Lab 5

~- · your . . f tion must be m Write a Pre-Lab assignment based on. the procedUre below, This In orma In your own notebook. Remember to Include a tltle, objective, and a summarized procedure written words. You must fill out the tables before coming to lab.

I th' · · · · f1Thra-nitrophenol n is set of experiments, you will examine how pH affects the spectral properties O

P (PNP).F irst, you will prepare five samples with known PNP concentrations, measure the absorba~ce of each,_ a

nd use the data to generate a standard curve. You will use the standard curve to det~~ me th

e ext,nct,on coefficient of the deprotonated form of PNP (para-nitrophenylate; PNPO_) t a wavelength close to its lambdamaJt (Amax). You will also ·use the standard curve to determine the unknown concentration of a PNP solution. Lastly, you will estimate the pK, of PNP. Materials

• 0.lmM para-nitrophenol (PNP) stock st>lution in water (15ml) • pH 10 buffer (30ml) ·

• PNP sample with unknown concentration (2ml) • 2ml microcentrifuge tubes • 4.5ml polystyrene cuvettes • BioMate 3S spectrophotometer

• pH buffers: 4 (or 5), 6, 7, 7.5, 8, 8.5, 9, and,10 (3ml each; O.lM) I

A. Determination of the Extinction Coefficient of PNP o- : Generation and use of a Standard Curve

1. Use the O.lmM PNP stock solution and RO water to prepare five 2ml PNP standards at the following concentrations: 10, 20, 30, SO, and 7SµM. Make sure they are mixed well. You can prepare them in 2ml tubes. These are your "standards" (your PNP solutions of known concentration). Fill out the table below before coming to lab (this must be included in your pre-lab):

Standard [PNP] Vol ofQ.lmM Vol of H20 Final Vol of PNP # (µM) PNP Stock (µL) (µL) Standard (µL) ,. 1 10 ' 2000 . 2 20 ,,

2000 - 30 ,.

.., 2000 3 ,, . .. ' - , - , . 4 so

2000 " . .. ·' - ·~

75 ' '

2000 s

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Obtain seven 4.5ml polystyrene cuvettes. Assume all cuvettes are dirty u_nless told otherwise. 2. Clean the cuvettes with Alconox and water. Rinse thoroughly and dry using a cotton swab.

. f standard and 3ml of pH 10 buffer. Do this for each of the five 3. In a 4.5ml cuvette, add ~ml o h . th f:nal [PNP] for each diluted standard? Fill out standards you prepared m Step 1. W ~t is e I •

the table below (this must be included m your pre-lab).

4 · Use the Basic ATC function to measure the absorbance of each diluted standard at 4oonm.

What will you use as the reference? ,, "

Note: When placing the cuvettes in the spectrophotometer, make sure they are oriented correctly. Notice that the cuvettes have an inverted triangle -along the top edge. Notice also that the cuvette holders inside the instrument

. _J Triangle

have two sides with holes for the light to pass through. Insert the cuvette such that the inverted triangle is directly above one of the holes in the holder. 8 Hole

5. Record at least three absorbance readings for each diluted standard {take three readings of .the same cuvette. You do not need to prepare each diluted standard three times). You will use this data to calculate the average absorbance for each standard.

B. Determining the unknown concentration of a PNP solution

1. Obtain a PNP sample with unknown:cdncentration; make sure you sign out your unknown. Be careful with your unknown, as we usually do not have enough to provide a new one in case of mishaps. Record your unknown number in your notebook and write your name on the unknown tube to prevent mix-ups.

2. Dilute 1ml of the unknown sample with 3ml of pH 10 buffer.

3. Measure the absorbance of the sample three times. What will you use as the reference?

C. Estimating the pKa of PNP

1. Transfer 1ml of 0.lmM PNP !to ei~~t separate cuvettes. Add 3ml of the following buffers: .no.minal pH values of 4 (or 5), 6, z, 7.5, 8, 8.5, 9, and 10, one to each cuvette. Record the exact pH of the buffers tha~were usei ' .

2. ·Read the absorbance of each sample three times. What will you use as the reference?

Ill. Data Analysis . . . , Part A to create a standard curve.

Before you write your assignment you must use the data from ti n of your unknown sample. ' PNP concentra o You will then use the standard curve to calculate the p

Lastly, you will plot your data from Part C to estl"1ate the pK. of PN ·

1. Parts A and B: . b b data you collected in lab. a. You will need a graphing program, like Excel. Use the a sor ance

· f h f the five diluted PNP b. Calculate the average absorbance and standard deviation or eac O I

b 1 standards. Put the average absorbance data in a table. Remember to include proper a e s,

units, and titles when making a table. c. Plot Abs,vg as a function of diluted [PNP] (square brackets denote concentration. [PNP] is a

short way of writing "PNP concentration"). For chart type, select scatter. This is your PNP standard curve. Remember to add labels (with units) and a title to your graph.

d. Perform a linear regression analysis to fit' th:e data. You must force the fit through the origin · (set the intercept to 0,0). It is not sufficient to simply add point 0,0 to vour data set. Include the equation of the trendline and,R2 value directly on the plot. To do this, once you plot your data, Add Trendline by right-clitking on any data point in the graph. A line should appear on the graph and a Format Trendline menu should open. In this menu, make sure the following are selected:

• Trendline Options: linear • Set Intercept 0,0 • Display Equation on chart • Display R2 value on chart .

e. Determine the extinction coefficient (E) from your pl ot. Where do you find this information? rlli ink of how the. Beer-Lambert equation (A=Ecl) relates to the equatio.n of the trend line that was used to perform the linear regression analys~.Jv.=mx);,.l reate a text box to display it on your graph. Remember to include units.

f. Add a caption with a title and a description that includes all the important information from the plot.

g. Use the standard curve to calculate the PNP toncentration of your unknown. Remember, this is the concentration of your diluted unkno

1 wn that was in the cuvette. ' )

h. Use the concentration of your diluted unk'nown to calculate the original {i.e., undiluted) concentration of your unknown. · ·

Points to consider for Parts A and B: '

• When performing a linear regression analysis on your data from Part A, why does it make sense to force the fit through the origin? This is a key concept.

• What would be a physical explanation for~ non-zero intercept? Again, this is a key concept. • Remember that you measured the absorbance of your unknown PNP sample after you diluted

it. What is the dilution factor?

2. Part C: a. Calculate the average absorbance and standard deviation at each pH. Put the average

absorbance data in a table. Remember to add a title and labels with units.

' b. Plot the Abs f .,· I ct scatter with smooth lines and ava as a unction of pH. For chart type, se e h

_markers. Remember to add proper labels (with units) and a title to your grap · c y 'II . . h K d'rectly on the plot. · · ou WI calculate the pk, from this plot. Use a text box to ind1ca~e t e P • 1 • d Re b I : . h . I des the important · . mem er to add a caption with a, title and a description t at me u

information from the plot.

Points to consider for part c: • · • _What does the shape of th.is plot remind you of? This will be key when identifying the pKa,

IV• Assignment: Results and Discussion For this assignment, you will write the Results and Discussion sections of a lab report. You will also include a title page, references, and an appendix with sample calculations. Below is a guide on how to write·these sections. More details on how to write a lab report are available in the Lab 3 folder on Blackboard. Your assignment must be typed using a word processing program that is compatible with Turnitin.com. Do not copy and paste the guide into your document.

1. Title Page f Spts) The title page must contain an original and descriptive title for your report (do not use the same one as the manual), your name, and _the date the report is due.

2. Results (40pts)

Write this section using paragraphs. Pay close attention to the flow of the information presented in this section. The transitions from point to point should be smooth; your descriptions should not read like you are answering short questions or bullet points.

You will present your analyzed data, results, and how they were obtained. Do not include explanations or interpretations of your results or limitations and importance of the study; that information is presented in the Discussion section.

Since you will be presenting different figures and resu!ts, use headings to separate the different parts of this section.

1. ·Determining the unknown concentration of a PNP solution

a. Start the Results section with a brief introduction that states the purpose the experiment and what was done. Include yqur unknown number (No more than 5 sentenc~s).

b. Include a table with the average absorbance values for each of the five standards. The table must include a title, labels,:and units .

. c. Include a figure showing your standard curve. The figure must include a proper title, labels with units, trendline equation, R2 value, and extinction coefficient. You must also include a caption below the fig,ure. The figure can be inserted in the text.

d. Describe the figure: how was it made, what information does it provide? How did you obtain that information?

e. Compare your extinction coefficient to the literature value for PNP at 400nm (calculate the% error).

f. State the concentration of your unknown (diluted and original).

g. Reference the calculations in the appendix ..

2. Determination of the pK, for PNP , '·

a. Include a brief introduction that states what you did. . b. Include a table with the absorbance ·values for each sample at different pH's. The table

must include a title, labels and units. · , c. Include a figure showing your graph of ·absorbance as a function of pH. The figure must

include a proper title and iabels (with units). Indicate the pK, directly on the graph. Include a caption below the figure. The figure can be inserted the text.

d. Describe the figure: how was it mad·e, what information does it provide? How did you obtain that information?

e. Compare the pKa that you obtained to the literature value for PNP (calculate the% error). f. Reference the calculations in the appendix.

3. Discussion (40pts) Write this section using paragraphs. Pay close attention to the flow of the information presented in this section. The transitions from point to point should be smooth; your explanations should not read like you are answering short questions or bullet points. You will recap your results in this section. You will also, and most importantly, explain the significance of your results, discuss why you obtained those results based on previously known information, compare your results to the literature, explain limitations and importance of study, etc. This section is usually filled with references. ' ·

Include a short introduction to spectrophot,ometry and para-nitrophenol. Since you will be presenting different figures and results, use:: headings to separate the different parts of this section. ·

1. Determining the unknown concentration of a PNP solution

a. Describe Beer's law and how it is used with a standard curve to determine the concentration of a sample. Relate Beer's law to the equation of a line.

b. Why is the line forced through zero in the standard curve? c. State the extinction coefficient for PNP at 400nm that you obtained from your data. How

does it compare to the literature value? Is it accurate? d. What does the R2 value of your linear regression analysis indicate about your data?

e. State the concentration of your unknown·. I '

f. Explain any deviations in your data t~a~ may have affected the accuracy of your results.

2. Determination of the pKa for PNP , a. State the pK1 you calculated. How does) t compare to the literature value?

b. Explain the shape of the graph and w~ere the pKa can be found. Why is the pKa found in that region of the curve? Is PNP mon9protic, diprotic, or polyprotic?

c. Exp.lain in detail the changes in structure, charge, and c~lor of PNP ~s the pH increases. If you use a figure to aid in your explanation, n1ake sure 1t has a caption.

d. Explain why increasing the ptrl causes a change in color. What is happening to the structure of the molecule that changes its spectrophotometric properties?

4. Reference (Spts}

You will use references for your literature values and to answer the questions above. When referencing literature for this lab you must:

a. Include at least three references, two of which must be outside references • Inside references: Any material given to you by the lecture or lab instructor

{Biochemistry text book, lab manual, PPT, etc.) • Outside references: Material not given to you by the lecture or lab instructor (other

class textbooks, research papers, reviews, etc.) b. Use in-text citations (MLA or APA is fine)

5. Appendix (10pts)

• Choose one sample and provide a sample calculation of the average absorbance and standard deviation.

• Include a sample calculation· for, the extinction coefficient, PNP concentration of your diluted and undiJuted unknownii p,Ka, and % error.

• Remember that everything that appears in the appendix should be referred to at some point in the Results section.

6. Observations (-Spts if not included)

You must include a copy for your handwritten observations and data collected during lab. Include a scan or a picture. Do not re-type your observations.

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I. Introduction

Because many biochemical assays incorporate spectrophotometry, it is important for an investigator to ~e w~ll-versed In its theory and application. Fundamentally, absorption spectroscopy measures how much !1ght IS absorbed by a sample at different wavelen:gths. A plot of absorbance as a function of wavelength is called an absorption spectrum. This technique can be used for quantitative estimation of compounds based on color intensities, qualitative studies (example: your elution profile from Lab 4), as well as measuring the spectral properties of molecules ·and atoms. Many biochemicals are colored; that is, they absorb visible light and therefore can be measured by colorimetric analyses. Colorless biochemicals can often be converted to colored products through i>rocesses called chromogenic reactions. It is importa~t to bear in mind that spectroscopy uses wavelengths outside the visible region of the electromagnetic spectrum as well.

The absorbance (A) at a given wavelength (A) is a function of the chemical properties of the sample-for UV-visible spectroscopy, this can correspond to molecular orbital electronic transitions-which are related to the molar absorptivity coefficient (E). Other factors that affect absorption are the path_ len~h (I) through the sample and the molar concentration of the compound of interest {c). The relationship between these terms is expressed mathematically in the Beer-Lambert Equation, also known as Beer's Law:

A;.. =,(E,J(c)(l)

The derivation of this relationship is sufficiently described elsewhere and it is recommended that you review this. The Beer-Lambert Equation is ofte~ used to determine the concentration of a compound in solution or to calculate the expected absorbant:e -of a sample with a known concentration. However, in this experiment we will try to expand our underst~nding of the application of this equation.

It is important to remember that the instrument m~asures the absorbance of a sample at a given wavelength by subtracting the absorbance of a 'reference sample (often referred to as a blank) from the absorbance of the sample of interest. In other words, at each wavelength the final, or net, absorbance is:

Anet = Asample Areference

= [ ( EAsample) ( Csample) (1)] - [ ( EAreference) ( Creference) (l)]

In most cases, the reference is one of the following: • Air • A solution that is identical to the sample, but does not contain the compound of interest • A solution of a non-absorbing spe~ies, suc1h as a buffer solution for which E= O at all wavelengths • A solution containing equimolar sample b~fore any chemical transformation has occurred

he method by which subtraction occurs depends on the instrument that is being used. For instance, ngle beam spectrophotometers are first calibrated (or "blanked") at a given wavelength using a cuvette

. bsorbance · ' · · ults m an a - .- otentiometer and res - nee are placed containing the reference solution. Setting the blank •~Justs a P t the sample and refere b am splitter,

of zero. In older non-computer Interfaced dual beam lnstrumen s, d chopping motor, or t uments, Inside the instrument at the same time. As the scan Is made, a mlrrore es In both types of m_s r . turn sends alternating beams of light through the sample and reference cuvett . • t be (PMTI, which m d light that is not absorbed Is transmitted and reaches a photomultiplier u . ultimately subtracte generates a signal In the form of an electri~al current. The signal of the reference IS from that of the sample.

. b measured Alth h • · · · · · d reference to e . oug some specialized spectroscopic methods require the sample an when osmg simultaneously, computerization of modern instrument~ has made that less necessary, even) . ollected dual b · st b 1· spectrum is c eam m ruments. Typically, the spectrum of the reference (i.e., the ase me db the PMT first. The hardware and software of the instrument converts the electrical current generate . . y • d to absorbance values that are stored in a numerical array When the spectrum of the sample is obtain~ ; th

e absorbance values of the reference are subtracted fr~m the values of the sample. The final data t a is actually obser.,ed is this difference, assuming. that a baseline correction was performed.

Based on the explanation above, every ~pectrum represents.the difference between the sample ~nd reference, even if the absorbance of the reference IS zero at every wavelength. However, spectroscop 1 s1;s

iefine the type of spectrum based on the identity of the reference solution. An absolute spectrum is >btained when the reference does not contain the compound of interest, or anaiyte, in any chemical form. rhat is, the reference cuvette contains the same solvent in which the analyte is dissolved, but it does not :ontain the analyte itself. A difference spectrum is obtained by using a reference that is identical to the ample, then subjecting only the sample to a reaction of some kind. The difference spectrum generated 1y this type of experiment represents the absorbance of the mc,dified sample minus that of the '•modified sample, or Delta (d) Absorbance, as a function of wavelength. As we will see in this and future xperiments, both absolute and difference spectroscopy can be informative and are often complimentary.

uvettes absorb light of varying wavelengths. The degree to which a cuvette contributes to the total 3sorbance depends on the light-absorbing propertieS of the cuvette material as well as scattering tifacts. The net result is that the light that passes through the sample and cuvette, called transmitted 1ht, is less intense than the incident light, or the light before it passes through the sample and cuvette. tis gives rise to an apparent increase in absorbance (or decrease in transmittance). It is important to sess the suitability of a cuvette based on the specific wavelength(s) that will be used during the periment. The most common cuvette materials and theifrespective suitable spectral regions are:

• Polystyrene (PS/plastic): 340-S00nm • Optical Glass: 320-2500nm

• Methylacrylate (PMMA/plastic): 300-S00nm (notfor all applications) • BrandTech UV-transparent Disposable Cuvettes: 270-900+nm (not for all applications) • Spectrosil• Quartz: 170-2700nm

Fo_r many applications that use wavelengths greater than 340nm, disposable polystyrene cuvettes are well suited, particularly when using solutions that can permanently stain glass. However, for measuring the absorbance of proteins at 280nm or DNA at 260nm, one must use Spectrosit• quartz cuvettes, regardless of BrandTech's claims.

Generally, light scattering artifacts are not a serious concern when using standard 4.5ml, semi-micro 1.5ml, or even micro (variable volumes} 1cm ·cuvettes. However, scattering artifacts can become significant when using small volume (< 1ml} cuvettes. Scattering is more likely to occur if the incident beam is rather wide or if it is not precisely orthogonal to the face of the cuvette. Typically, this contribution can be cancelled by using carefully matched cuvettes, or using the same cuvette for the reference and sample solutions. When scattering is significant, masked cuvettes should be used instead. These cuvettes are completely black, with the exception of the sample compartment.

Lastly, high precision cuvettes are expensive and should be handled with care. Clean the faces with lens paper only, NEVER use a Kimwipe on an optical quartz cuvette, handle the cuvette gently when placing it in the instrument, and thoroughly with tap water, 1% Liquinox, tap water, then deionized or reverse osmosis water. Rinse with 95% ethanol and dry as instructed to prevent water spots. You will be using quartz cuvettes later on in Lab 8.