UV Absorbance Measurement Solutions

Typical applications include:

• Peak Detection
• Chromatographic separation
• Fractionation
• Micro- and nano-filtration systems
• Concentration of aromatic solvents

Traditionally, online UV absorbance measurements were made with an adapted piece of lab equipment (often a spectrophotometer). Because lab units were generally not suitable for production environments, UV photometers were developed as production level instruments and designed to give absorbance values at one or two wavelengths. However, these photometers used mercury vapor lamps for their light sources and these are the source of the continuous drift you see.

Mercury vapor light sources are being phased out, in part because of the toxic nature of mercury, but mostly because LED technology has advanced and can now provide a much better alternative UV light source.

There are many areas where the DCP007-UV analyzer outperforms other measurement devices. To detail a few:

• The analyzer utilizes advanced digital electronics to allow for UV absorbance measurement of up to at least 4.5 AU (with a 1 cm optical path length) at up to two discrete wavelengths. A wide selection of shorter optical path-lengths allows for even higher absorbance measurements up to 90 OD or more.
• LED light sources have tight band emission rather than broad band line emission. Having light only at the wavelength of interest reduces stray light dramatically and increases linearity of measurement at higher absorbances.
• The DCP007-UV controls the energy output of solid-state light sources through a proprietary dual wavelength, four-channel measurement technique which ensures drift free measurement over long periods of time.
• Photodetector quiescent state output change due to ambient temperature change is constantly corrected, while low power drive to the light sources gives extended functional life (up to 10 years). Maintenance costs for DCP007-UV systems are exceptionally low.
• Smaller optical windows allow for zero-dead-volume hygienic measurement cells, and that means rapid detection of protein. Their non-diluting flow characteristics gives sharp peaks, allowing tighter control and increase batch yields.
• Non-liquid in-line verification and QA are made possible using an insertable cuvette style certified filter or liquid standard. This simple method utilizes the same traceable or prepared liquid standards used for laboratory benchtop instrumentation verification and calibration, providing a direct correlation to offline measurement techniques for added confidence in measurement.

As protein concentrations rise, absorbance values increase. Traditional UV analyzers often struggle to accurately measure at higher absorbances due to non-linear response. They are often setup with an optical pathlength that is too wide, so suffer from peak clipping at high absorbance when they are over-ranged. Their large internal hold-up volume causes dilution, slowing down response. These issues distort peak shape, reduce purity, and impact yield.

Modern multiwavelength deep-UV analyzers provide the tools needed to tackle these issues.

1. Equipped with high-resolution optics, they deal with stray light issues far more efficiently than traditional units, dramatically improving linearity of measurement at high absorbances.
2. High absorbance measurements need very short optical pathlengths, but the impact of mechanical tolerance on accuracy is much greater the shorter the path becomes. Modern absorbance instruments allow for an “off-peak” wavelength(s) to be selected that permits the use of a wider, more accurate optical pathlength while still being able to monitor and trend peak absorbance.
3. Zero-dead-volume cells prevent the dilution of fluid in the cell by fresh material flowing in and provide crisp, fast response to changes in absorbance.

Multiwavelength UV analyzers provide a much better solution to high titer processing that existing systems.

Advances and improvements in bioprocessing methods have led to protein products being expressed and concentrated to higher and higher levels. Higher protein concentrations mean higher absorbance measurements. Therefore, UV analyzers need the ability to measure to deep absorbance. While absorbance measurement is essentially linear, many light sources generate light at wavelengths other than 280nm; this can enter the measurement path as stray light, which creates artificially low absorbance readings and non-linear responses to protein concentration, particularly at high levels.

Accurate, reliable, and repeatable post column UV measurements are a minimum requirement during chromatographic separation to ensure good protein fraction purity and maximum yields. The Kemtrak DCP007-UV analyzer improves post column protein detection and measurement in two very specific ways:

• Zero volume hold-up measurement cells provide fast response to changing absorbance levels by preventing dilution of absorbing material by non-absorbing material already in the cell. This provides rapid response to changing absorbance and allows for more precise collection and cutoff, particularly during fractionation.
• The traditional Mercury vapor lamps used in absorbance photometers are noisy, hot, and constantly drift—this makes them less than ideal, particularly for fragile protein molecule work. The DCP007-UV uses solid-state light LED sources that do not produce heat and can be controlled dynamically to ensure constant light output, making them drift free. Furthermore, using UV LED sources dramatically removes the presence of stray light outside of the wavelength of interest, providing the ability to measure to 4.5A linearly, allowing monitoring of high concentration processes up to 90 OD.

In short, yes. Here’s why.

What tangential flow filtration (TFF) does

The main purpose of tangential flow filtration in bioprocessing is to concentrate a product previously filtered in an earlier step in the production process:

• The feed solution is circulated in a closed loop, passing through the filter.
• The filter retains the product being concentrated.
• Unwanted excess buffer and background material permeate through the filter material and drain away.

How UV absorbance monitoring supports TFF control

The installation of UV absorbance analyzers at strategic points in the filtration system provides:

• Real-time measurements of protein concentration
• Control of feed rates for maximum filter efficiency
• Reduction of product loss through filter failure
• Accurate determination of concentration end points

All of these measurements contribute to maximizing product yield in any given batch.

Retentate monitoring in the recirculation loop

Using a UV absorbance analyzer to monitor where retentate returns to a filtration system’s recirculation tank provides:

• A real-time concentration signature of product retained in the circulation loop
• Cascade control with feed absorbance to regulate the amount of feed into the recirculation system
• Stable loop concentration at an optimal level for filter operation

Once feed is complete:

• The monitor indicates retained product concentration change in the loop
• When the rate of change reaches a “plateau,” the process end point has been reached

Permeate monitoring and product loss detection

Permeate monitoring ensures that product is not being lost through the filter membrane:

• Under normal operation, the monitor provides a flat “zero” absorbance output
• If product begins to break through the filter, measured absorbance rises
• This indicates a loss condition caused by:
  • Filter media failure, or
  • Excessive concentration on the retentate side
• Once a breakthrough trend is observed, the process is typically halted

Importance of correct UV monitor configuration

Correct selection and installation of UV monitors is required for reliable results:

• There is no one-size-fits-all monitor suitable for all processes
• Each UV analyzer must be configured with the correct:
  • Optical wavelength
  • Optical path length
• Poor response or peak clipping prevents systems from operating at their optimum point
• As a general rule when measuring the same wavelength:
  • Retentate monitors use the shortest path length
  • Permeate monitors use the longest path length
  • Feed monitors fall between the two

Why the Kemtrak DCP007-UV fits TFF applications

The Kemtrak DCP007-UV provides:

• Drift-free measurement
• Low noise
• Exceptionally long light source life
• Zero cell hold-up volume
• Single, dual, or referenced wavelength measurements

Chromatography and TFF depend on crisp, real-time UV absorbance signals to determine cut points, control feed flow, detect breakthrough, and measure concentration changes. If the analyzer clips peaks, drifts, or dilutes the sample inside the measurement cell, operators lose visibility into separation quality or filtration efficiency. This can lead to lower purity, product loss, and inconsistent batch performance.

A modern analyzer should deliver deep-UV capability, multi-wavelength detection, real-time response, and high linearity at elevated absorbance levels. Zero-dead-volume hygienic cells help preserve peak shape, while mercury-free long-life xenon light sources reduce drift. Integrated, traceable verification supports regulated environments and ensures measurement stability over time.