previous 30 days. During the same time period, respondents reported that symptoms prevented them from doing what they wanted to do for one week (6.9 days). These missed activities include less time spent interacting with children, lowered productivity at work and a hindered ability to make plans.

“Sometimes even people with migraines don’t realize the impact of their disease,” Aurora says. “When I treated patients with migraines, I would ask them how many migraine headache days they had, and they would have to stop and think about it.”

In addition to causing patients to miss important events, migraine can have a significant negative impact on non-sufferers. Ninety percent of surveyed family members and caretakers of migraine sufferers said they feel “helpless” when their loved one has a migraine and that they wished that their loved ones could seek better care or treatment for their symptoms.

America’s biopharmaceutical researchers are working to unravel the underlying drivers of migraine and bring a new wave of treatment options to patients. With science moving rapidly, there is real hope on the horizon for patients.



Written by Oluwakemi Adi

www.aasnig.com, kemi@aasnig.com

08060874724, 07084594001

How To Calibrate Your Autoclave

“Calibration” is a word that is frequently used in the steam sterilization industry. This post will explore what it is, what is involved in doing it right, alternatives, and the potential effects on an existing sterilization process.

What is Calibration?

At its very basic premise, calibration is bringing the response of a sensor (e.g. transducer, thermocouple, etc.) to within a specified range relative to a primary reference standard. This is done by comparing the autoclave sensor’s response to a previously calibrated device (referred to hereafter as “standard”) whose response is traceable to a national reference standard, maintained in the United States by the National Institute of Standards and Technology (NIST).

As a rule of thumb, the standard should be five times as accurate as the device being calibrated. Therefore, when calibrating a temperature probe with a desired accuracy of ±0.5°C, the calibration standard should be accurate to ±0.1°C.

Why Calibrate?

The short answer is calibration ensures consistent results from a process. Steam sterilization efficacy is highly dependent upon actual temperature. For example, if a steam autoclave is running at 120°C for 15 minutes, the theoretical lethality of that cycle is only 82% of that of a cycle running at 122°C for the same amount of exposure time.

Since most laboratory autoclaves do not require temperature to be accurate to better than ±1°C, this variability can be more common than one would think. If your temperature transducers are calibrated, this problem will diminish.

Equipment Required to Calibrate

The proper way to calibrate an autoclave is with the use of a NIST-traceable device (standard) such as a dry block, oil bath, or temperature probe. If using a dry block or an oil bath  make sure it is designed to control to a constant temperature (±0.1°C). If the dry block or oil bath is not NIST-traceable, or its calibration has expired, then use a NIST-traceable temperature probe  with ±0.1°C minimum accuracy.

If the above-recommended equipment is not available one could use boiling water to help calibrate the autoclave’s temperature sensors. Boiling water can act as a constant-temperature (i.e. 100°C/212°F) bath that is somewhat near sterilization temperature. However, if the facility isn’t exactly at sea level then boiling water isn’t necessarily going to be 100°C/212°F. Check the atmospheric pressure in your area (obtained online at www.weather.gov) or use an absolute pressure manometer (mercury column or electronic) to obtain the exact pressure reading, then calculate the actual “pressure corrected” boiling temperature of water by using this steam table: (http://www.efunda.com/materials/water/steamtable_sat.cfm). If you place your sensor into boiling water (not in contact with the bottom of the vessel holding the water) and it isn’t within 1°C of the theoretical temperature, then you will need to carry out a calibration.

Calibration Methods

Calibration instructions can vary by the number of calibration points measured (i.e. 1-point, 2-point, or 3-point). What is the difference?

Single Point Calibration

A single point calibration is valid only within the accuracy at that specific point. When sterilizing at only one temperature, say 121°C/250°F, this is not too much of a problem, although you have no idea what is happening in any process excursions to higher or lower temperatures.

Two Point Calibration

Some calibration instructions recommend taking two measurements and calculating the slope (gain) and y-intercept (zero offset). Using two points for calibration is relatively fast and convenient; however, two points define a straight line and reveal nothing about any non-linearity in the probe’s reading. Also, any errors in the two measurements are not going to be evident.

Multipoint Calibration

A multipoint (more than two point) calibration will indicate if the probe is behaving in a nonlinear manner, which could be a good reason to replace it, and also allows any measurement errors to be averaged out over the greater number of points. These instructions suggest taking three or more measurements and performing a linear regression to get the slope and y-intercept. Any multipoint calibration should be done with points both outside the working range of the sterilization cycle(s) you will use. For example, a lab running at 121°C (and only 121°C) should calibrate at 116, 121, and 126°C or at 116 and 126°C for a two point calibration. This allows the response of the measurement and control system to be taken into account. If the lab is running cycles over a greater range, calibration should start and finish 5°C below the minimum temperature and 5°C above the maximum temperature used. A good rule of thumb is to calibrate against at least the number of decades in °C plus one. So if you run cycles at three different temperatures (e.g. 115°C, 121°C, and 134°C), you would make calibration measurements at four points (e.g. 110°C, 120°C, 130°C, and 140°C.

Sample Calibration Procedure for an Autoclave

  1. Record the as-found calibration data (zero and gain) for each sensor to be calibrated.
  2. Using appropriate caution (shut the steam off and wait for the pressure to go to zero!), remove the sensors to be calibrated from the sterilizer, leaving their cables connected to the control system.
  3. Set the zero and gain to 0 and 1, respectively.
  4. If using a NIST-traceable dry block or oil bath place the sensor in the dry block or oil bath.
  5. If using a NIST-traceable temperature probe, place the temperature probe into a central position in the dry block or oil bath and the sensors as close to it as possible.
  6. Measure at the selected temperatures and record the standard and transducer data. You should wait for at least one minute at each temperature to allow the measurement to stabilize. Don’t rush this step.
  7. Do a linear regression of the data collected. This is straightforward in Microsoft Excel [intercept () and slope() functions] with the standard’s data on the y-axis and the sensor’s data on the x-axis.
  8. Do a correlation as well [correl() function]. It should be at least 0.999999 (i.e. a very straight line).
  9. Enter the zero and gain values to the sterilizer controller to enter the calibration.
  10. Verify the calibration using at least one point like your process temperature. If more than one process temperature, then verify at each one. Then you will have an exact statement of the accuracy of the sensors.
  11. Re-install the sensors in the sterilizer.

If you have additional calibration questions or any questions related to autoclaves in general, please contact us for more information.


Jonathan A. Wilder, Ph.D., Stericert div. of H & W Technology

Written by Muyiwa Adebola

muyiwa@aasnig.com, www.assnig.com

07084594001, 07084594004

The LCGC Blog: GC Inlet Maintenance…have you really heard it all before?

May 15, 2014
By LCGC Editors

Many troubleshooting investigations in chromatography often don’t lead to a single causal factor. Often, the reason for problems or lack of method robustness are related to many small ‘contributory factors’ and this is particularly true of the problems associated with sample introduction in capillary Gas Chromatography.

I often find that, whilst most folks have a fair idea of what constitutes a good routine maintenance program for GC Split / Splitless inlets, the reasons for the routine are poorly understood. So, to add some deeper understanding to the problems associated with poor inlet maintenance, here is a quick multi-media blog on GC Inlet Maintenance.

Allows the sample syringe to enter the inlet and make the injection without depressurising the inlet or interrupting the carrier gas flow.

Out gassing – Septa are made from plasticised rubber or silicon and the plasticiser materials or silicon components ‘out-gas’ giving rise to increased baseline noise or the ‘hedgehog’ baseline appearance one associates with the elution of a homologous series of analytes. This is typically overcome by a gas which flows across the underside of the septum inside the inlet to carry away these outgassing products. This is called the ‘septum purge’ or ‘septum flush’ gas flow, and has a typical flow rate of a few mL/min.

Figure 1: Typical septum bleed profiles from splitless and split injections.

Septa ‘core’ or split through prolonged use, if the incorrect syringe point style is used or if the septum nut, which holds it in place, is over tightened, is badly fitting or if the septum is the incorrect size.

Figure 2: Coring of septa following repeated injections.

Figure 3: Needle tip styles such as type 5 cause less coring than type 3.

If the septum is split or cored, then the inlet may leak during the injection phase. With very badly cored septa, the carrier will leak as the inlet pressure is increased to maintain constant flow during the thermal gradient.

Septa ‘stick’ to inlets leaving behind material on the metal surfaces of the inlet, causing inlet sealing problems and bleed.

What you see:
From shards of septum which have fallen into the inlet or from a system where the septum purge flow is too low or not working, you will see the out-gassing products of rubber and plasticiser which are usually homologous series which cause noisy, rising and ‘hedgehog’ type baselines. The same goes for septa which leave residues on the inlet metal surfaces if they have become ‘stuck’ within the inlet. If you are using an MS detector – look out for the following ions in the baseline / background which are indicative of septum bleed:

m/z 73, 207, 281, 149, 167, 279

Note that the ions at 73, 207 and 281 m/z can also arise from column bleed (polysiloxanes), however the ions at 149, 167 and 279 m/z are due to plasticisers and are particularly indicative of septum bleed. Ions at 296 and 429 indicate septum breakdown products (rather than bleed) – usually from septum shards in within the liner which are in contact with hot quartz or metal surfaces.

From a system which leaks during the injection phase you will see a noticeable shift in baseline position across the solvent peak and poor peak area reproducibility.

Figure 4: Shift in baseline position after injection, indicating a leak through the septum during the injection.

Where the system cannot achieve the carrier pressures required at higher oven temperatures, the system may enter safety shut-down mode or you will experience a large baseline shift during the analysis with widely varying retention times.

How to stop it happening:
Ensure you have the correct septum material for the inlet temperature. Inlet temperatures above 350 oC generally require a special septum material. What temperature are your septa rated to? Why not go and check right now!

If you overfill the inlet with sample gas (sometimes called backflash), sample components will deposit onto the lower septum surface. Over time these bleed into the inlet, and onto the column, causing ‘ghost peaks’ or ‘memory effects’. What old timers like me would call ‘carry-over’. You should check that the conditions of the GC injection are compatible with the volume and solvent used for sample injection and that the volume of gas plasma formed on sample volatilisation, doesn’t exceed the inlet liner volume. See here for a nice downloadable tool to calculate sample gas volume: GC Calculators

The hole within a ‘pre-drilled’ septum acts as a centre guide and prevents coring provided that the septum is held under the correct torque.

Figure 5: Pre-drilled septa contain a needle guide which help to prevent coring / splitting.

Choose the correct septum size for your inlet. Over-sized septa leave a ‘scone cutter’ residues around the edge of the inlet which leads to leaks and septum bleed into the inlet.

Figure 6: Badly fitting or lower quality septa leave residues on the inlet and ‘cookie cutter’ residues.

Image courtesy of Agilent Technologies, Santa Clara, CA, USA.

Some septa have ‘non-stick’ coatings which can bleed into the column and cause baseline noise and discrete low level interfering peaks. Use only high quality septa.

Install the septum according to your manufacturer’s recommendations – paying special attention to the torque of the retaining nut. Too tight and the septum will split and core much more easily. Too loose and the nut may loosen further after repeated injection, causing loss of carrier pressure and instrument shut down.

Change the septum for each major batch of analyses, for critical or trace analyses. Over a very short time you will have a positive financial payback.

Check that the actual septum purge flow from your instrument matches the method set point or instrument readout. Since the inception of highly reliable Electronic Pressure Control (EPC) systems, flow meters have become much less commonplace. However EPC systems can fail, become blocked, give rise to variable or incorrect flows just like annual valves – it really is worth checking the flow manually from time to time (perhaps as part of your PM or OQ/PV schedule).

Some instrument designs feature an o-ring to isolate the carrier and spilt flow from other instrument flow paths. These are typically fitted around the inlet liner or as a sealing ring around the underside of the septum cap and are made from fluorocarbon or graphite.

Figure 7: Fluorocarbon o-ring seals used in certain instruments. Other instruments will use PTFE or graphite inlet seals.


Depending on quality, fluorocarbon o-rings can out-gas contaminating materials at higher temperatures

The o-ring can deform and cause leaks if not held under the correct torque.

The o-ring can stick to the liner or inlet metal surfaces, causing sealing problems.

What you see:

Discrete peaks on the baseline – typically due to the focussing of contaminants at lower over temperatures and subsequent elution of triphenylphosphine oxide, the principal outgassing component

Figure 8: Outgassing products from o-rings can lead to discrete peaks within the GC bleed profile, primarily due to the release of triphenylphosphine oxide.

Look for 277 m/z in the mass spectrum of your background or any interfering peaks!

If residues of the previous o-ring remain on the liner or on the inlet metal surface, this may lead to gas flows within the inlet leaking into one another. At worst this will lead to a safety shut down, however, the problem may be more insidious and cause poor quantitative reproducibility.

How to stop it happening:

Use high quality o-rings.

Change the o-ring every time you change the liner (see below).

Do not re-use liners or o-rings which have been stuck to the liner or inlet inner metal surface.

Liner, Packing and Inlet seals:

The liner is typically a tube of quartz glass which is used to constrain the gaseous sample so it does not contact the hot metal surfaces inside the inlet. In some instrument designs it acts to separate the column and split flows and is designed to be a replaceable consumable which will become contaminated with involatile sample residues over time. To facilitate sample mixing, provide a large surface area to allow volatilisation of high boiling analytes and to prevent column contamination, sometime the liner is packed with a plug of glass wool.

Figure 9: Quartz glass liner packed with deactivated quartz wool.

Some instrument designs have a lower seal within the inlet to allow efficient splitting of the sample, to act as a coupling device for the analytical column and to present an inert surface to the sample, especially during splitless injection where the analyte may be held within the liner for prolonged periods.

Figure 10: Metal liner seal.

Over time, the inlet liner, packing material and seal becomes contaminated with involatile sample material, which acts as a surface to adsorb analytes through inter-molecular physico-chemical interactions such as hydrogen bonding between polar sites.

Figure 11: Contaminated liners (left) and inlet seal (right).
Image courtesy of Agilent Technologies, Santa Clara, CA, USA.

Typically, the liner and packing materials will be constructed from quartz glass which contains a large number of surface polar (silanol) groups. To prevent unwanted secondary interaction with the analyte, these groups are chemically ‘derivatised’ to present a much less polar, less ‘active’ surface. Over time, the increased temperature and exposure to moisture will hydrolyse the derivatising species to the polar silanol groups and unwanted polar / polar interactions between the analytes and the liner and packing surfaces will be possible.

Figure 12: Typical liner / packing silylation reaction.

Typically the metal seals within the inlet will also be deactivated – and the hydrolysis described above is also possible.

What you see:
Poor quantitative reproducibility and peak tailing are the most common forms of problem when inlets become active.

Figure 13: Analyte peak tailing caused by unwanted secondary interactions with active sites within the liner, packing material or inlet seal.

In extreme cases, analytes may not appear within the chromatogram if they are quantitatively adsorbed within the inlet.

Sometimes, analytes are not thermally stable (pesticides, carbamates, explosives, brominated flame retardants etc.) and will degrade with prolonged exposure to higher temperatures. The presence of glass wool within the liner, and especially in cases where this glass wool is active in terms of polar / polar interactions, can exacerbate the thermal degradation of labile compounds.

How to stop it happening:

Ensure you use good quality, deactivated, liners and packing materials.
Ensure the liner design, packing amount, density and positioning are suitable for your application.
Ensure the liner, packing and seal are deactivated with modern deactivation process which will guarantee the highest levels of inertness.
Change the liner as often as is necessary. This will depend on your analytes and applications, and most importantly on the nature of your sample matrix and the amount of sample clean-up that you do prior to analysis. Most manufacturers recommend that the liner is changed ‘regularly’ – and you should satisfy yourself that the liner is clean and deactivated whenever you are running large campaigns of samples, critical analyses or trace level determinations. Remember that the o-ring should be changed with each liner change and any metal seals are also changed with every or every other liner change, especially where analytes are thermally labile, peak tailing is occurring, analytes are particularly polar or trace level determinations are being undertaken.

The split line:

A large bore (typically 1/8’’) tube which carries the split gas away from the inlet. The split line may also contain a filter or trap filled with adsorbent material such as deactivated charcoal, which acts as a trap for volatile species and reduces the emissions from the instrument split line vent.

Figure 14: (Top) Split line indicated by the yellow arrow. Typically a wider bore tubing which allows split gas to escape from the inlet. (Bottom) Typical in-line split vent filter.

Image courtesy of Agilent Technologies, Santa Clara, CA, USA.

The split line and filter (trap) become blocked over time with an accumulation of involatile material. The speed at which the tube and trap becomes blocked will be dictated by the number and cleanliness of samples.

What you see:
Contamination of the split line will result in ‘ghost peaks’ – broad baseline disturbances before or after all or certain analyte peaks. If a blank solvent is injected some or all of the peaks of the previous injection will be present, with low response and pronounced peak broadening.

Figure 15: Ghost peak deformations caused by, amongst other causes, re-injection of contaminants from the split line.
In extreme cases, retention times may vary, this is due to the EPC unit struggling to control and balance the various applied pressures and resulting flows against the back pressure created within the inlet by the blocked split line.

How to stop it happening:
Most preventative maintenance routines should contain a check / clean / replace action for the split vent filter and the split line should be removed, inspected and rinsed or replaced as necessary. For instruments with heavy usage or when analysing large numbers of dirty samples, this action should be carried out every 6 months or more frequently if required.

For more information – contact either
Bev (bev@crawfordscientific.com) or Colin (colin@crawfordscientific.com).

Wriiten by Opeyemi Aduroja

www.aasnig.com, opeyemi@aasnig.com

07O84594004, 08068129603


A cataract is a clouding of the eye’s natural lens, which lies behind the iris and the pupil.

Cataracts are the most common cause of vision loss in people over age 40 and is the principal cause of blindness in the world. In fact, there are more cases of cataracts worldwide than there are of glaucoma, macular degeneration and diabetic retinopathy combined, according to Prevent Blindness America (PBA).

Types of cataracts include:

  • subcapsular cataract occurs at the back of the lens. People with diabetes or those taking high doses of steroid medications have a greater risk of developing a subcapsular cataract.
  • nuclear cataract forms deep in the central zone (nucleus) of the lens. Nuclear cataracts usually are associated with aging.
  • cortical cataract is characterized by white, wedge-like opacities that start in the periphery of the lens and work their way to the center in a spoke-like fashion. This type of cataract occurs in the lens cortex, which is the part of the lens that surrounds the central nucleus.

Cataract Symptoms and Signs

A cataract starts out small and at first has little effect on your vision. You may notice that your vision is blurred a little, like looking through a cloudy piece of glass or viewing an impressionist painting.

How the same landscape looks to someone who sees normally.

Hazy, blurred vision may mean you have a cataract.

A cataract may make light from the sun or a lamp seem too bright or glaring. Or you may notice when you drive at night that the oncoming headlights cause more glare than before. Colors may not appear as bright as they once did.

The type of cataract you have will affect exactly which symptoms you experience and how soon they will occur. When a nuclear cataract first develops, it can bring about a temporary improvement in your near vision, called “second sight.”

Unfortunately, the improved vision is short-lived and will disappear as the cataract worsens. On the other hand, a subcapsular cataract may not produce any symptoms until it’s well-developed.

If you think you have a cataract, see an eye doctor for an exam to find out for sure.

What Causes Cataracts?

The lens inside the eye works much like a camera lens, focusing light onto the retina for clear vision. It also adjusts the eye’s focus, letting us see things clearly both up close and far away.

The lens is mostly made of water and protein. The protein is arranged in a precise way that keeps the lens clear and lets light pass through it.

But as we age, some of the protein may clump together and start to cloud a small area of the lens. This is a cataract, and over time, it may grow larger and cloud more of the lens, making it harder to see.

No one knows for sure why the eye’s lens changes as we age, forming cataracts. But researchers worldwide have identified factors that may cause cataracts or are associated with cataract development. Besides advancing age, cataract risk factors include:

  • Ultraviolet radiation from sunlight and other sources
  • Diabetes
  • Hypertension
  • Obesity
  • Smoking
  • Prolonged use of corticosteroid medications
  • Statin medicines used to reduce cholesterol
  • Previous eye injury or inflammation
  • Previous eye surgery
  • Hormone replacement therapy
  • Significant alcohol consumption
  • High myopia
  • Family history

One theory of cataract formation that’s gaining favor is that many cataracts are caused by oxidative changes in the human lens. This is supported by nutrition studies that show fruits and vegetables high in antioxidants may help prevent certain types of cataracts (see below).

Cataract Prevention

Though there is significant controversy about whether cataracts can be prevented, a number of studies suggest certain nutrients and nutritional supplements may reduce your risk of cataracts.

Good food sources of vitamin E include sunflower seeds, almonds and spinach. Good sources of lutein and zeaxanthin include spinach, kale and other green, leafy vegetables.

Other studies have shown antioxidant vitamins such as vitamin C and foods containing omega-3 fatty acids may reduce cataract risk.

Visit our Nutrition & Eyes section to read more about eye vitamins and how a healthful diet and good nutrition may help prevent cataracts.

Another step you can take to reduce your risk of cataracts is to wear protective sunglasses that block 100 percent of the sun’s UV rays when you are outdoors.

When symptoms begin to appear, you may be able to improve your vision for a while using new glasses, strong bifocals, magnification, appropriate lighting or other visual aids.

Think about surgery when your cataracts have progressed enough to seriously impair your vision and affect your daily life.

Many people consider poor vision an inevitable fact of aging, but cataract surgery is a simple, relatively painless procedure to regain vision.

Cataract surgery is very successful in restoring vision. In fact, it is the most frequently performed surgery in the United States, with more than 3 million Americans undergoing cataract surgery each year, according to PBA.

Nine out of 10 people who have cataract surgery regain very good vision, somewhere between 20/20 and 20/40.

During surgery, the surgeon will remove your clouded lens and in most cases replace it with a clear, plastic intraocular lens (IOL).

New IOLs are being developed all the time to make the surgery less complicated for surgeons and the lenses more helpful to patients. Presbyopia-correcting IOLs potentially help you see at all distances, not just one. Another new type of IOL blocks both ultraviolet and blue light rays, which research indicates may damage the retina.

Read more on this website about what to expect if you have cataract surgery and how to deal with rare cataract surgery complications.

Also, men should be aware that certain prostate drugs can cause intraoperative floppy iris syndrome(IFIS) during a cataract procedure.

Eyewear After Cataract Surgery

In most cases, unless you choose presbyopia-correcting IOLs, you will still need reading glasses after cataract surgery. You may also need progressive lenses to correct mild residual refractive errors as well as presbyopia.




written and editted by Oluwakemi Adi

kemi@aasnig.com, www.aasnig.com



Series 2


The Hydrogen gas is produced from deionised water using the exclusive 100% titanium Proton Exchange Membrane (PEM) technology for H2, which provides a very high reliability, new longer life and better H2 purity.

The exclusive cold dual dynamic regeneration dryer is completely maintenance free and eliminates all down time for maintenance that is typical of other systems on the market, guaranteeing the best hydrogen purity 24 hours a day.

The automatic checking for internal leaks whenever starting the units and constant control of operating parameters, guarantee maximum safety. The touch screen LCD interface provides simple and user-friendly management of all functions on the unit.


  • GC-MS • GC-FAST • GC-Carrier gas
  • Hydrogenation • ICP-MS • Fuel cell • THA


1 Improved chromatograph result

The use of hydrogen as a carrier gas allows:

– Faster and more sensitive than the more expensive helium

– Lower temperature elution, thus extending the life of the chromatograph column

– run time savings of 25% to 35% without a decline in resolution.

2 Increased laboratory efficiency

A constant, uninterrupted gas supply of guaranteed purity eliminates interruptions of analyses to change cylinders and reduces the amount of instrument re-calibrations required.

3 Improved safety

The vert limited internal volume (less than 50 ml) allows safe use of the gas generators where the use of cylinders is risky or prohibited. The application of tested safety technologies stops the unit in the event of leaks or malfunctions.

4 Ergonomic and compact design, simple installation

Can be installed in the laboratory, on or under the bench, eliminating the need for long gas lines from cylinders secured elsewhere.


  • Models available: 100, 160, 250, 300, 400, 500, 600, 1000, 1200, 1350 cc/min
  • Purity > 99.99999%
  • Exclusive cold dual regeneration dryer: removes both moisture plus oxygen and eliminates the need to monitor, change and purchase desiccant cartridges
  • Pressure up to 11 bar (160 psi): suitable for fast and high-speed GC methods
  • Option: Cold Palladium Catalyst reduces O2 <0.01 ppm and moisture < 1 ppm
  • Exclusive 100% titanium electrolytic cell: longer life / better purity of gas
  • LCD touch screen with indication in real time: H2 outlet pressure, H2 flow rate, water quality, water level, system status with auto-diagnostics of breakdown with alarms
  • Remote PC monitoring and diagnostic analysis via USB to interface the unit with customer’s PC software (allow to carry out checks and maintenance effectively, only via a remote connection)
  • Automatic checking for internal leaks to guarantee maximum safety
  • Capabilities allowing to work in cascading mode
  • Higher flow rates up to 10 L/min
  • Automatic flow compensation in the event of unplanned down-time
  • Continuous operation for critical applications


  • Hydrogen is produced using distilled or deionised water < 0.1 μS from hydrolysis, through a polymer membrane.
  • Electrolytic dissociation separates the water into its two main components: hydrogen ready for analytical use, and Oxygen that is released into the air.
  • No acid nor alkaline solutions are used in the hydrogen generation cycle.
  • No desiccant cartridge maintenance is required: there is a double column dryer with automatic regeneration.
  • This automatic drying system ensures the maximum grade of hydrogen purity.
  • An exclusif system allows to work in parralel mode and to connect until 10 units, to have flowrate until 10 L/min.

HYDROGEN GAS GENERATOR OPERATING PRINCIPLE (F-DGSi – Serie MF.H2 – Ver.2 2018 – Commercial documentation)


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


F-DGSi – Serie T.FID.MB.H2 – Ver.2 2018 – Commercial documentation

Website: www.aasnig.com

Email: aas@aasnig.com opeyemi@aasnig.com

Phone: 07084594004, 08068129603




Water Treatment Processes

Clean, safe water is vital for every day life. Water is essential for health, hygiene and the productivity of our community.

The water treatment process may vary slightly at different locations, depending on the technology of the plant and the water it needs to process, but the basic principles are largely the same. This section describes standard water treatment processes.

Coagulation / Flocculation

During coagulation, liquid aluminium sulfate (alum) and/or polymer is added to untreated (raw) water. When mixed with the water, this causes the tiny particles of dirt in the water to stick together or coagulate. Next, groups of dirt particles stick together to form larger, heavier particles called flocs which are easier to remove by settling or filtration.


As the water and the floc particles progress through the treatment process, they move into sedimentation basins where the water moves slowly, causing the heavy floc particles to settle to the bottom. Floc which collects on the bottom of the basin is called sludge, and is piped to drying lagoons. In Direct Filtration, the sedimentation step is not included, and the floc is removed by filtration only.


Water flows through a filter designed to remove particles in the water. The filters are made of layers of sand and gravel, and in some cases, crushed anthracite. Filtration collects the suspended impurities in water and enhances the effectiveness of disinfection. The filters are routinely cleaned by back washing.


Water is disinfected before it enters the distribution system to ensure that any disease-causing bacteria, viruses, and parasites are destroyed. Chlorine is used because it is a very effective disinfectant, and residual concentrations can be maintained to guard against possible biological contamination in the water distribution system.

Sludge Drying

Solids that are collected and settled out of the water by sedimentation and filtration are removed to drying lagoons.


Water fluoridation is the treatment of community water supplies for the purpose of adjusting the concentration of the free fluoride ion to the optimum level sufficient to reduce dental caries. Hunter Water is required to fluoridate water in accordance with the NSW Fluoridation of Public Water Supplies Act 1957.

pH Correction

Lime is added to the filtered water to adjust the pH and stabilize the naturally soft water in order to minimize corrosion in the distribution system, and within customers’ plumbing.

pH is an indicator of the acid or alkaline condition of water. The pH scale ranges from 0-14; 7 indicates the neutral point. The normal pH range of drinking water is 6 – 8.5. The pH is mostly a result of natural geological conditions at the site and the type of minerals found in the local rock. The pH can also be affected by acid rain. Water with a pH value less than 7 is acidic and tends to be corrosive. Acidic water (low pH) can leach metals from plumbing systems, which can cause pipes to leak. Metals that leach from the pipes (lead from lead pipes or copper from copper pipes) may also cause health problems. Water with a value greater than 7 indicates alkalinity and tends to affect the taste of the water. Alkaline drinking water may take on a “soda” taste. Corrosion problems also can occur in plumbing. The three types of pH adjustment devices are discussed below.






Written by Oluwakemi Adi



Mobile number: 08060874724