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If you’re a chromatographer, then it’s probably safe to say that one of your daily goals is to achieve great chromatographic results – chromatograms that feature well-resolved peaks with little-to-no band-broadening and a flat baseline…the kind of results that make your job easier and headache-free. To achieve that goal, you likely spend a lot of time on method validation, operational protocol, calibration, mobile phase preparation, and the like. Interestingly, what chromatographers sometimes overlook is the impact that the fluid path connections can have on the quality of the results obtained.
Fluid path connections are essentially comprised of two main components: the flow path tubing and the compression fittings. In this post, we will hone in on the flow path tubing and how to best prepare it for use in your system’s flow path.

• Choose the Best Inner Diameter & Length – One of the most important things that you can do with regards to the tubing you choose is to select the best inner diameter and length of tubing to pair with your application.

With regards to length, a good rule to follow is this: Keep your tubing as short as functionally possible. The tubing comprises the majority of the fluid pathway in the system. This means that longer lengths of tubing will create longer delays for gradient changes to reach the column, which can force your separations to take longer than necessary. Also, longer lengths of tubing force the sample components to remain in contact with the mobile phase for a longer period of time, which can lead to dilution of the sample and thus band broadening, which can lead to poorly resolved sample components, etc. Keeping tubing lengths as short as possible helps minimize the duration of time the sample is exposed to the mobile phase and can therefore help provide you with quality chromatographic results.

Another important thing to consider is the impact of tubing inner diameter on the results you obtain. Because the volume of the tubing is proportionally related to the square of the internal radius, that means the tubing inner diameter can have a dramatic impact on the residence time for the sample components in the flow path. For instance, if you use the standard .010” (~0.25 mm) inner diameter tubing as a “baseline” for tubing size, then simply changing to .005” (~0.13 mm) inner diameter tubing reduces the internal volume to ¼ of the volume in the larger inner diameter tubing. This results in an increased linear velocity, allowing the sample to reach the column more quickly, and the separated components to reach the detector more quickly as well. It also reduces the zone of interaction between the mobile phase and the sample components, thus reducing band-broadening effects as well as improving chromatographic results overall.

A key challenge when coupling mass spectrometry instruments to liquid chromatography equipment is how to best design the fluid pathway to ensure that optimal results are achieved. In fact, one of the most critical parts of the fluid pathway is the tubing, and as such, several things must be taken into account with regards to tubing selection and implementation:

• Tubing Internal Diameter– One of the advantages of mass spectrometry is the ability to obtain a lot of information about a sample in a short amount of time.  It is often assumed that the smallest possible internal diameter should be used for the flow path tubing to help reduce the time required for sample to transfer to the mass spectrometer,  and to minimize band broadening and sample dilution.  However, this is rarely the best option, primarily due to the relationship between internal diameter and back pressure created.Consider the following formula, which details the relationship between pressure drop and internal diameter:
  The pressure drop along a given fluid pathway is inversely proportional to the diameter of the fluid pathway taken to the fourth power! In other words, even small changes to the internal diameter can have a dramatic impact on the pressure drop experienced along that pathway.

  Additionally, as the tubing internal diameter reduces, the probability of a partial or complete blockage forming inside the tubing dramatically increases.

  Because of these and other factors, rather than choosing the smallest internal diameter possible, it is usually a better strategy to balance the benefits of having a smaller internal diameter (e.g., lower band broadening, less delay time, etc.) with the pressure-drop increase that will occur along with the increased likelihood of a tubing blockage.

• Tubing Material– In addition to the internal diameter, another major factor that must be considered is the material from which the tubing has been manufactured.  This material can impact a number of characteristics, each of which may have an impact on the success of any given analysis:
  • Chemical compatibility – Is the material compatible with both the mobile phase passing through as well as with the components that comprise the sample?
  • Pressure holding ability – Based upon the system and application parameters, will the tubing be able to withstand the anticipated pressure in the area of the system where it is located?
  • Electrical conductivity – Is the tubing part of the source assembly, and if so, will its electrical conductivity (or lack thereof) impact the expected performance?
  • Temperature stability – Can the tubing withstand the temperature to which it will be exposed during the run without having its physical properties altered dramatically?

  Other things – including gas permeability, overall flexibility, dimensional tolerances, etc. – help make choosing the right tubing material critical to designing an optimal fluid pathway.

• Tubing Preparation– Once the optimal tubing internal diameter and the best tubing material are chosen, if the tubing isn’t cut properly, it can lead to less-than-optimal results.All too often, analysts don’t exercise much care in the way the flow path tubing is cut.  However, poorly cut tubing can result in partially occluding the opening—even pushing the flow path opening off-center.  In some cases, these problems can lead to higher pressures and even the complete blockage of a fluid pathway.The best way to cut flow path tubing is to circumscribe the tubing – that is, to rotate the cutting tool around the outer diameter of the tubing, slowly cutting through the tubing wall and terminating the cut once the blade breaches the internal diameter.  Preparing the tubing this way helps to not only maintain a quality surface on the end of the tubing, but it also helps ensure the thru-hole is centered and fully open.

  As can be seen in the picture below, with the tubing on the left being cut properly and the tubing on the right being prepared incorrectly, cutting the tubing can have a dramatic impact – either positive or negative – on the overall fluid pathway.

  

  Certainly there are other aspects that should be considered when designing an optimal fluid pathway with LC-MS applications.  However, because of the tremendous impact that tubing internal diameter, tubing material, and tubing preparation can have on the results achieved, investing some time and effort in this area will often lead to dramatic improvements in both using the equipment and the results obtained.

About the author: John Batts is a Technical Specialist and chromatography expert at IDEX Health & Science. He is also the author of the “All About Fittings Guide,” which you can download at idex-hs.com.

The challenge many researchers face is how various pumping technologies negatively impact cellular material:

• Reciprocating Pumps – Two common styles of reciprocating pumps used in analytical-scale life science work are diaphragm pumps and piston / syringe pumps. Diaphragm pumps typically employ the use of check valves and a flexible diaphragm mounted to the motor shaft of a driving motor, and fluid moves in and out of this pump style through the “pulsing” action of the diaphragm. Piston / syringe pumps work through combining a positive displacement piston or a plunger with some type of rotary shear valve, with fluid movement occurring by the movement of the piston or plunger.
  Both of these styles of reciprocating pumps create problems for cellular viability due to a moderately strong vacuum force and the shear forces to which the cells are exposed. These forces can cause cells to undergo lysis, thus dramatically reducing cellular viability and the opportunity to conduct longer-term testing. Additionally, these pumping technologies are more difficult to clean, increasing the possibility of sample carryover and cross-contamination.
• Gear Pumps – Gear pumps work through the rapid rotation of two (or more) intermeshing gears. As the “driving” gear and the “driven” gear(s) interface with one another at high rotational speeds, fluid is moved along between the gears’ teeth. This style of pumping can create problems with biological samples due to the physical forces involved as the fluid transfers at high linear velocities. The gears’ teeth will often shear cellular material, rendering the sample or fluid being analyzed useless. Additionally, because fluid touches the mechanical portion of these pumps, it is difficult to avoid cross-contamination between samples.

One pumping technology that is finding increased popularity is Peristaltic Pumps. Peristaltic pumps transfer fluid using a series of rollers that facilitate the compression and expansion of soft-walled tubing. This pumping technology offers several advantages to help maintain cellular viability while also reducing sample-to-sample cross-contamination through several unique features:

• Low Vacuum Force – Peristaltic pumps typically work with soft-walled elastomeric tubing. This tubing is easily compressed but also returns to its initial shape quickly. Peristaltic pumps employ the use of rollers, which completely compress the flow path tubing as they rotate under a tubing bed. Once compressed, as the rollers continue their movement, the tubing expands back to its original shape, creating a low vacuum that helps pull fluid into the tubing before the next roller compresses the tubing once again. The small amount of vacuum created by the re-expansion of the tubing is sufficient to move fluid without typically damaging the cellular material.
• Low Shear Force – Peristaltic pumps maintain fairly consistent flow rates (except for the pulsation effect inherent in the pumps) and avoid having the fluid come into direct contact with the mechanical part of the pump. Both of these work to minimize the shear force the sample might experience and help lead to increased sample viability.
• Minimal Tubing Compression Points – Because the soft-walled peristaltic tubing is only fully compressed at finite points – with a majority of the tubing left open – the number of opportunities for biological material to become compressed and damaged is limited.
• Tubing-Only Flow Path – One of the unique design features of peristaltic pumps is that only the tubing comes into contact with the material being transferred – the material never comes into contact with the mechanical portion of the pump. This feature allows the tubing to be either cleaned and sterilized or replaced between analyses, virtually eliminating the possibility of sample-to-sample contamination.

An additional feature unique to most single-channel Ismatec^® pumps is the actual design of the rollers and the tube “bed” against which the tubing is pressed by the rollers. While many pumps employ the use of flat surfaced rollers and tube beds, most of the Ismatec single-channel pumps have been designed with convex-shaped rollers and a tube bed with a variable level of concavity. As the rollers make initial contact with the tubing, only the center of the tube is compressed, allowing biological material to escape through the gap towards the tubing wall and avoid being damaged or destroyed. (See Figure 1 Below)

Figure 1 – Convex Rollers & Concave Tube Bed

Independent research has been conducted, comparing the impact of this roller / tube-bed design with other options, resulting in clear evidence of both increased cellular concentration (during incubation) and increased cellular viability. (See Charts 1 & 2 Below)

Chart 1 – Comparison of Cell Concentration Over Time
(black is “no pump”, red is Ismatec pump with convex rollers and concave tube bed, other colors are less-effective pump options)

Chart 2 – Comparison of Cell Viability
(black is “no pump”, red is Ismatec pump with convex rollers and concave tube bed, other colors are less-effective pump options)

There are disadvantages to peristaltic pumps that should be considered. Some of these disadvantages include the limited pressure differential against which the pumps may operate. Also, the tubing itself can create challenges, as the chemical compatibility of elastomeric tubing is not universal and the tubing can wear over time, creating flow inconsistencies and/or changes during its lifetime.

But perhaps most importantly, peristaltic pumps experience pulsation, a phenomenon inherent in their operation. Pulsatile flow can lead to effects such as “splashing” of fluid as it leaves the flow path tube as well as inconclusive real-time flow analysis due to constantly-changing flow rates inside the analysis chamber.

While these limitations to peristaltic pump technology can hinder the use of the technology in some applications, for many applications – especially those classified as life science where biological samples are involved – peristaltic pumps are the pumps of choice.

About the author: John Batts is a Technical Specialist and chromatography expert at IDEX Health & Science. He is also the author of the “All About Fittings Guide,” which you can download at idex-hs.com.

Three years ago, a customer came to IDEX Health & Science with a problem: they were encountering a build-up of bubbles in a line connecting a salt water reference solution to a sensor head. The line was one meter long and had originally been made of PTFE fluoropolymer. Other fluids were connected to the sensor head through identical lengths of tubing and were kept at the same place in the instrument. Early development of the instrument had shown that unless all the fluids were degassed, they would develop bubbles in the tubes as they moved from the reservoirs to the sensor head. To ensure the fluids were properly degassed, the design engineers developed a multiple layer bag which had near-zero air permeability.

By using this bag, all the fluids could be degassed thoroughly by the instrument manufacturer, then supplied to the end-use facility where the reagents could be placed on the machine when needed. Although costly, the method of providing pre-degassed reagents was found to eliminate bubbles from all lines… except the one containing the salt water reference solution. Measurements of the amount of dissolved air remaining in the salt water reference solution showed that the salt water was becoming saturated with air overnight in the meter-long transfer line, but the formation of bubbles in the line was very puzzling. Even though the system engineers changed the transfer line from PTFE to a lower permeability fluoropolymer, FEP, they found the bubble problem still occurred at nearly the same rate.

Since the conductivity of the salt water was referenced prior to each sample in a “standard, sample, standard” protocol, a deviation in conductivity caused by an air bubble was causing the results from a test to be rejected by the software. A purge cycle was implemented in the software to assist in removing the bubbles from the reference side of the conductivity bridge, which improved results but didn’t completely eliminate them. Rejecting the results from the test due to bubbles in the reference standard was costing the end-user of the instrument considerable money—not only for the test but by requiring re-sampling of the patient in some instances. The reference solution error rate before– and after– the instrument software change to flush the bubbles is presented in figures 1 and 2.

Figure 1: Original sample data obtained without a software recognition of bubbles followed by a flush. Salt water reference solution showing noise and conductivity changes due to bubbles.

The patient samples processed through the system under the above conditions resulted in the unacceptable rate of errors as seen below.

Figure 2: Post software change which included bubble recognition and reference solution flush. Again, sample errors result from salt bridge bubbles; Original FEP tubing

After reviewing all the data, IDEX Health & Science engineers made a Transfer-Line Degasser to match the flow restriction and the format of the original line connecting the degassed reagent to the sensor head. The instrument’s on-board vacuum was used to create the vacuum inside the lumen of the Poridex™ tube, such that the entire length of the line from the connection to the bag to the sensor head was being actively degassed. Three features of the Transfer-Line Degasser were vital to the installation: Firstly, the Transfer-Line Degasser was flexible, allowing easy retrofitting to installed instruments. Secondly, the entire contents of the line could be degassed within minutes of an instrument re-start. Thirdly, the Transfer-Line Degasser took up no additional space in the instrument.

Following the initial testing of the installed Transfer-Line Degasser, the following results demonstrated the efficacy of the application.

Figure 3: Conductivity measurements of salt water reference after installation of Poridex™ Transfer Line Degasser.

As expected, the elimination of bubbles from the salt water reference standard resulted in a near-zero number of failed samples.

Figure 4: Conductivity measurements of Patient Samples following the installation of Poridex™ Transfer Line Degasser

Since the instrument had already passed FDA certification, the modification to the instrument was made to actively degas the salt bridge transfer line, then to re-certify. Although it would have been possible to degas all of the reagents instead of incurring the continuing costs of the associated degassing and packaging of each individual reagent, recertification was a barrier to this potential cost-saving effort. Where the other reagents spent only two or three hours in their respective transfer lines, the salt bridge solution could spend as much as 24 hours or more. Since air is hydrophobic, any bubble which formed in the salt solution within the lines would preferentially adhere to the fluoropolymer tubing inner wall and therefore act as a further nucleation site for increasing the bubble size. Purging the line could only remove the larger bubbles, which adhered to the inner surface. The Poridex tube that makes up the inner space of the Transfer- Line Debubbler offers a porous surface through which bubbles contacting its surface can escape into the vacuum. It also presents a vacuum to the salt solution as it rests in the space between the Poridex inner tube and the outer wall of the transfer line. The OEM customer graciously allowed us to use their figures, which accurately represent the action of the Poridex transfer line.

Active Degassing Transfer Line–Epilogue

It is possible to prevent bubble formation by degasssing the system fluid in the transfer tubing itself. The Systec® Tranfer-Line Degasser employs a unique co-axial approach to remove dissolved gases before they can form bubbles and affect critical results. Vacuum is applied to the inner tube, which pulls bubbles and dissolved gases from the solution in the outer tube.

Figure 5: Poridex™ Transfer-Line Degasser section and explanation of the process

The application we have described above is one of many different opportunities to use a transfer line to remove both bubbles and dissolved gas along its entire length. Wherever temperature, pressure or solution concentration changes may occur, so too may bubbles caused by the change in solubility of atmosphere in the fluid. Because Transfer-Line Degassers are light-weight, flexible and made using inert polymers, they are uniquely suited for removing gas and bubbles while transferring fluids long distances without additional volume inside instruments and have the added feature of being able to do so between fixed and moving stages.

The author of this post, Carl Sims, is a Senior Scientist at IDEX Health & Science. He specializes in chemical instrumentation, analytical chemistry methods, and gas-liquid transfer.

To sign up for the upcoming Degassing & Debubbling webinar, or to view our archived
webinars, visit us here: http://www.idex-hs.com/about/webinar_video_library.aspx

Now you have a choice of components for actively removing bubbles with or without also removing dissolved system gases. Online vacuum degassing offers operating convenience, high efficiency, and low operating costs compared to other common degassing technologies.

Watch to learn more about using these components to optimize fluidic delivery!

Debubbling

Downloads:
http://idex.cachefly.net/IHS/debubbler.mp4
http://idex.cachefly.net/IHS/debubbler.ogv

Degassing

Downloads:
http://idex.cachefly.net/IHS/degasser.mp4
http://idex.cachefly.net/IHS/degasser.ogv

Carl Sims will be back with Part 3 in early December!

Visit www.idex-hs.com, follow us on Twitter @IDEXHS, or leave a comment here to receive more information or to chat with our fluidic experts!

In today’s medical analyzers, bubbles can interfere in ways previously not encountered in flowing systems. Bubbles can interfere with dispensing when air is included in an aspirated liquid sample, causing direct volumetric failure. An air bubble entrained somewhere in the dispensing system can cause an indirect volumetric failure during sample aspiration and dispensing due to pressure changes.

Fig. 1: A single syringe pump drew in and dispensed water that was air saturated. The sampling tubing had sufficiently small ID to generate a moderate flow restriction. Dispensing anomalies (errors) can be seen to occur approximately 2% of the time.

Since air is hydrophobic, bubbles will adhere to nearly every part of a dispensing system, requiring high velocity or induced turbulent flow to displace and discharge the bubble from the flow stream and into a waste area. This process is time consuming, unpredictable, and may require designing the system to recognize bubbles are present. Simply looking at the number of sensors available to detect bubbles demonstrates the depth of the issue. Optical sensors, ultrasonic sensors, video sensors, thermal sensors all exist to detect bubbles in fluids. Regardless of the design, aqueous systems are still subject to outgassing due to changes in temperature, pressure, or when mixed with other fluids. In these cases, degassing the fluid streams may be the only sure way to eliminate bubble formation.

Fig. 2: When the water was degassed, bubbles formed by cavitation ceased to cause dispensing errors.

Degassing the fluid does add cost to any dispensing system, but the cost of a single erroneous sampling and dispensing event may be greater than the cost of adding degassing.

Degassing for HPLC applications is an absolute requirement dictated by the solubility of air in mixed solvents. With HPLC, engineers can easily justify the use of a proportioning valve and a degasser versus the cost of adding another solvent pump to form a mixture. Even when a second high pressure pump is required, such as in UHPLC systems, degassing of the solvent prevents pumping failures caused by random bubbles.

As we learned in this post, we can see the random nature of bubbles entering an analysis. Since it is possible to operate diagnostic IVD instruments in a relatively closed environment with fluids supplied to the instrument in full equilibrium with the environment and the instrument, degassing the wash water or other fluids under these conditions may not be a requirement. When environmental conditions change away from the equilibrium, degassing the fluids in the instrument will ensure proper operation and ensure patient samples are analyzed with the fewest errors possible.

In the third section of this blog, we will address a customer application wherein the engineers recognized the requirement for degassing the fluids on the IVD diagnostic instrument, but in spite of all the great engineering design, additional degassing was found to be a necessity.

The author of this post, Carl Sims, is a Senior Scientist at IDEX Health & Science. He specializes in chemical instrumentation, analytical chemistry methods, and gas-liquid transfer.