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UNIT: Preparation of Standard Curves

# UNIT: Preparation of Standard Curves

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07/21/2013

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MLAB 2401 - Clinical Chemistry Lab Manual
C
B 33
UNIT:Preparation of Standard Curves
5curves.wpd
Graphing standard curves and obtaining accurate patient and control values from them.
Objectives
Upon completion of this exercise, the student will be able to:1.Properly set up a standard curve whether using linear or semi-log paper.a.Absorbance vs. concentrationb.% transmittance vs. concentration2.Using the prepared curve, determine the concentrations for control and patient specimens.
Materials
1.Linear graph pape2.Semi-log graph pape3.Absorbance and % transmittance values for standards, controls and patients.
Principle
Many laboratory tests require the outcome of a carefully controlled chemical reaction be evaluatedor read in a photometer (colorimeter or spectrophotometer). Since these instruments are capableof only measuring the amount of light being allowed to pass through the cuvette, their readoutdevices display % of light transmitted or mathematically derived absorbance. One method of obtaining concentration from % transmittance or absorbance is through the use of a standardcurve.For our purposes, standard curves are defined as a graphs with absorption or %T plotted on theY axis, and increasing concentrations of standard along the X axis. If Beer’s Law is followed, theresulting line representing absorbance vs concentration will be straight. A standard curve is constructed after obtaining the %T/Abs readings from a number of solutionsof known concentration (standards) used in a reaction or procedure. After the readings areobtained each is plotted on semi-log (% transmittance) or linear (absorbance) paper against thecorresponding concentration. If the procedure follows Beer's Law, the points plotted will generallylie such that a straight line can be drawn through them. The concentration of controls and other unknowns (patient samples) can be determined by locating their %T/Abs reading on the line, thendropping an imaginary line down from that point to intersect the concentration axis.Once a standard curve is developed for a particular test method on a particular spectrophotometer, it should be checked periodically to determine that it is still good. A new curveshould be constructed when there is a change in:
!
reagent lot numbers

UNIT: Preparation of Standard Curves (continued)
B 34
C
MLAB 2401 - Clinical Chemistry Lab Manual
!
methodology/procedure
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an instrument parameter (change bulb, optics cleaned, etc.)Once the curve is drawn, a number of things must be considered to determine its acceptability.The majority of the curve’s points should be on or close to the line. There could be many reasonsfor a point not being on the line. If the standards are formed from a series of dilutions, theaccuracy of the dilutions must be suspect. Calculations of the dilutions and spectrophotometer errores are other possibilities. Whether or not the curve passes through the point of origin (the“0"), varies with the procedure. If Beer’s law is followed and the procedure is linear at the lower concentrations, the curve’s line generally goes through the zero.
Procedure I
Basic Standard Curve Characteristics1.Neatness counts. Preparing a good standard curve takes time and practice. A sharp pencilshould be used during the early construction period.2.Use the X axis for concentration. Determining how to space between the individualconcentrations is done by trial and error and will also depend on the individual procedure.3.The Y axis is labeled either %T (semi-log paper) or Absorbance (linear paper). The amountof spacing for absorbance readings is often times determined through trial and error.4.Centered on top of the graph (such that it doesn't interfere with the readings) should be thename of the analyte measured (i.e., glucose).5.On the upper right portion of the graph (again, such that it doesn't interfere) should belabeled as follows:Your NameDate Analyte/ProcedureInstrumentWavelength6.The following is an abbreviated list of errors or problems encountered by students in thepast. It is provided for you to consider as pitfalls to avoid. You should keep this/thesepage(s) handy when preparing your chemistry lab curves.a.Bottom of Y axis did not start at 0.000.b.Compressed Y or X axises.c.Uneven spacing of Y or X axises.d.Not labeling correctly/in the right place.e.Drawing curve point to point.f.Required information is missing/in the wrong place.g.“Fat” pencil lines/double/smeared lines.h.Making dots on curve’s line for unknown’s absorbance value.I.Drawing dotted lines on graph representing how the concentration of unknown wasdetermined. j.Drawing circles around dots on the curve line.

UNIT: Preparation of Standard Curves (continued)
MLAB 2401 - Clinical Chemistry Lab Manual
C
B 35
k.Not using the long axis for concentration.
Procedure II

Preparing a Glucose Standard CurveIn this experiment, a standard curve for the analysis of glucose will be constructed. In order to dothis, a number of solutions of known concentration (standards) will be used in the reaction, andthe absorbance of each will be measured.This method of assaying glucose is based on the reaction of aldohexoses (i.e., glucose) with o-toluidine in acetic acid to form an equilibrium mixture of glycosylamine and the correspondingSchiff Base. The green-colored product is measured spectrophotometrically at 630 nm.Reaction Parameters1.Reaction TypeEndpoint2.Reaction Temperature100°C3.Sample/Reagent Ratio1:804.Wavelength630 nm5.Fasting Serum NV65-100 mg/dL6.Linearity15-500 mg/dL1.Turn on spectrophotometer and allow to warm up for 15 minutes.2.Using the
stock
glucose standard solution, prepare 10 ml each of the following glucose
2
standards using DI HO as a diluent. Each tube should be labled with name andconcentration (mg/dl glucose).3.Follow manufacturer’s instructions for the glucose procedure.
STOCK:
10 g/L - 10,000 mg/L = 1000 mg/dl
Desired ConcentrationDilution NeededVolumeStock (ml)Volume
2
DIHO (ml)TotalVolume(ml)1.0 mg/dlnone010.010.02.25 mg/dl1:400.259.7510.03.50 mg/dl1:200.59.510.04.100 mg/dl1:101.09.010.05.200 mg/dl1:52.08.010.06.500 mg/dl1:25.05.010.07.1,000 mg/dlnone10.010.0(Tube #1 is the
reagent blank
)