Gradient Profiling

Gradient Profiling

Gradient Profiling has matured. It used to be simply a means to monitor the effluent from a gradient so you could locate your particles in the fractions created during the run.  Lately, in studies of polysomes for instance, users have found the traces it produces to be a valuable diagnostic tool on their own. This makes more demands on the flow cell generating the traces: it must not only create an accurate picture of the gradient, it also needs to preserve the separation attained in the gradient so that downstream analysis is not compromised. This is where our new, patent pending Triax™ flow cell fits in. We have coupled the most sensitive and accurate light sensing technology available to a unique flow cell geometry that minimizes the mixing, providing beautiful profiles while preserving resolution.

The Triax™ is all new. We started with a list of things we wanted to improve over existing flow cells and another list of new features. Let’s start with the improvements. The first target was to eliminate flatlining of the profile, either at high ODs or below zero OD. These two problems are related. All existing flow cells used in gradient fractionation have an AUFS setting (absorbance units full scale). Before you start, you must set the maximum OD you are likely to see, for example 2 OD. If you are unlucky, and the OD exceeds the AUFS setting, the graph and the data flatline until the OD falls back into the range you set. On the low end, you must zero the instrument before you start, and should the data fall below your zero value, the trace will flatline on the bottom of the graph. Both of these issues arise because the output from the flow cell is an DC voltage between 0.0 and 1.0 V at whatever zero and AUFS you set. A third issue then arises: to avoid flatlining, users tend to be conservative in their selection of the AUFS, setting it well above their highest OD, for example, using AUFS = 2 OD, when the largest peak is .04 OD. This results in a trace with “steps” in the data because only 4% of the available resolution is being used for the entire profile. Our solution is simple: start with 1,000,000 arbitrary units of light and record the loss of this signal as absorbing particles pass through the flow cell. There is no AUFS setting and sub-zero OD values are accurately recorded.  Better still: the absorbance data is linear from 0 to 5 OD at .0001OD resolution, with a non-linear response to an absurd 20 OD.

The improvements the Triax carries are related to the geometry of the flow cell itself. The design eliminates bubbles and automatically aligns the profiles at the start of the run. Bubbles on the wall of the flow cell can cause severe disturbance of the profile, so they must be removed before the run starts. They arise from the design of the flow cell itself: a well-known one has the shape of a flattened bubble of quartz with small orifices on opposing ends where the gradient enters and exits. The flow runs straight through the center of the flow cell, leaving bubbles that are difficult to remove. This makes it impossible to align the trace at the start without first driving out all the bubbles. The solution to both issues was to design a flow cell that gives bubbles no place to hide. With the Triax, you drive all the water out of the flow cell with air before you start, and let the data acquisition begin when the gradient's meniscus enters the flow cell, bubble-free and precisely aligned. 

The other important improvement is the level of automation we have achieved. A single key retrieves all the settings you need and another key starts the run. You are now a spectator. Your gradients will be precisely aligned at the meniscus, the fraction collector delivers your numbered fractions seen on the profile, and there is even a way to recover the last fraction from the tubing using the air pump. 

Lastly, the Triax can pulse the flow cell with visible light to excite your favorite fluor. EGFP, mCherry, Alexa 488, 555, 568, Cy2, 3, 3.5, 5, mKate2 and many others, simultaneously recording the UV and fluorescent profiles, a first in biological research.