Module 5: Cal Curves

Question 1

Calibration curve

Answer 1

AKA Standard graphs
plot A of a minimum of 3 standards against their known concentrations
Used to determine the contraption of unknowns and controls
Should use more than half of graph
X axis is always concentration
Y axis is always A
Line of best fit going through origin and stopping at last point on graph

Question 2

Labeling requirements of cal curves (6)

Answer 2

Name of analyte
Name of method of analysis
Instrument name and #
Wavelength
Date of analysis
First initial and last name of tech

Question 3

Samples with readings above the linear range

Answer 3

Dilute sample and reread on graph to determine concentration. Then multiply concentration by the dilution factor
1/2 dilution would have a factor of 2

Question 4

Linear range synonyms

Answer 4

Analytical or reportable range

Question 5

New cal curves are required when: (2)

Answer 5

a component of the instrument is changed (ex. new light bulb)
new or different lots of reagents are used

Question 6

Increasing concentration values often results in

Answer 6

a loss in linearity

Question 7

Standards

Answer 7

AKA calibrators

are PURE SOLUTIONS of an analyte with known concentrations

Question 8

Stock standard

Answer 8

high concentration often prepared initially

Higher concentration = more stable = longer shelf life

Question 9

Working standard

Answer 9

are dilutions of the stock standard that are prepared in smaller volumes just before use
Prepared in volumetric glassware

Question 10

Stock standard calculation

Answer 10

Want 100mL of 40mmol/L urea
Urea = 60.06g/mol

grams of urea = (40mmol/L)(0.100L)(1mol/1000mmol)(60.06g/mol)
Grams of urea = 0.24g
Final statement: 0.24g of urea is weighed and diluted it up to 100ml in a 100ml volumetric flask

Question 11

Working standard calculation

Answer 11

C1V1 = C2V2
Final statement: 2mL of 40mmol/L stock solution is diluted up to 10mL in a volumetric flask to make an 8mmol/L working standard

Question 12

Reagent blank

Answer 12

a cuvet with reagent used in the method and diluent in place of the serum sample
Any absorbance of light is due to the color of the reagent
Accounts for the color of the reagent
Often used as the zero concentration standard

Question 13

2 ways to used reagent blank as zero concentration

Answer 13

1) measured and subtracted rom other test solutions

2) subtracted by using it to set 0A (100%T) on the spectrophotometer

Question 14

Sample blanks

Answer 14

Contains same amount of sample used for the test but an equal amount of diluent instead of reagent
Accounts for the color of the sample
Absorbance of the sample blank is read against the reagent blank and then subtracted from the test solution containing that sample
Not all methods require use of sample blank

Question 15

Controls

Answer 15

Detect error in the procedure
Commercially prepared with known concentrations
When a control falls out of range, it indicates a problem has occurred with he procedure
Same matrix as patient samples

Question 16

Control evaluation (3)

Answer 16

1) in or out of control range
2) control is valid or not valid
3) entire run (QC and patients) is accepted or rejected
If rejected, explain next step (ex. repeat control)

Question 17

Slope

Answer 17

refers tot he direction and steepness of the curve
slope = rise/run
(change in y/change in x)

Question 18

Intercept

Answer 18

point at which the curve intercepts or meets the x or y axis of the graph
For methods that use a reagent blank, the intercept will be at the origin (0 A, 0 concentration)

Question 19

Sensitivity

Answer 19

ability to detect or measure small changes in concentration
aka small change in concentration will cause big change in absorbance on a graph
The steeper the slop of a cal cure, the better the sensitivity

Question 20

Using max A will give

Answer 20

Maximun sensitivity

Question 21

times when wavelength other than max A will be used (2)

Answer 21

1) to extend the linear range (may be used over wider range of concentrations)
2) to reduce absorption caused by interfering substances that also absorb at the max A wavelength
Both of these will decrease the sensitivity

Question 22

3 ways to reduce sensitivity without changing the wavelength

Answer 22

1) decrease amount of sample relative to amount of reagent (less product formed and lower absorbance)
2) decrease the distance of the light path (smaller cuvet)
3) diluting the final reaction mixture

Question 23

MDL

Answer 23

Minimum detection limit
smallest concentration of an analyte that can be distinguished from zero (expressed in units of concentration)
Influenced by sensitivity of a method
Reported as “

Question 24

Basic procedure for measuring patient samples

Answer 24

1) standards prepared. Concentrated stock standard can be made and diluted into working standards
2) standards are measured on instrument
3) readings are plotted against known concentrations of standards to make cal curve
4) controls are measured (ensure they are in range)
5) patient samples measured and compared to standards/cal curve to determine concentration