Creating a calibration curve with Adobe Photoshop

Contributors

Faculty

LaKeisha McClary, Ph.D.
Assistant Professor of Chemistry 
Science and Engineering Hall
The George Washington University

Team Members

Richard Graham, Ph.D.
Instructional Designer
Center for Teaching Excellence 
The George Washington University

Cristabel Ocasio Ilarraza, M.A.
Instructional Designer
Center for Teaching Excellence 
The George Washington University

Leah Sims
Multimedia Producer
Center for Teaching Excellence 
The George Washington University

GW-Adobe Science Education Initiative Program Leads

Jason Torres, Ed.D.
Director, Digital Learning
Center for Teaching Excellence
The George Washington University

Sylvain Guiriec, Ph.D.
Physics Innovation Lab
Assistant Professor of Physics
Columbian College of Arts and Sciences, Department of Physics
The George Washington University

Project information

Audience

The intended audience for this lesson is undergraduate chemistry students. 

Learning objectives

• Demonstrate competency with acid-base calculations.
• Gain proficiency with using Adobe Photoshop to process images for RGB values.
• Calculate rations of R, G, and B values for images of known pH values to generate a calibration curve.
• Interpret the calibration curve to determine pH measurements for experimental solutions.

Purpose

Using image processing software as an alternative to a pH meter requires that we have a reliable method to create a calibration curve. The calibration curve will use RGB ratios as a function of pH values,¹ and we will use Adobe Photoshop to process the images and Excel to generate the calibration curve. You will have an opportunity during the design-it-yourself (DIY) experiment to explore other image processing software (e.g., GIMP), methods to process images prior to analysis, and pH indicators besides those we will use in this activity.

Background

CHEM 2123W includes an embedded research experience that is largely guided by your interests within a topic selected by the instructor. This academic year, we will continue to explore using household ingredients for acid-base titrations. The work is inspired by Andrews et. al. who reported an at-home protocol to guide their students through general chemistry experiments that were safe to do unsupervised in their homes; GIMP histograms were generated and used to determine the intensities of red, green, and blue pixels in images of students’ experimental solutions, and those intensities were used to generate a calibration curve for subsequence experiments.¹ Students were able to use their calibration curves to report pH measurements that were within 0.3 pH units of known values.¹ 

Figure 1. RGB ratios of the red cabbage pH indicator as a function of pH. R:G, G:B, and B:R ratios are shown as the red, green, and blue lines, with pH values indicated above the close circles. The three open squares show the R:G (red), G:B (green), and B:R (blue) ratios of a sample analyzed by a student, indicating the pH was approximately 7.4-7.6.¹

One limitation of GIMP, however, is that calculating the intensities of red, green, and blue pixels requires numerous steps. We will use Adobe Photoshop to simplify image processing.

At GW, students have explored using vinegar and a variety of bases (e.g., baking soda, borax, ammonia) and pH indicators (e.g., red cabbage, red beets, black beans, turmeric) with varying success. The primary limitation students faced was not having a reliable calibration curve. The aim of this activity is to alleviate this challenge and open the possibilities of using Adobe Photoshop to analyze acid-base titrations with a variety of acids, bases, and indicators. For now, though, we will focus on replicating Andrews et al.¹ 

Red cabbage is a universal indicator so follow these steps for each image corresponding to one of the 14 pH values (1-14) that you took during the titration of vinegar with KOH(aq). In Excel, you will input your RGB ratios and generate a calibration curve, a necessary tool for your DIY experiment when using imaging software.

General assignment

Lesson activity: Creating a calibration curve using Adobe Photoshop

The objective of this lesson is to equip learners with the essential skills to proficiently use Adobe Photoshop to process and create a calibration curve. This lesson will enable learners to accurately adjust and manipulate various calibration techniques and construct a reliable calibration curve to analyze and interpret data accurately.

Step 1

Open Adobe Photoshop and upload the images that you plan to use to create your calibration curve. It helps if you have the images in one folder and named according to their pH value. To upload images, select from Open > from the file selection menu. 

On your computer select the folder where your image file(s) are located. Choose an image file to upload.

Step 2

Select an area of your image that does not have shadows or reflections. Choose the Marquee Tool 

 and select one pixel and zoom in by selecting (View > Zoom In) on that region until you see pixels. 

Figure 2 Use Marquee tool to zoom in to a specific section of a picture

Step 3

View the RGB histogram (Window > Histogram). Change the channel and record the mean value for red, green, and blue.

Figure 3 Select Histogram to display RGB values

Step 4

Input the raw values into the appropriate cells in an Excel file. The Excel template will automatically compute the ratios that you need and generate the calibration curve. The calibration curve consists of 2 sets of x, y data points at a given pH for R:G, G:B, and B:R. Thus, a single pH value, X, will be represented 3 times.¹ Remember that your values will be different from that of your peers because of the different hardware, the different area of the pixels selected for export, and also perhaps due to errors in preparing solutions.

Figure 4 RGB data input in excel

Step 5

Submit your calibration curve and Excel files via Blackboard. We will debrief the assignment after the next pre-lab discussion. Begin to jot down potential ideas for how to use this process to answer a research question of interest to you during the DIY portion of the course.

Project output

Defined success 

This exercise was designed to demonstrate and provide essential skills to proficiently use Adobe Photoshop in processing and creating a calibration curve, equipping learners with valuable tools for accurate data analysis and interpretation. This exercise provided learners with key steps to analyze and adjust images using Adobe Photoshop and export histogram values to Excel. By understanding the importance of calibration in accurate data analysis, learners can implement appropriate adjustments to ensure the reliability and validity of their calibration curve. By analyzing data, learners can draw meaningful conclusions about the relationship between pH values and image measurements, enabling more informed decisions in scientific and analytical contexts.

Additional resources

• Photoshop scripting basics
• Online interactive platform to create plots of pH indicator colors.
• Photoshop essential training