Application Note
BioPharma/Pharma

Concentration Analysis of
Ultra‑microvolume Samples by
UV-Vis Spectroscopy
Simple, precise, and nondestructive concentration
analysis with Agilent Cary 60 UV-Vis and TrayCell 2.0

Authors
Geethika Weragoda,
Wesam Alwan, andFabian Zieschang
Agilent Technologies, Inc.

Abstract
The Agilent Cary 60 UV-Vis spectrophotometer fitted with a TrayCell 2.0
Ultra‑Microvolume Cell was used for the analysis of protein and nucleic acidsamples. The non-destructive measurement of microliter-sized sample volumes
is beneficial in terms of sample preservation, sample handling, and eliminating theneed to perform tedious and error-prone dilutions. Using the TrayCell 2.0 is quick andeasy, resulting in workflow improvements for users.

Introduction
Modern labs are increasingly looking for accurate, precise,and easy-to-use methods to analyze samples such as
DNA, RNA, and proteins without dilution. The Agilent Cary
60 UV-Vis spectrophotometer with a TrayCell 2.0 UltraMicrovolume Cell fitted with a suitable pathlength cap
provides a convenient and easy-to-use platform for the direct
measurement of ultra‑microvolume quantities of sample.
High and low concentration samples can be analyzed usinga short and long pathlength cap, respectively, ensuring awide dynamic range. The method is nondestructive, allowing
recovery of precious samples, and the TrayCell 2.0 is easy toclean, increasing the usability of the technique.

Towards a more sustainable lab
The Cary 60 UV-Vis has been independently audited
for its environmental impact and has received the ACT(Accountability, Consistency, Transparency) Label,verified by My Green Lab. The label provides
information about the environmental impact of the
Cary 60 UV-Vis throughout its entire life cycle (Figure 2).
The Cary 60 UV-Vis improves the environmental
impact of laboratories without impeding productivity or
scientific progress.

The Cary 60 UV-Vis spectrophotometer (Figure 1) is designed
for repetitive, routine applications as well as more advanced
applications. It is a double‑beam instrument equipped witha powerful, highly focused xenon flash lamp. The lamp
maximizes the level of light that passes through the sample,
ensuring high-quality photometric results. The Cary 60 UV‑Vis
is therefore ideal for measuring small sample volumes
accurately and reproducibly. The xenon flash lamp only
illuminates the sample when data is acquired, protecting
sensitive samples from photodegradation and reducing power
consumption. Also, the Cary 60 UV‑Vis spectrophotometer
is immune to the distorting effects of room light. Room light
immunity of the Cary 60 UV-Vis allows the operation with
an open sample compartment, enabling easy access, andreducing the risk of compromised data due to handling errors.
Compared to existing methods, which can produce inaccurate
or unrepeatable results due to instrument limitations, thehigh-performance Cary 60 UV-Vis provides better quality data.

Figure 2. The ACT label provides information about the environmental
impact of manufacturing, using and disposing of a product, andits packaging.

Figure 1. The Agilent Cary 60 UV-Vis spectrophotometer.

2

In a typical absorbance measurement, the TrayCell
2.0 Ultra‑Microvolume Cell (part number G6871C) is
placed in the standard cell holder of the Cary 60 UV‑Vis
spectrophotometer. The TrayCell 2.0 can be fitted withexchangeable caps with four different pathlengths, 2.0(part number G6871-68005), 1.0 (part number G6871‑68004),
0.2 (part number G6871-68003), and 0.1 mm(part number G6871-68002), providing a wide dynamic range.
An ultra‑microvolume aliquot of the sample (0.7 to 10 μL,
depending on the pathlength) is pipetted onto the measuringwindow of the TrayCell 2.0 and the corresponding cap withthe selected pathlength is placed on top. The highly focusedlight beam from the Cary 60 UV-Vis is directed through thesample via fiber optics in the TrayCell 2.0 (Figure 3). The lightbeam is then reflected by the mirror in the cap back to thespectrophotometer detector, repassing through the sample,
as outlined in Figure 4.

Cap with reflecting mirror

SampleOptical fiberLight passing
through thesamplePrismsLight beam

Figure 4. Optical design of the TrayCell 2.0 Ultra‑Microvolume Cell.

Before the next sample is introduced onto the measuringwindow of the TrayCell 2.0, the window and the mirror inside
the cap need to be cleaned. There is no need to remove
the TrayCell 2.0 from the cell holder during cleaning of
the window. The precisely defined spacing between thewindow and the mirror inside the cap ensures that the
optical pathlength is accurate and remains constant forevery measurement.
In this study, the Cary 60 UV-Vis spectrophotometer fitted witha TrayCell 2.0 Ultra-Microvolume Cell was used to measureultra-microvolume samples of protein and DNA. Different
pathlength cap options for the TrayCell 2.0 were used toreduce dilution and extend the measurable concentration
range of the sample. The photometric performance of the
Cary 60 UV-Vis spectrophotometer with the TrayCell 2.0 was
demonstrated using Bovine Serum Albumin (BSA) and herring
sperm DNA samples.
Figure 3. Schematic of the Agilent Cary 60 UV Vis spectrophotometer fittedwith a TrayCell 2.0 Ultra-Microvolume Cell for the direct measurementof ultra-microvolume quantities of sample. Representation of the highly
focused beam of the Cary 60 UV-Vis passing through the TrayCell 2.0.

3Instrumentation and materials

Results and discussion– Agilent Cary 60 UV-Vis spectrophotometer

Photometric reproducibility at low concentrations– TrayCell 2.0 Ultra-Microvolume Cell
– BSA protein: to prepare a 400 mg/mL stock solution,a known amount of BSA protein (Sigma-Aldrich, CAS
9048‑46-8) was dissolved in PBS buffer (phosphatebuffered saline). The stock solution was serially diluted toprepare a set of samples at different concentrations.
– Herring sperm DNA: to prepare 5 mg/mL stock solution,a known amount of herring sperm DNA (Sigma-Aldrich,
CAS 438545-06-3) was dissolved in PBS buffer. The stock
solution was serially diluted to prepare a set of samples atdifferent concentrations.
An ultra-microvolume (3 µL) of sample was placed on themeasuring window using a pipette, and data acquisition
was carried out using the Agilent Cary WinUV software,version 5.1.3.1042.

To assess the usable absorbance range of the Cary 60
UV-Vis spectrophotometer with the TrayCell 2.0, both
high- and low‑concentration samples were measured. For
low concentration measurements of limited volume samples,
the TrayCell 2.0 is ideal, as it requires only ultra‑microvolume
aliquots of sample. Also, the Cary 60 UV-Vis is sensitive
enough to measure low concentrations, whereas many
spectrophotometers do not have the sensitivity to measureconcentrations less than 25 ng/µL. The performancecapabilities of the spectrophotometer are vital at these low
concentrations, so wavelength scans need to be performed toensure that a typical sample curve is observed. Ten repeatedwavelength scans of a 20 ng/µL herring sperm DNA sample
were collected using the Cary 60 UV-Vis with the TrayCell 2.0
fitted with a 1.0 mm cap. Absorbance for this sample is 0.04,
which is equivalent to 0.4 Abs in a standard 10 mm cuvette.
As illustrated in Figure 5, the Cary 60 UV-Vis produced highly
reproducible wavelength scans, which is important for
ultra-microvolume analysis.

Figure 5. Ten repeat wavelength scans of herring sperm DNA (20 ng/µL, 0.04 Abs at 260 nm) acquired using the Agilent Cary 60 UV-Vis spectrophotometer with
the TrayCell 2.0 Ultra-Microvolume Cell fitted with a 1.0 mm pathlength cap.

4

Photometric linearity

Photometric linearity measurements for BSA protein

The quantitative and qualitative measurements of herring
sperm DNA and BSA protein were acquired using the Cary
60 UV-Vis fitted with a TrayCell 2.0. The 1.0 mm pathlength
cap is designed for the measurement of sample volumes
in the range of 3 to 5 µL, which is ideal for routine working
concentrations of DNA, RNA, and proteins.

Single wavelength absorption measurements for BSA protein
were collected at 280 nm (1 to 400 mg/mL concentration
range) using the Cary 60 UV-Vis spectrophotometer. Like forthe herring sperm DNA sample, excellent photometric linearity
up to 2.4 Abs was obtained using the 1.0 mm pathlength cap(Figure 7A). At 2.4 Abs, the corresponding concentration of
the BSA protein was 40 mg/mL. However, highly concentrated
BSA protein samples were accurately analyzed by simply
swapping the TrayCell 2.0 cap with shorter pathlength
options. BSA protein concentrations up to 200 mg/mL wereanalyzed using the 0.2 mm pathlength cap (Figure 7B). The
corresponding absorbance for the 200 mg/mL BSA protein
was 2.3, which is equivalent to 115 Abs for a standard 10 mm
cuvette. The 0.1 mm pathlength cap, which is the shortest
pathlength cap available within the TrayCell 2.0, was used tomeasure concentrations up to 400 mg/mL (Figure 7C).

Photometric linearity measurements for herring
sperm DNASingle wavelength absorption measurements at 260 nm weretaken for the herring sperm DNA samples in the concentration
range of 5 to 1,500 ng/µL. As shown in Figure 6, excellent
photometric linearity up to 2.4 Abs was obtained using the
TrayCell 2.0 with 1.0 mm pathlength cap. At 2.4 Abs, thecorresponding concentration of the herring sperm DNA was
1,500 ng/µL. Excellent photometric linearity was observedfor extremely low concentrations of the DNA sample, as low
as 5 ng/µL (0.008 Abs, equivalent Abs 0.08), with 1.0 mm
pathlength cap (Figure 6, inset).

Abs (1 mm cap option)

2.5

0.105

2.0

These results demonstrate that direct measurement of
BSA protein samples up to 400 mg/mL can be easily
performed using the Cary 60 UV-Vis with TrayCell 2.0, withouttime‑consuming and error‑prone dilutions (Table 1).
Table 1. Comparison of equivalent absorbance for BSA protein using 1.0, 0.2,
and 0.1 mm pathlengths.

0.055

1.5 0.005

5

20

35

TrayCell 2.0
Ultra-Microvolume Cell

50

1.0
0.5
0.0

0

250

500
750
1,000
DNA concentration (ng/µL)

1,250

1,500

Figure 6. Photometric linearity for herring sperm DNA samples up to
1,500 ng/µL. The inset shows the results for low concentration samples in
between 5 to 50 ng/µL.
2.5

2.5

A

1.0

0.2

0.1

Highest Concentration Measured (mg/mL)

40

200

400

Measured Absorbance

2.4

2.3

2.0

Equivalent Abs to 10 mm Standard Cuvette

24

115

200

Note: Equivalent Abs is the absorbance value calculated for a 10 mm pathlength.
The benefit of being able to measure up to 400 mg/mL is that highly concentrated
protein solutions can be measured directly, eliminating dilutions.

2.5

B

1 mm pathlength
2.0

Pathlength (mm)0.2 mm pathlength2.0

y = 0.0609x - 0.0006
R² = 0.9997

0.1 mm pathlength2.0

y = 0.0109x + 0.0366
R² = 0.9989

Abs

Abs
1.0

1.0

1.0

0.5

0.5

0.5

0.0

0

10
20
30
BSA concentration (mg/mL)40

y = 0.0051x - 0.0958
R² = 0.9983

1.5

1.5

Abs

1.5

C

0.0

0

50
100
150
BSA concentration (mg/mL)

200

0.0
100

150

200

250

300

350

400

BSA concentration (mg/mL)

Figure 7. Photometric linearity measurements for BSA concentration up to 40 mg/mL using the 1.0 mm pathlength cap (A), up to 200 mg/mL using the 0.2 mm
pathlength cap (B) and up to 400 mg/mL using the 0.1 mm pathlength cap (C).

5

The Cary WinUV Concentration module for quick andeasy data acquisition and analysis

1) Open the Concentration module and click the Setup tab toopen the instrument setup window.

The Cary WinUV software includes powerful features andstreamlined methods for data collection, analysis, storage,
and display, while reducing complexity. It also includes
different modules that are designed to cover a range ofapplications including qualitative wavelength scan or reads,
concentration analysis, enzyme kinetics, and many more.

2) In the Cary tab, enter the wavelength for single wavelength
measurements in Wavelength option (for BSA proteins,enter 280 nm).

In this study, full wavelength scans were performed using theScan module, while photometric linearity measurements were
performed as single wavelength absorbance measurements
using the Concentration module. To demonstrate theinstrument setup and related results‑report generated
by the Concentration module, the photometric linearity
measurements were carried out for BSA protein samples
using a 0.1 mm pathlength cap. (The same results are shownin Figure 7C). Setting up the Cary WinUV software is quick andeasy, requiring a few simple steps as follows and illustrated in
Figure 8:3) Enter the number of replicates that are needed foreach standard using Replicates or Sample/Std
Averaging. In this example, data were collected foreach BSA standard solution as the average of threeconsecutive measurements.
4) In the Standards section, enter concentrations of standard
samples in ascending order and select the Fit type. In thisexample Fit type was selected as Linear with minimum R2(Min R2) of 0.9500.
5) Both the software and the instrument are ready for
analysis. Click the Start button to start the analysis andsimply follow the standard/sample loading instructions tocontinue the analysis.

Figure 8. Instrument setup for data acquisition and analysis using the Concentration module in the Agilent Cary WinUV software.

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Following the data acquisition, the corresponding
concentration versus absorbance graph (calibration curve)
is automatically generated by the software (Figure 9),
reducing time-consuming data workup procedures. The
calibration curve is saved in the Concentration module. When
analyzing an unknown sample, the software automaticallyuses the calibration curve to calculate and report the
sample concentration.

Cleaning of TrayCell 2.0 Ultra‑Microvolume Cell
Figure 10 outlines the simple steps needed to clean and loada sample in the TrayCell 2.0. The sample window and cap
are wiped clean with a lint-free swab or lint-fee lab cloth andthe next sample is loaded with a pipette. These procedures
eliminate time-consuming cleaning steps commonly
associated with conventional cuvettes and reduce the risk ofsample carryover.

Fully flexible solution
A standard 10 mm pathlength cuvette is recommended for
extremely dilute samples, which cannot be measured using
different cap options available with the TrayCell 2.0. Standard
10 mm pathlength cuvettes are available in a range ofvolumes, from 40 µL to 3.0 mL.
To perform a wider range of measurements, the Cary
60 UV‑Vis spectrophotometer can be fitted with long
pathlength cells, automated multicell changers, aswell as temperature‑controlled cuvette holders. The
range of measurements include quantitation, sample
automation, or analyzing kinetic processes. Components
are easily interchangeable, making the Cary 60 UV-Vis
spectrophotometer a widely used instrument for routine
biological UV-Vis measurements.

Figure 9. Calibration curve and the concentration analysis report generated automatically by the Agilent Cary WinUV software for the BSA protein using the
TrayCell 2.0 with 0.1 mm pathlength cap. The same graph is shown in Figure 7C.

7

Figure 10. Schematic of cleaning and loading a sample in the TrayCell 2.0.

Conclusion

Further information

The Agilent Cary 60 UV-Vis spectrophotometer fittedwith the TrayCell 2.0 Ultra-Microvolume Cell provides
a convenient and easy‑to‑use platform for measuring
ultra‑microvolume samples such as nucleic acids andproteins. Repeated qualitative wavelength scans of a
20 ng/µL herring sperm DNA sample demonstrated thesensitivity and reproducibility of the Cary 60 UV-Vis for the
analysis of ultra-microvolume samples. Using four different
caps for the TrayCell 2.0 (and therefore pathlengths),
extended the analytical range of the method, alleviating theneed to perform tedious and error‑prone dilutions. A linear
photometric range was observed for herring sperm DNA andBSA protein samples in the range from 5 to 1,500 ng/µL and
1 to 400 mg/mL, respectively. The streamlined workflow,
versatility, and photometric performance of the Cary 60
UV‑Vis spectrophotometer with the TrayCell 2.0 make itsuitable for accurate measurements of undiluted nucleic
acids and proteins.

– Agilent Cary 60 UV-Vis spectrophotometer

www.agilent.com/chem/cary-60-uv-vis
DE21781667
This information is subject to change without notice.
© Agilent Technologies, Inc. 2022
Printed in the USA, December 20, 2022
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