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CHEM 241L

Terms in this set (186)

Exp 2 objectives
-perform a slab electrophoresis on a thin membrane
-stain a protein sample on an electrophoresis membrane
-calculate Bovine Serum Albumin isoelectric point using migration distances at different pH values
Which solution is corrosive and a potential safety hazard in this lab? (Exp 2)

The trichloroacetic acid staining solution.
The basic (pH 8.00) buffer solution
The acetic acid solution
The acidic (pH 4.00) buffer solution
The trichloroacetic acid staining solution (Ponceau stain)
True/False: If you have 3 strips in one electrophoresis tray connected to a power supply, the current should be set to 12 mA. (Exp 2)
False- should be set to 3mA/strip
In this experiment you will allow the migration to proceed for exactly ____ . (Exp 2)

A. 20 min
B. 40 min
C. 10 min
D. 35 min
E. 15 min
F. 25 min
G. 45 min
H. 30 min
45 minutes
Electrophoresis is useful for determining the _____________ of the sample. (Exp 2)

A. polarity
B. molecular charge
C. affinity for the stationary phase
D. pKa
E. solubility
molecular charge
A molecule will migrate toward the anode if it has a ____ charge. (Exp 2)
negative
Calculate the isoelectric point for Protein B, which has two ionizable protons (pKa1 = 3.9 and pKa2 = 7.7). (Exp 2)
pI = (3.9+7.7)/2 = 5.8
Electric field is mathematically defined as: (Exp 2)

A. V = E/d
B. E = V x d
C. V = E x d
D. E = V/d
E=V/d
(Exp 2) slab/membrane electrophoresis
-uses thin, flat membrane strips whose ends are immersed in electrolyte buffer, which can be pH specific
-sample is injected onto sample origin, or center of strip
-provides power source that sends current through strips
-mobility and migratory direction of analyte is proportional to its charge, electric field strength, and frictional coefficient
(Exp 2) capillary electrophoresis
-applies analyte onto capillary tube, usually fused with silica, with ends in electrolyte buffer
-this version has high resolution because there is no stationary phase and it eliminates time it takes for analyte to equilibrate between mobile and stationary phases
-sample is injected by placing pressurizing capillary in sample vial
-provides power source that sends current through strips
-mobility and migratory direction of analyte is proportional to its charge, electric field strength, and frictional coefficient
(Exp 2) electrophoretic mobility (µ)
µ=q/f
q=total charge
f=frictional coefficient

µ isn't included in the equation sheet
(Exp 2) positively charged molecule migrates to
cathode, which has a negative charge

molecule's distance will be positive
(Exp 2) negatively charged molecules migrates to
anode, which has a positive charge

molecule's distance will be negative
(Exp 2) larger molecules will travel
slower and shorter distances
(Exp 2) smaller molecules will travel
faster and longer distances
(Exp 2) isoelectric point
the pH that leads to electric neutrality, or net charge of zero, and leads to no migration

(pI=pKa1+pKa2/2)
the two pKas are those pH values between which the molecule has a net charge is zero
check out this link if that's confusing! http://www.chem.ucalgary.ca/courses/351/Carey5th/Ch27/ch27-1-4.html
(Exp 2) application of electrophoresis other than determining isoelectric point
separation of charged molecules as they will migrate in different directions at different rates
(Exp 2) Can you perform the same calculation with a single molecule of BSA?
No, BSA is too big so you can't perform the same calculation that you did with aspartic acid
(Exp 2) If we looked at a solution of aspartic acid instead of a single molecule, would there be any difference in calculating the pI?
No, because aspartic acid wouldn't have changing pKas.
(Exp 2) electrophoresis setup
-strips placed in buffer filled tray glossy side up and are left to soak for 10 minutes
-remove one strip at a time and apply BSA to sample origin (remember only to apply a bead onto the applicator wire and not to press too hard or strip will tear)
-lay strips on brown bridge with ends in buffer, make sure to note which side is positive and negative
-cover the tray before turning on power supply
(Exp 2) power supply settings
-voltage: 300V
-current: 3mA/strip
-time: 45 minutes
(Exp 2) staining BSA
move strips in this order:
1. Ponceau stain (leave for 5 minutes)
2. 5% acetic acid
3. D.I. water
(Exp 2) measuring migration
measure in millimeters (mm)
if the stain moved to the (+) end, or the anode, then distance is NEGATIVE, the molec is negative.
if the stain moved to the (-) end, or the cathode, then distance is POSITIVE, the molec is positive.
(Exp 2) how was the isoelectric point found?
-electrophoresis with BSA was run in various pH buffers (4.0, 4.5, 5.0, 6.0, 7.0)
-when BSA was run in pH buffers more acidic than 5.0 (like 4.0, 4.5) the molecule migrated towards the (-) end or cathode, meaning it had a NET positive charge
-when BSA was run in pH buffers more basic than 5.0 (like 6.0, 7.0) the molevule migrated towards the (+) end or anode, meaning it had a NET negative charge
-at pH of 5.0, half the trial showed movement to (+) and half to (-); average movement was about zero indicating that the pI was close to a value of 5.0
(Exp 2) literature versus calculated pI value
literature: 4.70
calculated: 4.98
(Exp 2) how was the experiment pI calculated
-average distance migrated v. pH was graphed
-the points had a parabolic relationship and the three points creating the parabola were used to create a parabolic equation (x=0.217y2-0.731y+4.98)
-since the y was distance migrated and we know that pI occurs with zero observed migration, by plugging in y=0, the x would be the pI value
(Exp 2) what happened when the buffer was 4.0 v 4.5 or when it was 7.0 v 6.0?
-buffer of 4.0 resulted in larger migration (+d)
-buffer of 7.0 resulted in larger migration (-d)
-this shows that the net charge got larger as you moved farther from pI value
(Exp 2 EOL) Can you separate a mixture of the following three proteins using membrane electrophoresis?
Protein A has a pI > pH of the run buffer, Protein B has a pI < the pH of the run buffer, and Protein
C has a pI = pH of the run buffer. If so, describe the movement of each protein.
-Membrane electrophoresis can separate the three proteins in the mixture
-protein A pI > buffer pH: would have a net positive charge and migrate to cathode end (-)
-protein B pI < buffer pH: would have a net negative charge and migrate to anode end (+)
-protein C pI = buffer pH: would have a net neutral charge and migrate zero
-three proteins --> display three different bands migrating in varied directions
-could determine which band and protein corresponded based on direction of migration
(Exp 2 EOL) Based on the chromatographic methods you learned this semester, state which separation
technique would be the best choice for:

Separation of volatile components found in a perfume and that result in its characteristic
fragrance.
Gas Chromatography
-in GC, analyte is a volatile liquid or gas sample -mobile phase is a carrier gas
-stationary phase is usually a nonvolatile liquid bound to a solid support
-the volatile analyte can pass through this nonvolatile phase and its retention times, displayed in chromatographs, will separate the different components, i.e. one compound in perfume from another
(Exp 2 EOL) Based on the chromatographic methods you learned this semester, state which separation
technique would be the best choice for:

The determination of molecular weight of a polymer that was synthesized by a friend in a polymer synthesis lab.
Size Exclusion
-can be used to separate molecules of based on molecular weight
-column can be calibrated using molecules of KNOWN weight and the retention times (tr) from these molecules can be used to create a calibration curve of the logarithmic molecular weight (log(MW)) versus retention times (tr)
-then, the unknown analyte is run through the column
-its retention time can be substituted into the linear regression of the calibration plot of log(MW) v tr to find its estimated molecular weight
(Exp 2 EOL) Based on the chromatographic methods you learned this semester, state which separation technique would be the best choice for:

The separation of nonvolatile pollutants found in drinking water.
Gel Electrophoresis
-technique separates molecules by charge and size
-applying a voltage through strip or tube creates a cathode (-) and anode (+)
-if a water sample is placed onto strip, it would separate components of sample and one could identify contaminant pollutants based on their migration distances
(Exp 2 EOL) Based on the chromatographic methods you learned this semester, state which separation
technique would be the best choice for:

Separation of two similarly sized amino acids.
Liquid Chromatography
-the amino acids, although similar in size will likely have R groups with different properties (for example one might be polar and the other nonpolar)
-the mobile phases used can be personalized to each analyte in LC
-using reverse liquid chromatography, which uses nonpolar stationary phase, one could elute the polar amino acid with a polar mobile phase and the nonpolar amino acid with a nonpolar mobile phase
(Exp 2 EOL) Based on the chromatographic methods you learned this semester, state which separation
technique would be the best choice for:

The separation of Albumin and Gamma-Globulin
Size-Exclusion
-Albumin has a molecular weight of 66.4 kDa
-Gamma-Globulin's molecular weight ranges from 155 to 160 kDa
-using LC, a Sephadex G-75 stationary phase could be used (range from 3,000 Da to 80,000 Da)
-Albumin falls in this range and would travel through the bead, while Gamma-Globulin would travel around it
-Gamma-Globulin would elute first and Albumin would elute second, allowing one to separate the two into different tubes
(Exp 2 EOL) Based on the chromatographic methods you learned this semester, state which separation
technique would be the best choice for:

Separation of a mixture of elicit drugs.
Liquid Chromatography
-pharmaceutical companies often use liquid chromatography to separate similar drugs, especially those which are enantiomers or similar in structure to each other
-for example, naproxen has two enantiomers that each has a different effect
-using a (S,S) stationary phase:
the (R) enantiomers would be eluted first because it has a weak binding to the stationary phase
the (S) enantiomer second due to stronger binding
-such qualities in drugs can be exploited for separations
(Exp 2 EOL) In this lab you used Ponceau stain to identify the protein band on the strip. Research and present
at least 3 other methods scientists use to detect proteins in membrane or gel electrophoresis
I think we can ignore this questions bc not all labs had to know it.

1. Radioactive Stains
-can be applied to gel after electrophoresis is ran and observing which molecules were bound using a "gamma scintillation counting technique or radioautography"
-one technique to make stain is to saturate normal stain with thermal neutrons
-this staining method identifies and binds to proteins

*The next two detection methods can be specifically use SDS PAGE

2. Metal Ions
-metal ions, such as Zinc ions, can visualize SDS PAGE results
-the Zinc metal precipitate is applied to gel, turning bound proteins opaque

3. Antibodies
-antibodies, including primary and secondary, can visualize specific proteins run or present in SDS PAGE
-primary antibody will be applied, followed by a secondary antibody that emits fluorescence when bound to primary ab
-photodetector is then used to visualize fluorescence

summary of SDS PAGE: 1) insert analyte into wells onto polyacrylamide gel 2) add SDS 3) transfer onto nitrocellulose paper 4) add primary and secondary antibodies, and observe through photodetector
(Exp 2 EOL) Compare and contrast membrane electrophoresis and capillary electrophoresis. What are the similarities and what are the differences.
Membrane Electrophoresis:
-thin, flat membrane strips, ends are immersed in electrolyte buffer
-sample is injected onto sample origin, or center of strip

Capillary Electrophoresis
-applies analyte onto capillary tube (usually fused with silica), ends in electrolyte buffer
-this version has high resolution because there is no stationary phase and it eliminates time it takes for analyte to equilibrate between mobile and stationary phases
-sample is injected by placing pressurizing capillary in sample vial

Both:
-place electrodes in buffer reservoirs and supply a voltage to each end, creating a current.
-mobility and migratory direction of analyte is proportional to its charge (q), electric field strength (E), and frictional coefficient (f) (remember that rate (u)=(q/f)*E
(Exp 2 EOL) Research and provide one pertinent clinical analysis example (be specific) as to how cellulose
polyacetate electrophoresis is used.
-cellulose polyacetate electrophoresis is used to separate and detect serum proteins (proteins in blood plasma)
-electrophoresis with this membrane provides a simple technique that is easily executed and reproducible
-these qualities are significant in making clinical assays, employed in areas with less access to health resources
-provides clear results without a complicated method
-a specific clinical analysis example is detection of Hb Bart, abnormal hemoglobin accumulating in erythrocytes, at birth
-ideally, "alpha-genotyping" is used for detection, BUT in countries with limited access this simple screening suffices
(Exp 2 EOL) A protein with a net negative charge migrates 3.2 cm in 45 min when 150. V is applied to a 10.0
cm long electrophoresis strip
a. calculate electric field strength
b. calculate migration rate of the protein
c. calculate the mobility of the protein
a.
E=V/d
E=(150V)/((10 E-2 m) **Note: d is the strip length not migr. d
E=1.5E3 V/m

b.
u=d/time
u=(3.2E-2 m)/(45min×(60 sec)/(1 min))
u=1.2E-5 m/s OR 1.2E-8 mm/s

c.
u=μ×E
(1.2E-5 m/s)=(μ)×(1.5E3V/m)
μ=8.0E-9 m^2/V∙s
(Exp 2 EOL) Gas Chromatography
-Analyte: volatile compounds
-Mobile Phase: carrier gas
-Stationary Phase: non-volatile liquid bound to solid
-Detection: absorbance values against time from chromatograph at set wavelength
*Note: the LOWER the boiling point, the higher the vapor pressure is, the FASTER the molecule will be eluted because it will be in gas phase faster
-Function: can separate mixture of compounds based on differential qualities; separates different compounds as a function of time or elution order
(Exp 2 EOL) Liquid Chromatography (Reverse)
-Mobile Phase: customizable (nonpolar, polar, chiral, etc.)
-Stationary Phase: nonpolar solvent
-Detection: spectrophotometer (absorbance), mass spectrometer; the detection method is dependent on differential qualities of analyte that you're analyzing
-Function: can separate mixture of compounds based on differential qualities; separates different compounds as a function of time or elution order
-*Can personalize with mobile phase to separate analytes based on qualities (polar MP, etc.)
(Exp 2 EOL) Electrophoresis
-Analyte: mixture or sample of compound(s) with different charges and/or sizes
-Mobile Phase: NONE
Run on: column, tube, or strip
-Stationary Phase: strip (unless capillary electro- then it doesn't have a stationary phase either)
-Detection: after running current through, strip is stained and distance traveled from origin is measured
-Function: can separate mixture of compounds based on differential qualities; separation of analytes is weight dependent
(Exp 2 EOL) Size-Exclusion Chromatography
-Analyte: mixture or sample of compound(s) with distinct MW
-Mobile Phase: buffer
-Stationary Phase: beads/pores
-Detection: retention time of each analyte (elution time)
-Function: can separate mixture of compounds based on differential qualities; separation of analytes is weight dependent
-*Can personalize stationary phase to separate certain molecules based on size
(EXP 3) Objectives
- Determine the pKa of a compound
- Construct the absorption spectra using a UV-VIS spectrophotometer
-Use Beer's Law to calculate molar absorptivity and concentration
-Perform a solvent-solvent extraction to qualitatively analyze solubility.
EXP 3 Technique
Used UV-Vis spectrophotometer to measure absorbance
(EXP 3) T or F: Ethyl acetate is flammable so needs to be handled with caution.
TRUE
(EXP 3) In today's lab experiment you will construct what type of plot to determine the pKa of bromothymol blue?
Derivative Plot
(EXP 3) The main goals of todays experiment are (select all that apply)
- To determine the wavelength of maximum absorbance of the basic form of bromothymol blue.
-To determine the pKa of bromothymol blue.
-To determine the wavelength of maximum absorbance of the acidic form of bromothymol blue.
(EXP 3) The part of a molecule that absorbs light from the spectrophotometer is the:
chromophore
(EXP 3) Why is it important to chose the wavelength of maximum absorption for a spectrophotometric analysis?
Both A and B
A. This is the wavelength that will give the maximum response for a given concentration of analyte.
B. At this wavelength, there will be minimal variations from any monochrometer shifts because it is a semi-flat region of the spectra.
(EXP 3) Calculate the molar absorptivity of bromothymol blue at 620 nm if the percent transmittance (% T) is determined to be 15.1% at this wavelength. The pathlength of the cuvet is 10.0 mm and the concentration of bromothymol blue solution is 5.00 x 10-5 M.
1.65 x 104 M-1 cm-1
(EXP 3) If the Ka of a weak acid is 6.6 x 10-5, what is the pKa?
4.2 or 4.18
EXP 3 Safety:
Use secondary containment. Ethyl acetate is flammable.
EXP 3 Goal:
Determine pKa of bromothymol blue, a common acid-base indicator.
EXP 3 Key Terms:
- Absorbance
-Spectrum
-Beer's Law
- A= log (I0/I)
I0 is the irradiance of the light measured in the absence of an absorbing solution, I is the irradiance of the light measured after it passes through a solution of interest. In general measure absorbance b/w 0 and 2.
-a plot of absorbance vs. wavelength
- A=ebC
EXP 3 Transmittance converted to Absorbance
A= log (I0/I)= -log T
EXP 3 Graphs
- abs vs. wavelength spectrum fro bromothymol blue in its acidic (pH 5.00) and basic (10.00) forms
- absorbance vs pH graph with values for each pH (5.00, 6.00, 7.00, 7.30, 7.60, 8.00, 9.00, 10.00)
at inflection point [HIn-]=[In-] and pH=pKa
- dA/dpH vs. avg pH plot where max in first derivative plot is pKa of bromothymol blue
EXP 3 Experimental Parameters
Measure mode: ABS
Wavelength Range: 800nm-340nm
Rec. Range: -0.10A to +1.70A
Scan Speed: Fast
No. of scans: 1
Display mode: Sequential
position cuvet so that light passes through clear side.
EXP 3 Procedure
-First two test tubes:
1. 2mL of stock bromothymol blue solution and 1mL of pH 10.00 buffer
2. 2mL of stock bromothymol blue soln and 1ml of pH 5.00 buffer
-Next 6 test tubes:
In each added 2mL of bromothymol blue soln and 1 mL of corresponding buffer (6.00, 7.00, 7.30, 7.60, 8.00, 9.00)
-Last 2 test tubes:
In each added 2mL of bromothymol blue soln and 1ml of ethyl acetate. In first added 1mL of pH of 10.00 buffer. In second added 1mL of pH 5.00 buffer. Once in UV-Vis room, let these two test tubes sit undisturbed so layers could separate.

For first 2 test tubes, recorded absorbance values in 10nm increments from 340nm-800nm, and recorded peak wavelength. pH of 5.00 had one peak, while pH of 1.00 had 2 peaks.
For next 6 test tubes, recorded absorbance at wavelength at which In- absorbs maximally
For last 2 test tubes: in pH 5.00 buffer aqueous layer was on bottom while in pH 10.00 buffer, aqueous layer was on top. Extracted aqueous layer for each and measured absorbance at max wavelength. For pH 10.00 buffer, measure at max wavelength for In-, and pH 5.00 buffer, measure at max wavelength for HIn.
EXP 3 Discussion Main points
the acidic form (pH 5.00) of bromothymol blue was more soluble in the organic species. The basic form (pH 10.00) of bromothymol blue was more soluble in aqueous species.
EXP 4 Objectives
- Use a glass pH electrode to measure pH of solutions
-Select a conjugate acid/base pair to prepare a buffer at a specific pH
-Prepare buffer solutions using two methods.
-Analyze buffer capacity and understand its significance.
(EXP 4) T or F: Buffer capacity is at a maximum when pH = pKa
TRUE
(EXP 4) In today's lab you will measure the change in pH of each of the prepared buffer solutions by adding which of the following drop-wise?
B. 1.0 M NaOH and 1.0 M HCl
(EXP 4) Calculate the pH of a solution in which [OH-] = 2.5 x 10-4 M. pH = 4
10.4 or 10.40
(EXP 4) In HF+ H20 --> H30+ + F-
____ is an acid and ____ is its conjugate base.
HF or H3O+, and F-or H2O
(EXP 4) Which of the following combinations would be best to buffer the pH to 9.00?
NH4+ and NH3 (pKa = 9.24)
EXP 4 POST LAB WORKSHEET QUESTIONS
...
Buffer capacity. Calculate the change in pH (ΔpH) for each buffer you prepared after the addition of NaOH and HCl. The initial pH is the pH of the solution before any added NaOH or HCl.
Equation used for the calculations:
Buffer capacity=((#moles of OH- or H3O+ added)/((change in pH)(volume of buffer in L)))
Did the two methods used in this experiment produce buffer solutions with the same measured pH?
No, the two methods (theoretical and practical) used in this experiment produced buffer solutions with different pH's.
Discuss reasons why your measured pH values would be different from the target pH. Discuss other possible sources of error.
Reasons for the difference primarily include "activity coefficients not being considered" [Harris 2010], but also errors in measuring like contamination of one sample to another by not rinsing the electrode in between trials or uncertainty in certain factors like the stock concentration on the bottles having an approximate concentration that isn't the exact concentration of the strong acid or strong base added. Basically, deviations from ideal conditions could all cause differences between these values, and in this experiment we just assumed everything is under the ideal conditions but didn't make 100% sure that everything was ideal.
Materials and Methods Section write-up. (These are specific to what I did but I tried to generalize it as much as possible, basically each pair was assigned a pH and we had to choose two reagents that were within 1 pH unit of our assigned pH to create the desired buffer solution)
Used PASCO SW 500 interface box to obtain pH values. Calibrated the pH electrode using 2 standard system (pH of 4.00 and 8.00). Recorded pH values to 2 decimal places.
-Made the theoretical buffer by adding 6.50 mL of CH3CO2H (or your first reagent) and 0.240 grams of CH3CO2Na *3H2O (your second reagent) to a volumetric flask and diluted to the line marked on the flask with DI water and mixed the contents. -Made the practical buffer by adding 10 mL of CH3CO2H to a beaker then diluted with 80 mL of water and mixed contents. Rinsed, and dabbed dry the electrode, then placed it in practical buffer. Drops of 0.5 M NaOH were added using a disposable pipet until a pH close to 4.5 (to your assigned pH) was reached.
-Two 100 mL beakers were set up: one labeled theoretical, other labeled practical. A sample of 25 mL of theoretical buffer was obtained and 2-4 drops of 1.0 M HCl were added. Once close to changing the pH by 1 pH unit, drops were added by 1 drop increments. All increments and corresponding pH values were recorded. Stopped once the pH had changed by 1 pH unit. These steps were repeated for the practical buffer.
Obtained another 25mL of theoretical buffer and added 2-4 drops of 1.0 M NaOH using a pipet and record the pH alongside how many drops were added. Once close to changing pH by 1 pH unit, added drops in 1 drop increments, and stopped once the pH had changed fully by 1 pH unit. These steps were repeated for the practical buffer.
EXP 4 Goal
Prepare 2 buffers (practical and theoretical) to a pH of 4.5. Then, test the effectiveness of each buffer by seeing which can resist pH changes more.
EXP 4 Experimental Parameters
Set sampling rate to 2.00s
EXP 4 Calibrating electrode
-calibrate to 2 point system,
-calibrate to first point with a pH solution of 4.00 and then to second point with a pH solution of 8.00
(EXP 4) Match each of the following with bases with the correct conjugate acid (A. Cl-, B. NO3-, C.NH3)
1. HCl
2. HNO3
3. NH4+
4. H2Cl
5.NH2-
1. A
2. B
3. C
4. NONE OF THE ABOVE
5. C
(EXP 4) Before you can make any pH measurements, you must calibrate the pH electrode using standard buffer solutions. In today's lab you will use which of the following standards to calibrate the pH electrode?
A. 4.00 and 8.00
EXP 4 EQUATIONS
-pH=pKa +log ([A-]/[HA])
- know how to go from moles to grams
Exp 5 objectives
-perform a chromatographic separation utilizing size exclusion chromatography
-analyze proteins based on retention volume
-calculate the distribution coefficient for four proteins
Where should you place your backpack when you come to lab? (exp 5)
A. In your lab drawer.
B. In the cabinets under the white board labeled for backpack storage.
C. On the ledge above your lab bench.
D. On the floor next to your lab drawer.
E. There is no designated area for backpacks.
In the cabinets under the white board labeled for backpack storage.
What do you do with the phosphate buffer in today's lab? (exp 5)
A. Pour it into the liquid waste container in the hood.
B. Pour it back in the container for re-use.
C. Pour it down the sink and rinse down the drain with running water.
D. Neutralize with sodium bicarbonate and pour into the liquid waste containe
C. Pour it down the sink and rinse down the drain with running water.
Which compound will elute from the column first, Dextran Blue (2 x 10^6 Da) or Phenol Red (354.38 g/mol)? (exp 5)
Dextran Blue
In Gel Permeation Chromatography, small (low molecular weight) molecules will pass through the column _______. (exp 5)

A. more slowly than large (high molecular weight) molecules
B. more quickly than large (high molecular weight) molecules.
C. unaffected by the stationary phase material.
D. Both A and C.
A. more slowly than large (high molecular weight) molecules
The G-75 stationary phase has a fractionation range of 3,000 - 80,000. A solution containing 4 proteins (each MW is listed below) is introduced to the top of this column and allowed to pass through to separate the proteins.
Protein A: 6,000 Da
Protein B: 2,000 Da
Protein C: 60,000 Da
Protein D: 29,000 Da

Given the above proteins, what is the correct elution order (from 1st to last) that would be observed? (exp 5)

A. Second
B. First
C. Third
D. Fourth
B 1. Protein C
A 2. Protein D
C 3. Protein A
D 4. Protein B
In SEC theory, Vm is equal to what? (exp 5)
A.
Vm = 0 when the molecule is smaller than the fractionation range of the gel stationary phase.

B.
Vm = Vi when the molecule is smaller than the fractionation range of the gel phase.

C.
Vm = Vo when the molecule is larger than the fractionation range of the gel stationary phase.

D.
Vm = Vo + Vi
D. Vm = Vo + Vi
If the retention volume is 53 mL, the void volume is 14 mL and Vi = 11 mL, the partition coefficient (K) = ? (exp 5)

Hint K = (Vr - V0) / Vi
3.55|3.5
(Exp 5) Size Exclusion Chromatography (SEC)
-form of liquid chromatography that utilizes a gel stationary phase to separate molecules according to their molecular sizes in solution
-if the mobile phase is aqueous, then the technique is gel-filtration
-if the mobile phase is organic, then the technique is gel-permeation chromatography
(Exp 5) In SEC, large molecules
Cannot penetrate the small pores of the gel phase and are thus excluded
(Exp 5) In SEC, smaller molecules
Penetrate into the interstices the Sephadex gel phase and are retained
(Exp 5) Distribution coefficient
K = (Vt - Vo) / (Vm - Vo), where
Vt = elution volume of a molecule larger than gel pore sizes
Vo = void volume
Vm = Vo + Vi
Vi = internal pore volume of gel
(Exp 5 EOL) 1. What would be a different way by which you could determine the retention volume for the proteins and molecules analyzed in today's lab, besides visually looking for the most intense fraction? Explain why your alternative approach would work for the experiment you just ran.
1. Besides visually looking for the most intense fraction, spectroscopy is a viable option in order to determine the retention volume for the proteins and molecules analyzed in this lab. Spectroscopy leads to more accurate data because the data can be recorded more often. This approach would work for the experiment because it would be almost the same, the main difference being having to keep an eye on the volume of solution that has run through the column, so a graduated cylinder would be useful.
(Exp 5 EOL) 2. Based on your experimental data and observations of the separations you performed, and how you determined your retention volumes, could you have run one separation using a solution that contained phenol red, Dextran Blue, Cytochrome c, and Hemoglobin and determined their retention volumes? Explain why or why not.
2. One separation would not have worked for all four of these solutions because the retention values are so similar; cytochromes c and phenol red were only four mL different in terms of elution volumes. It would be hard to tell where one compound is most intense because the colors would be mixed in. Therefore, the experiment broke the separation up into two separations in order make each separation clearer.
(Exp 5 EOL) 3. The below graph was constructed from size exclusion chromatography data. A newly isolated protein was run through the same size exclusion column as the compounds that were used to construct the below graph, and this protein was found to have a retention volume of 13.5 mL. Calculate Kav value for this protein. Show all your work.
3. Kav = (Vr - Vo) / (Vt - Vo) = (13.5 - 11.0) / (16.0 - 11.0) = 2.5 / 5.0 = 0.5
(Exp 5 EOL) 4. In the plot shown in Question 3, explain why Proteins A, B, and C all elute at Vr = 11 mL even though they all have different molecular weights? Was a mistake made in the experiment?
4. Proteins A, B, and C must have molecular weights that are greater than the fractionation range of the gel. Each protein may have different molecular weights, but they just happen to all be outside the fractionation range. Thus, an error was not made in the experiment.
(Exp 1 p1) Objectives
-create standard calibration and addition plots
(Exp 1 p1) gas chromatography
-stationary phase: Carbowax- strongly polar
interacts with polar molecules
interacts with nonpolar molecules via dipole-dipole interactions
-mobile phase: carrier gas
-molecules distribute between the stationary and mobile phases
(Exp 1 p1) calibration method
-use known solutions (standards) with 2%, 4%, 6% ethanol in pentanol
-run through GC to get chromatogram
-find average area under peak for each solution type
-plot "ethanol peak area v. % ethanol"
(Exp 1 p1) equipment
-use graduated 1.0 mL glass pasteur pipet
-use three valve bulb (A=air remove, S=suck it in, E=eject it out)
-use 10 mL volumetric flask (remember the bottom meniscus at the line is 10 mL)- KEEP THE FLASK CAPPED because the solution is volatile
(Exp 1 p1) GC settings
He pressure= 30-40 psi
He flow rate= 16-20 mL/min
Injector/Detector temp= 170 C
Column temp= 120 C
Filament current 140 mA
Column length= 5 ft
Attenuation= 1
(Exp 1 p1) digital flow meter
-hold on/off button until you hear clicking
-attach tubing to filter and hold
(Exp 1 p1) chromatogram
-peak 1: ethanol (if small peak is present after this, it is from water)
-peak 2: pentanol
-graphs: voltage against time
(Exp 1 p1) syringe
-2.5uL
-clean with 100% pentanol
-don't bend plunger
-make sure there aren't any bubbles
-depress syringe and immediately remove
(Exp 1 p1) extraction sample
-aqueous layer (bottom): water
-organic layer (top): pentanol

*during extraction trials you take from the organic/top layer of pentanol
(Exp 1 p1) When obtaining reagents from the main lab hood for your experiment, which of the following indicated the proper technique?
Pour a small amount into a beaker or flask, cover and bring it back to your lab bench.
(Exp 1 p1) You are instructed to make a 2% ethanol in pentanol solution using a 10 mL volumetric flask. What are the correct steps to take (select all correct steps - NOTE the steps are not necessarily listed in the correct order).

A. Measure out 0.2 mL of ethanol and add to the 10 mL volumetric flask.
B. After adding the ethanol to the 10 mL flask, fill the flask with pentanol to the mark.
C. After adding the ethanol to the 10 mL flask, add a small amount of pentanol to the flask and swirl to mix the solution. Then add pentanol to fill up to the mark.
D. Measure out 9.8 mL of pentanol and add to the 10 mL volumetric
A. Measure out 0.2 mL of ethanol and add to the 10 mL volumetric flask.
C. After adding the ethanol to the 10 mL flask, add a small amount of pentanol to the flask and swirl to mix the solution. Then add pentanol to fill up to the mark.
(Exp 1 p1) In today's experiment you will make how many injections?

A. 4 if you have time, otherwise at least three of each solution.
B. 2 if you have time, otherwise at least one of each solution.
C. 1
D. 3 if you have time, otherwise at lease two of each solution.
E. As many as you can fit in during the lab period.
E. 3 if you have time, otherwise at lease two of each solution.
(Exp 1 p1) Gas chromatography (GC), can be used for which of the following: (Select all that apply)

A. to determine the purity of a substance.
B. to separate the components of mixtures.
C. to quantitate relative amounts of the components in a mixture.
D. to qualitatively identify compounds in a mixture.
All of the above.
(Exp 1 p1) Solvent-Solvent extraction is used to do the following: (Select all that apply)

A. Separate the compound of interest (analyte) from the rest of the sample (the sample matrix).
B. Avoid interference with other compounds in the sample.
C. Analyze a particular component within a complex mixture.
D. Analyze every compound within a mixture.
A & C
(Exp 1 p1) For a solute (S) partitioning between aqueous and organic solvents you can write K = _______
K= [S]organic/[S]aqueous

Note: this is used after extraction
(Exp 1 p1) True/False: The lower the partition coefficient, the better the solvent is able to extract analyte from the sample matrix.
False- the higher the K, the better the solvent (pentanol) was able to extract the analyte from the sample matrix (water)
(Exp 1 p1) Calculating K
-from the calibration plot, find the linear regression (ex: y = 0.1008x + 0.791)
-the extraction initially had 5% ethanol so you substitute 5% into x and get a peak area of 1.295, this is the extracted amount in organic layer
-K=(% extracted)/(% total)-(% extracted) ****this equation can only be used when given % of analyte not concentration*****
the % extracted is that in the organic detected by chromatogram, the total is that which started off in the aqueous before the extraction
-K=(1.295)/(5-1.295)=0.40
-this K value isn't that good because you want more to have been extracted into the org layer/pentanol
q
fraction of ethanol remaining in aqueous layer which is the sample that the analyte came from
Exp 1 part 2 objectives
-using standard addition method to determine the percent ethanol in each of the three samples
Calculate the partition coefficient if 1/4 of a 10% ethanol sample is extracted from the aqueous phase with 1-pentanol (Exp 1, part 2)
A. 0.33
B. 0.66
C. 0.5
D. 1.0
A. 0.33
In today's lab you will use what type of pipette bulb will you use when measuring your standard solutions? (Exp 1, part 2)
A. No bulb is used because autopipettes are used.
B. 2-Valve Pipette Bulb
C. 3-valve pipette bulb
D. Yellow Pastuer pipette bulb
C. 3-valve pipette bulb
In today's experiment you will prepare which of the following series of standards? (select all that apply) (Exp 1, part 2)
A. beverage A + 8% ethanol.
B. 2%, 4% and 6% ethanol standards.
C. Beverage A + 2% ethanol,
D. beverage A + 4% ethanol
E. Beverage A + 0% ethanol
F. beverage A + 6% ethano
C. Beverage A + 2% ethanol,
D. beverage A + 4% ethanol
E. Beverage A + 0% ethanol
F. beverage A + 6% ethanol.
What types of samples are ideal for GC? (Exp 1, part 2)
A. Non-volatile compounds
B. Volatile compounds.
C. Proteins and carbohydrates
D. Aqueous samples
B. Volatile compounds.
What are some common reasons to carry out an extraction? (Exp 1, part 2)
A. isolate the desired analyte
B. concentrate the desired analyte
C. to modify the desired analyte
D. separate the analyte to avoid interference with other species
E. All of the above
F. A, B & C
G. A, B & D
H. A, C & D
G. A, B & D
A. isolate the desired analyte
B. concentrate the desired analyte
D. separate the analyze to avoid interference with other species
If a standard addition plot was constructed for a given analysis and the linear regression gave you a slope of 1.25 and a y-intercept of 3, what is the percentage of ethanol in the sample? (Exp 1, part 2)

A. 6%
B. 0.5%
C. 2.4%
D. 4.5%
C. 2.4%
(Exp 1, part 2) Standard Addition Method
-used to directly calculate the amount of analyze in the original sample
-does not require any knowledge of partition coefficient or fraction of ethanol left behind
-used when matrix effect is involved
(Exp 1, part 2 EOL) 1. Consider the gas chromatographic separation of the esters methyl acetate, methyl propionate, and methyl n-butyrate on a column containing a stationary phase of intermediate polarity.
• What would be the retention order on this column?
• Would this retention order change if a non-polar stationary phase had been used instead? Explain.
• Would the retention times change on the non-polar column? Explain.
-the order of elution is methyl acetate, methyl propionate, and methyl butyrate due to boiling points since they are all not very polar
-if a non-polar stationary phase had been used instead, then the retention order would be the same because boiling point is what determines the retention rate in a GC separation, not polarity since their polarities are very similar
(Exp 1, part 2 EOL) 2. If the peaks in your chromatogram did not separate well enough for you analyze them, would it possible to increase the resolution (separation) of these two peaks? If so, how could the resolution be increased?
-if the resolution of the separation is poor, then the flow rate of the carrier gas can be adjusted to increase the resolution. By decreasing the flow rate, the separation between each peak would be much easier to see. Unfortunately, decreasing the flow rate too much can impact the results, so the flow rate can only be decreased by a certain amount. Furthermore, using a non-polar versus polar stationary phase can lead to clearer results based on the polarity of the solutes.
(Exp 1, part 2 EOL) 3. Compare and contrast the calibration method and the method of standard addition for determining the amount of alcohol in a sample.
-the calibration method uses known amounts of solute, followed by the use of data and known solute amounts to create a linear equation. This equation can then be applied to an unknown sample (of similar solute) to find the amount of solute. On the other hand, standard addition known amount of solute is added to a mixture. An equation is then constructed from the data. This equation allows for the x-intercept to be determined, leading to the unknown concentration of the original sample to be determined as well. In both of these methods, a known amount of solute is used to make a linear equation. However, these methods require different calculations. For example, only the standard addition needs a partition coefficient or the amount of solute remaining in the aqueous phase. Also, the standard addition method takes into account matrix effects.
(Exp 1, part 2 EOL) 4. 1. What are two characteristics that make up a good carrier gas in GC?
-two good characteristics of carrier gases are that there should be no reactions between the gas and the sample. If the carrier gas reacts with the sample, then the chromatogram created using the GC machine and Capstone would not be accurate. Also, if the carrier gas reacts with the stationary phase in the column (based on polarity), then the reaction would not run properly. If the stationary phase and carrier gas interact, then the sample will not elute properly. This would interfere with the data, leading to the chromatogram being incorrect as well.
(Exp 6) Objectives
-Perform solvent-solvent extraction to isolate plant pigments from spinach
-Separate these pigments with liquid chromatography
-analyze the separation of the pigments with a spectrophotometric detection.
-Evaluate the relative retention times and the resolution values
(Exp 6) Background
Plant Pigments
-Porphyrins
- Chlorophylls a (green) and b (yellow-green)
- Produced in response to a sunlight and play a crucial role in photosynthesis
-Leaves change color in fall because chlorophyl changes colors.

-Carotenoids
- Carotenes (orange-yellow)
-Participate in photosynthesis, but do not break down rapidly and are present in the plant leaf during entire lifetime

-Flavenoids
- Flavone (yellow)
- Flavonol (yellow)
(Exp 6) Major Steps Involved in Procedure
1. sample preparation
2. chromatographic separation
3. Spectrophotometric detection
(Exp 6) Preparation of Spinach Extract
1. Grinded in blender with acetone, removes nearly all plant pigments
2. Mixed with NaCl solution resulting in two-phase system (aqueous/organic)

*Aqueous (polar)- flavonoids, xanthophylls
*Organic (non-polar)- chlorophylls, carotenes, xanthophylls,
(Exp 6) Least Polar to Most Polar
Carotenes, Chlorophylls, and Xanthophylls
(Exp 6) 3 Primary Variables in Liquid Chromatography
1. Composition of mobile phase and stationary phase
2. polarity of mobile phase and stationary phase
3. Composition of mobile phase as a function of time
(Exp 6) Phases of Liquid Chromatography
1. Normal-phase LC: stationary phase is polar while mobile phase is non-polar
2. Reverse-phase LC: stationary phase is nonpolar while mobile phase is polar
(Exp 3 EOL) Describe molar absorptivity and how is it related to the measurements made in UV/Vis spectroscopy. Why is it important to know the molar absorptivity? (Be specific).
Molar absorptivity "provides information on how much light a molecule can absorb at a particular wavelength. It is essential to understand what molar absorptivity is to understand what a UV spectrum is, and how it measures absorbance, in order to determine the maximum wavelengths. It is important to know molar absorptivity to use Beer's Law, in which we are able to relate absorbance values, obtained using spectrophotometry, to concentration.
(Exp 3 EOL) At what wavelength does the isosbestic point occur for bromothymol blue? What is the significance of this point?
The isosbestic point occurred at a wavelength of 500 nm. At this point, both the acidic and basic species have the same absorbance, thus one can't tell the difference between the two species in solution.
(Exp 3 EOL) Is it possible to perform an acid-base titration to determine the pKa of Bromothymol Blue? If so, explain how you would carry out the experiment and how the pKa for Bromothymol blue would be determined from this type of analysis. This is different from what was done in this lab.
Yes, it is possible by taking the acidic form of bromothymol blue (HIn) and titrating it with a strong base, such as NaOH. Monitoring the pH with a pH sensor, the pH and corresponding added volumes of strong base are recorded. The titration is stopped once a basic pH is reached (around 9 or 10). Then, a titration curve is created with pH on the y-axis and volume of strong base added on x-axis. You would then find the volume at the equivalence point (EP) and divide it by 2 to get the volume at the half EP. At the half EP, pH=pKa, so the pH corresponding to the volume at the half EP is the pKa of bromothymol blue.
(Exp 3 EOL) What properties are needed for a good solvent-solvent extraction? Give one example (different from the use in lab today), of why you would need to use a solvent-solvent extraction
Ideally, a good solvent-solvent extraction would be able to separate one analyte from the remaining mixture, in order to easily extract that particular analyte. Properties needed for a good solvent-solvent extraction include immiscible liquids [to each other] that would ideally separate into two independent layers, and being able to separate and solely extract the analyte of interest. An example of why we would use solvent-solvent extraction is to extract DNA from a cell.
(Exp 6 EOL) In this experiment, if you were not able to see the colors as they eluted and were not told the elution order, how could you have distinguished the chlorophylls from the other pigments in the chromatogram?
...
(Exp 6 EOL) When would you use a gradient elution and an isocratic elution? How are both methods carried out generally? Why is a gradient elution performed in this experiment?
...
(Exp 6 EOL) What comprises the stationary phase in this experiment and why is it important to know this? Is it polar or non-polar? What about the molecular structure leads you to this conclusion?
...
(Exp 6 EOL) Briefly describe reverse phase and normal phase LC. What would the elution order be if the sample were run in normal phase conditions instead of reverse phase conditions?
...
(Exp 7) Is it possible to utilize the enzymes used in this experiment to analyze the amount of sucrose and/or fructose in a solution? Why or why not (describe specific reasons regarding the enzymes used)?
...
(Exp 7) Based on the differences/similarities found when comparing the reaction at 0°C temperature to the reaction at 40°C, what would you predict would happen if the reaction temperature were increased to 15°C? What does this tell you about the relationship of temperature to reaction time?
...
(Exp 7) In this experiment, you monitored the reaction of ferrocyanide to ferricyanide to measure the amount of glucose in the system. People with diabetes have to frequently monitor their glucose levels and do so using a blood glucose monitor. Briefly describe how these commercial glucometers measure the amount of glucose in blood and how they differ from the method utilized in this experiment.
...
(Exp 7) For the last part of the experiment, you analyzed sugar solutions that were digested and undigested using the same enzyme system as you did to analyze glucose in all other solutions. Discuss the components of the sugar solution and how the digestion process affects those components.
...
Isocratic vs. Gradient Elution
Gradient: Changes composition of mobile phase over time.

Isocratic: One mobile phase

Step-Gradient Elution- goes from most polar mobile phase to least polar mobile phase.
(Exp 6) What is relative retention and resolution and how are they important in determining how effectively compounds are separated?
-Relative retention: compares retention times for two compounds

-Adjusted retention time: subtracts time for unretained peak (baseline correction)

-Resolution: bandwidth of two peaks
(Exp 6) When obtaining reagents from the main lab hood for your experiment, which of the following indicates the proper technique?
Pour a small amount into a beaker or flask, cover and bring it back to your lab bench.
(Exp 6)Given what you know about the mobile phases in this experiment, rank mobile phase 1, mobile phase 2 and mobile phase 3 in order from most polar (1) to least polar (3)
(1) Mobile Phase 1
(2) Mobile Phase 2
(3) Mobile Phase 3
(Exp 6) How can you most accurately determine the actual flow rate of the mobile phase through the column?
Collect mobile phase from the column in a graduated cylinder for a specified period of time.
(Exp 6) After filtering your extracted spinach extract through the glass wool, you should add which solvent to the extract?
Acetonitrile
(Exp 6) In what order should the plant pigments elute from the C-18 column in today's experiment?
xanthophylls → chlorophylls → carotenes
(Exp 6) In a normal phase LC experiment, the stationary phase is non-polar (polar/non-polar) and the mobile phase is polar (polar/non-polar).
polar, non polar
(Exp 6) Which of the below is the correct mathematical expression for the partition coefficient? A represents the analyte of interest.
K = [A]2/[A]1
(Exp 7) Objectives
-To use a spectrophotometric enzyme assay to detect changes
-Varying the temperature, reaction time and amount of enzyme affect a reaction.
(Exp 7) Background
-Simple sugars: monosaccharides, glucose, and fructose

-Disaccharides and polysaccharides must be broken down into monosaccharide units

-Enzymes- biomolecules that catalyze specific reactions by changing the rate of the reaction without being consumed, very selective structure/shape

-Biosensors- small portable analytical devices that combine the selectivity of biomolecules to some type of signal transducer
(Exp 7) Enzymes
Glucose Oxidase and Horseradish Peroxidase

Reaction 1: Glucose oxidase catalyzes oxidation of glucose with O2 to produce B-D-gluolactone and hydrogen peroxide (H2O2)

Reaction 2: Horseradish peroxidase catalyzes reaction of hydrogen peroxide (H2o2) from reaction 1 with ferrocyanide, which is then oxidized to ferricyanide

* Amount of oxidized ferricyanide produced is the same amount of glucose in the solution*
(Exp 7) What is being investigated?
Effects of temperature (0 degrees celsius vs. 40 degrees celsius), volume of enzyme mixture added (.10mL vs. 0.50 mL enzyme mix), and the reaction time (5 min vs. 45 min)
(Exp 7) Sample Calculations

Glucose Concentration
use calibration curve (A vs. mM) from standard solutions 1-7
(Exp 7) Sample Calculations

Dilution Concentration
M1V1= M2V2
(Exp 7) Spectrophotometric Enzyme Assay
In this experiment, a spectrophotometric enzyme was used for the detection of glucose in Gatorade (a digested sugar solution and an undigested sugar solution)
(Exp 7) If a piece of glassware breaks during your experiment, the first thing you should do is:
Inform the TA and ask for assistance.
(Exp 7) When using an adjustable 0 - 1000 microliter pipette, how many times would you have to draw up and dispense if you needed 4 mL of a reagent?
4 times set at 1000 microliters.
(Exp 7) Which of the following enzyme(s) will you use in today's lab? (select all that apply)
Glucose oxidase and Horseradish peroxidase
(Exp 7) A _____________ translates the recognition event into a physically measurable value in a sensor.
transducer
(Exp 7) Unlike glucose or fructose, sucrose is a
disaccharide
(Exp 7) You have a solution that you wish to dilute 50 times (50x). Which of the below represents a 50x dilution?
5 mL solution diluted to 250 mL
(Exp 7) You take 10.0 mL of your unknown solution and dilute it to 100. mL. You then determine that the concentration of this diluted sample is 0.25 M. What was the concentration of the original (undiluted) sample?
2.5 M
(Safety Quiz/ Glassware)
You put a flask containing a solution of benzoic acid and organic solvent on a hot plate at a medium setting in order to evaporate off the organic solvent. Your TA has told you it will take 15 min. After 5 min you realize you have to use the restroom. You should do which of the following?
Ask your TA to watch your experiment so you can go to the restroom.
(Safety Quiz/ Glassware)You are permitted to have a bottle of water outside your book bag in the lab room as long as it is securely capped or has a lid?
False
(Safety Quiz/ Glassware)
When obtaining reagents from the main lab hood for your experiment, which of the following indicated the proper technique?
Go to the main hood, wait your turn and pipette directly out of the stock bottle.
(Safety Quiz/ Glassware)
Where should you place your backpack when you come to lab?
In the cabinets under the white board labeled for backpack storage.
(Safety Quiz/ Glassware)
You remember that you left your lab manual in your lab room yesterday after leaving Morehead. You stop by the lab room the next day before labs start and find that your lab room is open and no chemical work is being done. Which of the below statements are true?
1) You must find someone on the lab staff to accompany you into the lab room to pick up the manual that you left behind.
2) You may email your TA and ask if they can go to the lab room and pick up your manual or meet you to escort you to the lab room.
(Safety quiz/ Glassware) Safety is always my number 1 priority in lab?
True
(Safety Quiz/ Glassware) My cell phone should remain put away at ALL times while in the lab room.
True
(Safety Quiz/ Glassware) Which of the following statements is correct about eating in the lab?
I am not allowed to eat anything while in the lab room.
(Safety Quiz/ Glassware) What is the correct way to mix water and acid
Always add acid to water
(Safety Quiz/ Glassware) There are many hazards associated with chemicals. Which of the following are the proper procedure to learn about the hazards you may encounter in lab (select all that apply).
SDS, lab manual, check with your TA if you are unsure.
(Safety Quiz/ Glassware) In which of the following locations are you required to have your safety goggles covering your eyes at all time?
In your lab room, in GC room, and UV/Vis Spec room
(Safety Quiz/ Glassware) Your lab coat must stay in the lab room the entire semester?
True
(Safety Quiz/ Glassware) It's five minutes before your lab period begins and you realize that you are not properly dressed for lab. You could (choose all correct options):
1. Return to your residence to get the proper clothing, if time permits.
2. Go to the Student Stores to purchase the proper clothing.
3. Ask a friend to bring proper clothing, if time permits.
(Safety Quiz/ Glassware) Proper lab attire is required while in your chemistry lab. Select all that are considered appropriate lab attire.
Long pants, sneakers, and one other that I did not get
(Safety/ Glassware) While taking your pre-lab quiz and listening to your pre-lab lecture, you are not required to wear your safety goggles.
False
(Safety/ Glassware) Which of the following statements is/are correct:
1. When leaving the lab room with chemicals, I must place them on a cart, or in one of the rubber black carriers to prevent chemicals from spilling in the hallways/elevators.
2. When exiting the lab room I must first take off my gloves and dispose of them in the glove recycling containers provided on the same lab bench where I got my clean gloves.
(Safety/ Glassware) If there is no chemical waste container in the main lab hood, what should you do?
You should immediately let your TA know that there is no waste container and always verify with them whether any chemical waste should be poured down the drain.
(Safety/ Glassware) When you are finished using a disposable glass Pasteur pipette, where should you dispose of it?
In the solid waste container in the hood.
(Safety/ Glassware) If a piece of glassware breaks during your experiment, the first thing you should do is:
Inform the TA and ask for assistance.
(Safety/ Glassware) When using a volumetric flask to make a solution, which statement(s) represent the correct procedure for making the given solution. (select all that apply)
1) When making a solution of a weak acid from a solid, first weigh the solid, then add it to the volumetric flask. Add a small amount of water to first dissolve the solid, then once dissolved, fill the flask the rest of the way with water so that the bottom of the meniscus is at the line on the neck of the flask.
2)When preparing 10 mL of a 2% ethanol in pentanol solution, first 0.2 mL of ethanol, then add a small amount of pentanol to mix. Once mixed, add pentanol until the bottom of the meniscus is at the line on the neck of the volumetric flask.
(Safety/ Glassware) When getting approximate amounts of a reagent from the hood, what piece of glassware would you typically use?
a beaker
(Safety/ Glassware) Which of the following statements lists the correct order in which the light passes through a uv/vis spectrometer?
lamp ~> grating(monochromator) ~> sample ~> detector
Sets found in the same folder