Chemistry Lab Report

Experiment 1 – quantitative measurements.

1. Loading samples.

You will be given a series of concentrations of fluorescein solutions in PBS buffer. The first set contains concentrations of 100μM, 50μM, 25μM, 12.5μM, 6.25μM, and PBS as a blank control. The second set contains the concentrations of 10μM, 5μM, 2.5μM, 1.25μM, 0.625μM, 0.3125μM, and PBS as a blank control. You will be given a 96 well plate for loading solutions (Figure 1).

You will be given two unknown samples labeled as “Low” and “High”. Their concentrations will fall in the range of 0 to 10 mM.

· -  Pipette 100 μL of each concentration from the first set in row A. Record the well number and corresponding concentration, e.g A2 – 10 μM, A3-5 μM, etc. Leave the first column (i.e. column 1) empty or filled with 75 μL of PBS buffer. 


· -  Rows B and C should be duplicates of what is filled in row A. 


· -  Pipette 100 μL of each concentration from the second set in rows D, E and F. 


· -  Now pipette 100 μL of one unknown sample in 3 empty wells in rows from A to F. For 
example, you select wells A9, B9, and C9. Perform the same for the other unknown sample. 
(Hint: The unknown concentration (labelled as “High”) may be in the order of mM, 
what would you do in order to measure it?) 


· -  Now take the solution of 0.625 μM fluorescein. Fill different volumes in row H as below: 25 μL- H12, 50 μL – H11, 100 μL - H10, 200 μL –H9, 200 μL -H8, PBS buffer- H7. Make a duplicate in the same row, i.e. 10 μL - H1, 25 μL – H2, 50 μL – H3, 100 μL – H4, 200 μL –H5, 200 μL –H6, PBS buffer- H7. 
Note: Generally, you need at least three duplicates for quantitative measurements. You should give the average number from the three measurements and report the standard deviation.
Try to pipette as accurate as possible. This will affect your results.
Don’t use the same tips if you change to another solution. 
Figure 1. a 96 well plate. 


2. Perform quantitative measurements

· -  Switch on the Microplate reader (if it is not on). 


· -  Double click “Gen 5” software, this brings up the interface window, Figure 2. 


· -  Click “Read now” in Figure 2, this brings up a new window, Figure 3. 


· -  Click “Synergy” and “OK” in Figure 3, this moves to Figure 4. 


· -  Click “Read” in Figure 4, this brings up “Read steps” window, Figure 5. 


· -  In the “Read step” window, you can now set up the parameters for the measurements. 


· -  The first measurement will be absorbance measurements. You can follow the settings listed in the window. [ Discuss in your report, why we choose 490 nm for the absorbance measurement?] 


· -  After you choose the parameters, Click “OK”, this brings up the new window for loading sample (Figure 6). 


· -  Now, load your well plate and click “Ok” after you have done this. 


· -  The instrument will perform the measurements. After it completes, it brings up a new 
window (Figure 7), asking “ Do you want to execute “PowerExport (Excel)” for “plate 1”? 
Click “yes”, it will save the data in excel. Otherwise, you will loose the data. 


· -  Now, repeat the steps from Figure 4, and change the setting in Figure 5 to perform “fluorescence measurements”. Note, choose the right filter set for fluorescein probe. 
Continue the rest steps as described above. 
Figure 2 Figure 3 
Figure 4 Figure 5 


·

Figure 6 Figure 7

3. Data analysis

You have now obtained both absorbance and fluorescence values of the solutions of known concentrations and of the unknown samples.

· -  Work out the relationship between absorbance and concentrations from the two sets of samples 


· -  Work out the relationship between fluorescence intensity and concentrations from the two sets of samples 


· -  Work out the relationship between absorbance and the volume of solutions (i.e the height of the solutions or the optical path) from the data in row H. 


· -  Work out the relationship between fluorescence and the volume of solutions (i.e the height of the solutions or the optical path) from the data in Row H. 


· -  Work out the concentration of the two unknown samples. 
What are the relationships (i.e curves) like for the two sets of samples? Discuss the difference between them in the report. 
How do the variations in the height of the solutions affect fluorescence and absorbance measurements? Are the influences the same? Discuss this in the report. 
What did you learn from the quantification of the two unknown samples? 


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The results

This is the result below that I had it in the lab from the devices that has been used in the lab.

Note: could you please answer the question that at the end of lab sheet in the discussion section.

figure 4 showing the rows that have each solution.

	1	2	3	4	5	6	7	8	9	10	11	12
A	118	OVRFLW	OVRFLW	OVRFLW	85410	52123	119	41	OVRFLW	OVRFLW	2707	120	Read 2:485/20,528/20
B	130	OVRFLW	OVRFLW	OVRFLW	84880	51287	120	43	OVRFLW	OVRFLW	2199	132	Read 2:485/20,528/20
C	126	OVRFLW	OVRFLW	OVRFLW	85015	51229	121	42	OVRFLW	OVRFLW	2208	131	Read 2:485/20,528/20
D	125	71890	43982	26617	14782	6515	3500	125	43	58072	41	42	Read 2:485/20,528/20
E	232	76154	44995	28439	14867	6579	3642	130	43	58151	43	43	Read 2:485/20,528/20
F	2869	77734	45964	27532	14702	6698	3586	129	43	57990	43	42	Read 2:485/20,528/20
G	449	1933	3763	5456	10750	11382	211	42	43	43	44	42	Read 2:485/20,528/20
H	924	2087	3292	5177	11343	10934	195	43	43	43	40	44	Read 2:485/20,528/20

	1	2	3	4	5	6	7	8	9	10	11	12
A	0.038	1.541	0.853	0.415	0.232	0.134	0.037	0.043	2.553	0.323	0.043	0.038	Read 1:490
B	0.038	1.482	0.848	0.414	0.237	0.139	0.038	0.044	2.542	0.323	0.042	0.039	Read 1:490
C	0.038	1.546	0.854	0.415	0.236	0.139	0.038	0.044	2.556	0.323	0.043	0.038	Read 1:490
D	0.038	0.18	0.12	0.082	0.061	0.048	0.041	0.04	0.046	0.165	0.046	0.045	Read 1:490
E	0.039	0.206	0.12	0.082	0.061	0.048	0.043	0.039	0.046	0.166	0.046	0.045	Read 1:490
F	0.038	0.2	0.12	0.079	0.059	0.047	0.043	0.037	0.043	0.165	0.046	0.045	Read 1:490
G	0.047	0.246	0.045	0.045	0.057	0.059	0.039	0.046	0.044	0.045	0.044	0.045	Read 1:490
H	0.109	0.596	0.041	0.044	0.057	0.057	0.038	0.045	0.045	0.043	0.045	0.045	Read 1:490

	1	2	3	4	5	6	7	8	9	10	11	12
A	3.633	3.736	3.686	3.655	3.657	3.657	3.647	3.648	OVRFLW	3.662	3.657	3.619	Read 1:260
B	3.619	3.722	3.679	3.673	3.663	3.665	3.663	3.617	OVRFLW	3.657	3.645	3.639	Read 1:260
C	3.638	3.727	3.662	3.65	3.661	3.664	3.674	3.627	OVRFLW	3.664	3.651	3.638	Read 1:260
D	3.622	3.659	3.664	3.646	3.643	3.657	3.63	3.663	3.629	3.673	3.632	3.632	Read 1:260
E	3.626	3.66	3.661	3.679	3.662	3.647	3.656	3.651	3.646	3.664	3.638	3.643	Read 1:260
F	3.636	3.656	3.651	3.631	3.65	3.64	3.651	3.645	3.646	3.663	3.624	3.623	Read 1:260
G	3.636	3.784	3.657	3.665	3.672	3.665	3.686	3.633	3.616	3.61	3.632	3.628	Read 1:260
H	3.601	3.934	3.632	3.646	3.688	3.665	3.659	3.625	3.64	3.632	3.628	3.62	Read 1:260