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Notes & Tips

Quantitative pH assessment of small-volume samples using a universal

pH indicator

 Jeffrey D. Brown a,⇑, Nathaniel Bell a,1, Victoria Li b,1, Kevin Cantrell b

a Department of Biology, University of Portland, Portland, OR 97203, USAb Department of Chemistry, University of Portland, Portland, OR 97203, USA

a r t i c l e i n f o

 Article history:

Received 12 December 2013

Received in revised form 26 May 2014

Accepted 2 June 2014

Available online 11 June 2014

Keywords:

Isoelectric focusing

pH indicator

pH determination

Wnt

a b s t r a c t

We developed a hue-based pH determination method to analyze digital images of samples in a 384-well

plate after the addition of a universal pH indicator. The standard error of calibration for 69 pH standards

was 0.078 pH units, and no sample gave an error greater than 0.23 units. We then used in-solution iso-

electric focusing to determine the isoelectric point of Wnt3A protein in conditioned medium and after

purification and applied the described method to assess the pH of these small-volume samples. End users

may access our standard to assay the pH of their own samples with no additional calibration.

 2014 Elsevier Inc. All rights reserved.

Although pH determination using standard electrodes is useful

for many applications, electrode fouling, small sample volume, anda desire to not contaminate an electrode with surfactant-contain-

ing analytes can limit the utility of this approach. In such cases,

universal pH indicator solutions may be used. These multichromat-

ic pH indicators consist of a dye cocktail that undergoes a contin-

uous change in color as the solution pH changes. pH assessment

relies on a qualitative comparison between the solution color and

a color reference card. Due to the subjective nature of such assess-

ments and variability among users, pH determination using

universal indicators can be unreliable; the resolution, reproducibil-

ity, and precision of such determinations are subject to substantial

and variable user errors that limit the confidence of pH determina-

tion. Other methods rely on spectrophotometric measurements of 

the absorbance at a particular concentration of pH indicator (e.g.,

see Ref. [1]). These methods, although accurate, require expensive

equipment, large sample volume, and extensive sample prepara-

tion. To address these shortcomings, we developed a quantitative

hue-based method to assay the pH of small (100 ll) samples in a

384-well plate after the addition of a universal pH indicator.

Ubiquitous digital  cameras or scanners are used to capture RGB

(red–green–blue)2 color intensities in TIFF or JPG digital images.

RGB values are converted into HSV or HSL color spaces, which quan-

tify and separate the color, or hue, from the other parameters such assaturation and lightness. The hue is measured on a cylindrical coor-

dinate system (a color wheel) and, due to its insensitivity to concen-

tration and illumination variations, is a robust measure of the optical

properties of color-changing indicators [2]. The relationship between

the easily measured hue and the pH is quite stable for a particular pH

indicator cocktail, and once established the hue values from any dig-

ital image can be transformed into a good estimate of pH. Investiga-

tors employing the same commercial pH indicator solution used in

our work can apply our hue calibration data with no additional

manipulation to assess the pH of their small-volume samples. Due

to the reliable nature of the hue parameter, this approach provides

a transferable, rapid, inexpensive, and easily scalable means to

determine the pH of numerous small fractions using only a con-

sumer-grade digital camera or scanner and data processing using

free and/or common software.

To generate a standard curve that relates hue and pH, we pre-

pared a buffer solution containing a (1:50 dilution of) universal

pH indicator (Fluka, cat. no. 36828, Sigma–Aldrich, St. Louis, MO,

USA) and monitored pH with a Ross micro pH electrode (Thermo

Scientific, cat. no. 8220BNWP, ThermoFisher Scientific, Waltham,

MA, USA) as we titrated the solution with acid or base. Images of 

the buffer solution were acquired approximately every 0.1 pH unit

with a Canon EOS Rebel T1i digital SLR camera. Other work demon-

strated the efficacy of various cameras or scanners in generating

the images used in the hue-based approach   [3]. Images of these

standards were processed in MATLAB and used to calculate the

http://dx.doi.org/10.1016/j.ab.2014.06.001

0003-2697/  2014 Elsevier Inc. All rights reserved.

⇑ Corresponding author. Fax: +1 503 943 7784.

E-mail address:  brownje@up.edu (J.D. Brown).1 These authors contributed equally to this work.2

 Abbreviations used:  RGB, red–green–blue; IEF, isoelectric focusing; pI , isoelectric

point; BSA, bovine serum albumin.

Analytical Biochemistry 462 (2014) 29–31

Contents lists available at   ScienceDirect

Analytical Biochemistry

j o u r n a l h o m e p a g e :   w w w . e l s e v i e r . c o m / l o c a t e / y a b i o

http://dx.doi.org/10.1016/j.ab.2014.06.001mailto:brownje@up.eduhttp://dx.doi.org/10.1016/j.ab.2014.06.001http://www.sciencedirect.com/science/journal/00032697http://www.elsevier.com/locate/yabiohttp://www.elsevier.com/locate/yabiohttp://www.sciencedirect.com/science/journal/00032697http://dx.doi.org/10.1016/j.ab.2014.06.001mailto:brownje@up.eduhttp://dx.doi.org/10.1016/j.ab.2014.06.001http://-/?-http://-/?-http://-/?-http://-/?-http://crossmark.crossref.org/dialog/?doi=10.1016/j.ab.2014.06.001&domain=pdfhttp://-/?-http://-/?-

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coefficients of a sigmoidal curve fit that relates the measured hue

to the pH of the solution, as shown in Fig. 1. This standard curve

was used in subsequent analyses and is transferable to other digital

devices and applications using the same indicator cocktail. The

freely available application ImageJ (http://rsbweb.nih.gov/ij) can

be used to manually select a region of interest in an image and cal-

culate the mode of the hue values in the selection as described in

the  supplementary protocol   (see online supplementary material).

A simple Excel spreadsheet (http://wordpress.up.edu/phindicator/

excel) can be used to calculate the pH based on this hue using

the pre-established curve fit parameters that are usable with any

image of the Fluka indicator cocktail. A MATLAB script that identi-

fies all of the filled wells in an image and automatically calculates

the pH in each one from a single image is available on request.

To validate this pH assessment method, we analyzed fractions

generated by a MicroRotofor free-solution, carrier ampholyte-

based isoelectric focusing (IEF) system (Bio-Rad Laboratories,

Hercules, CA, USA) to characterize the isoelectric point (pI ) of 

the bovine serum albumin (BSA), Wnt3A, and a soluble Friz-

zled8CRD-IgFc fusion protein   [4]   under native conditions. The

MicroRotofor yields 10 approximately 250-ll fractions, and the

pH of these samples must be assessed in order to understand

the pI   of the proteins that focus to the fractions. Although this

apparatus is not well suited to high-resolution pI  determination,

the 3-ml focusing chamber can accommodate a relatively large

analyte volume, and recovered fractions are suitable for subse-

quent analyses, including functional assays. IEF analysis can indi-

cate post-translational modifications that alter a protein’s charge

[5] or biomolecule complex formation [6].

Initial assays were performed using broad-range ( pH 3.5–9.5)

Bio-Rad BioLyte 3/10 ampholytes (cat. no. 163-1112), and pH was

monitored with a Ross micropH electrode. As shown in Fig. 2, puri-

fied Wnt3A focused to approximately pH 9.0 fractions, consistent

with a predicted pI   of 8.26 (http://web.expasy.org/compute_pi;

see Ref.   [7]), whereas unpurified Wnt3A protein in conditioned

medium from expressing cells focused to an approximately pH

5.0 fraction. Post-translational modification by anionic groups,including protein phosphorylation   [8]  and sialic acid-terminated

oligosaccharides   [9], can decrease protein pI ; however, such pI 

shifts tend to be on the order of

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We also subjected BSA to MicroRotofor fractionation. Fraction pH

was monitored with the Ross pH electrode and the universal pH

indicator as described. Electrode and hue methods were in good

agreement, and BSA focused to approximately pH 5.0 fractions

(data not shown), consistent with the reported pI  of the native pro-

tein [13].

We noticed a substantial depression in hue-based pH calcula-

tion for pH > 7.0 samples from MicroRotofor runs that included

1% Chaps detergent. This deviation was not observed using the

Ross micro pH electrode, indicating that the universal indicator

suffers from Chaps-dependent error in this range. The deviation

may be analogous to the  protein error  exhibited by some pH indi-

cator dyes used for protein quantitation assays. Suzuki   [14]

reported that inclusion of non-ionic detergents in such assays

results in an upward shift in the pH required for a chromatic

change of the protein-associated dye molecule. A similar phenom-

enon affecting a dye in the universal indicator in the presence of 

Chaps could result in the observed depression in the hue-based

pH determination. To quantify this surfactant effect, a second stan-

dard curve was generated in the presence of 1% Chaps (Fig. 1) and

reveals a substantial shift relative to the surfactant-free curve.

Although the technique that we described here can be applied

to most aqueous samples, deeply colored analytes (e.g., chromo-

proteins) and carrier ampholytes can be a source of significant

errors; however, the Bio-Rad BioLyte ampholytes used in our

experiments have little color at working concentration, and the

standard error of pH assessment using the hue method was only

0.2 pH units for analyzed fractions. In addition, hue-based pH

assessment of BSA—which has a modest reddish-brown hue—did

not suffer from any greater error from electrode-based pH assess-

ment than other proteins.

In summary, we developed a quantitative method to assess the

pH of small-volume samples that is inexpensive, rapid, and non-

destructive and that scales easily to numerous analytes. Although

our individual experiments generated only 10 fractions, we rou-

tinely stored frozen fractions and prepared several dozen samples

for hue-based pH assessment in a single plate. This method will beof particular utility to assay large numbers of fractions generated

by techniques that include chromatofocusing and free-flow IEF.

We then used a Bio-Rad MicroRotofor IEF apparatus to document

a dramatic pI  shift in Wnt3A protein during purification, a differ-

ence that may correspond to the inclusion of Wnt3A in exosomes

before purification in the presence of detergents. Finally, we

uncovered a substantial Chaps-dependent error in pH assessment

using a universal indicator in solutions of pH > 7.0 and recommend

caution when using universal indicators in the presence of 

surfactants.

 Acknowledgments

The authors thank the University of Portland for supporting this

work. This work was funded in part by M.J. Murdock Life Sciences

(Grants 2008354 and 2010196). The authors thank Brianna Brown,

Calli VanderWilde, Michelle Thomas, Blair Pearson, and Keri Jack-

son for their assistance in these investigations.

 Appendix A. Supplementary data

Supplementary data associated with this article can be found, in

the online version, at  http://dx.doi.org/10.1016/j.ab.2014.06.001.

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