Laboratory Report #11

Quantitative Analysis of Urine Creatinine

Name: Quitalig, Aloysius M. Group No. 1 SCORE

Course, Year, Section: BS PSYCH 2-A

Instructor: Evelyn Enriquez Date: April 24, 2021

I. MATERIALS

Part A
Micropipette and tips 10% NaOH
Test tubes 1% Picric acid
Test tube rack Distilled H2O
Glass Pipette Urine sample
Vernier Spectrophotometer Standard Creatinine sample

II. PROCEDURE

1. Prepare 5 test tubes and add the following:

Test Tube 1: Unknown sample
50µL urine sample
Test Tube 2: Standard solution (0.4 mg/mL)
10µL standard solution + 40 µL dH2O
Test tube 3: Standard solution (1.0mg/mL)
25µL standard solution + 25µL dH2O
Test tube 4: Standard solution (2.0mg/mL)
50µL standard solution
Test tube 5: blank
50µL dH2O
2. Add 2mL picric acid to each of the test tubes.
3. Followed by the addition of 150 µL NaOH to each test tubes.
4. Mix and let it stand for 15 minutes.
5. Dilute each solution to 10mL (add 7.80mL dH2O) (Blank solution)
 Creatinine Standard Solutions Concentrations:
o 0.4 mg/mL
o 1.0 mg/mL
o 2.0 mg/mL
6. Transfer the solution to a cuvette and read the absorbance of a solution in a UV –
vis spectrophotometer at wavelength 520nm.
III. RESULTS AND DISCUSSION

Weight of patient: 59kg

Estimated total volume of urine in 24h: 1,500 mL

Concentration Corrected
Sample Absorbance at 520 nm
(mg/mL) Absorbance
blank 0 0.012 0.012
Standard solution 1 0.4 0.225 0.213
Standard solution 2 1.0 0.571 0.559
Standard solution 3 2.0 1.055 1.043
urine sample 0.1164 0.082 0.07

Concentration (mg/mL) Corrected Absorbance

blank 0 0.12
Standard solution 1 0.4 0.213
Standard solution 2 1.0 0.559
Standard solution 3 2.0 1.043
urine sample 0.1164 0.07

a. Equation of the line: y = mx + b

y = 0.5231057x + 0.0091101
b. Creatinine coefficient:

GIVEN:
m = 0.5231 𝑦−𝑏
𝑥= 𝑚
b = 0.00911 0.07 − 0.00911
y = 0.07 = 0.5231
R² = 0.9984 = 0.1164022175 ≈ 0.1164

Weight of patient = 59kg

Estimated total volume of urine in 24h = 1,500 mL
Urine concentration = 0.1164
Creatinine Coefficient Urine sample =
𝑚𝑔 𝑐𝑟𝑒𝑎𝑡𝑖𝑛𝑖𝑛𝑒 𝑝𝑒𝑟 𝑚𝐿 𝑢𝑟𝑖𝑛𝑒 ×𝑒𝑠𝑡𝑖𝑚𝑎𝑡𝑒𝑑 𝑣𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 24−ℎ𝑜𝑢𝑟 𝑢𝑟𝑖𝑛𝑒 𝑠𝑎𝑚𝑝𝑙𝑒
𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑝𝑎𝑡𝑖𝑒𝑛𝑡 𝑖𝑛 𝑘𝑔
𝒎𝒈
𝟎.𝟏𝟏𝟔𝟒 ×𝟏,𝟓𝟎𝟎 𝒎𝑳
𝒎𝑳
= 𝟓𝟗 𝒌𝒈

Creatinine coefficient = 2.9593 ≈ 2.96 mg/kg

c. Attach the screenshot of the calibration curve computed in the excel file

d. Discuss the spectrophotometer, Beer-Lamberts law

Spectrophotometer is an instrument that “counts” the number of
photons that enters a sample and compares it with the number of pre-existing
photons. It is also able to take white light and separate it into its constituent
colours which allows the user to examine the absorption of light of individual
wavelengths. The amount of a known chemical substance (concentrations)
can also be measured by this device. Its basic principle is that over a certain
range of wavelength, each compound can absorb or transmit light. UV-visible
and IR spectrophotometer are the two classifications.

Beer-Lambert’s Law (Beer’s Law) states that there is a linear

relationship between absorbance and the concentration of a sample, molar
absorption coefficient and optical coefficient of a solution:
𝐴 = 𝜀𝑐𝑙
𝐴 = 𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒
𝜀 = 𝑀𝑜𝑙𝑎𝑟 𝑎𝑏𝑠𝑜𝑟𝑝𝑡𝑖𝑜𝑛 𝑐𝑜𝑒𝑓𝑓𝑖𝑐𝑖𝑒𝑛𝑡 = 𝑀
𝑐 = 𝑀𝑜𝑙𝑎𝑟 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 = 𝑀−1 𝑐𝑚−1
 𝑙 = 𝑂𝑝𝑡𝑖𝑐𝑎𝑙 𝑝𝑎𝑡ℎ 𝑙𝑒𝑛𝑔𝑡ℎ = 𝑐𝑚
The law enables to concertation of a solution by measuring its absorbance to
be calculated. The sample dependent property, Molar absorption coefficient,
is a measure of how strong an absorber the sample is at a certain wavelength
of light. The moles L-1 (M) of the sample dissolved in the solution is the
concentration in question for this formula. As for the length, it is the length of
the cuvette used in absorbance measurement. Usually this exists at 1cm.

e. Show the calculation for the creatinine in the urine sample

𝑦−𝑏
𝑋=
𝑚
0.07 − 0.00911
𝑋=
0.5321
0.06089
𝑋=
0.5231
𝑚𝑔 𝑐𝑟𝑒𝑎𝑡𝑖𝑛𝑖𝑛𝑒
𝑋 = 0.1164022175
𝑚𝐿 𝑢𝑟𝑖𝑛𝑒
IV. POST LAB QUESTION

1) What are the sources of creatinine excreted in the urine?

Creatinine is a waste product of the body that is derived from muscle creatine.
It is a nonprotein nitrogenous substance that the body produces daily during
muscle movements and when digesting meat. It moves through the body water
slower than urea. As for its transport path throughout the body, it is filtered
through the kidneys and excreted in urine. Higher creatinine levels than normal
may be indicative of diabetes or kidney problems.

2) Why is creatinine excretion used to estimate the completeness of a 24-hour urine

collection and as a basis for normalizing urinary concentrations of various
metabolites and estimating the lean body mass of individuals?

The clearance rate of creatinine is measured in amount per unit time, which is
why a 24-hour urine sample test is asked in order to determine the amount of
creatinine that the human kidney is clearing. Medical professionals are able to
make conclusions if one’s metabolism and urinary excretion is normal or not
through identifying the amount of creatinine that the individual's kidney could
release.

3) How is creatinine excretion affected by dietary meat intake?

Creatinine is a product of our daily muscle activities and digestion of proteins,

specifically meat. Thus, a meat intake has an effect in our creatinine excretion.
The more we consume meat the more protein our body digests, yielding a large
amount of creatinine to excrete.

4) What are the other uses of measuring creatinine excretion?

Measuring creatinine cannot only determine if the renal of an individual is

functioning normally or not but it can also be used in determining the normality of
the blood. If creatinine products are left in the blood and are determined when
tested, this could be another indicator that there is something wrong in the
functioning of the kidneys of an individual.
 V. REFERENCES

Module 12: Quantitative Analysis of Urine Creatinine. (n.d.). NEO LMS. Retrieved

April 20, 2021, from

https://usa.neolms.com/?from=%2Fstudent_team_assignment%2Fshow%2F2

1034506&log_in_required=true

University of San Agustin - CLASE - Department of Chemistry and Physics. (n.d.).

Quantitative Analysis of Urine Creatinine Video. NEO LMS. Retrieved April

20, 2021, from

https://usa.neolms.com/?from=%2Fstudent_lesson%2Fshow%2F2460997%3

Flesson_id%3D10878204%26section_id%3D37711993&log_in_required=true

IUPAC, Compendium of Chemical Terminology, 2nd ed. (the “Gold Book”); Compiled
by A. D. McNaught and A. Wilkinson. Blackwell Scientific Publications (1997)

Edinburgh Instruments Ltd. (2021, April 15). Beer Lambert Law | Transmittance &

Absorbance. Edinburgh Instruments. https://www.edinst.com/blog/the-beer-

lambert-

law/#:%7E:text=The%20Beer%2DLambert%20law%20states,calculated%20b

y%20measuring%20its%20absorbance.

Libretexts. (2020, August 15). 2.1.5: Spectrophotometry. Chemistry LibreTexts.

https://chem.libretexts.org/Bookshelves/Physical_and_Theoretical_Chemistry

_Textbook_Maps/Supplemental_Modules_(Physical_and_Theoretical_Chemi

stry)/Kinetics/02%3A_Reaction_Rates/2.01%3A_Experimental_Determination

_of_Kinetics/2.1.05%3A_Spectrophotometry