Flashcards
(374 cards)
1
Q
What is “statistics” according to Dictionary.com?
A
The practice or science of collecting and analysing numerical data in large quantities, especially for inferring proportions in a whole from a representative sample.
2
Q
How else is “statistics” defined in mathematics?
A
A branch of mathematics dealing with the collection, analysis, interpretation, and presentation of masses of numerical data.
3
Q
Why do we need statistics in large datasets?
A
To distinguish between randomness and systematic features or patterns in the data.
4
Q
What are the four main tasks of statistical analysis?
A
1) Design experiments and collect data; 2) Describe data (summary stats, graphs, tables); 3) Tests of hypotheses; 4) Model fitting (associations, prediction, classification).
5
Q
Name two online video resources to learn statistics basics.
A
StatQuest with Josh Starmer (YouTube) and GraphPad Tutorials (YouTube).
6
Q
What book helps non-mathematicians learn statistics?
A
“Statistics without Tears” by Derek Rowntree.
7
Q
What software will you learn for statistical analysis in this course?
A
GraphPad Prism.
8
Q
Give an example of how statistics appear in daily life.
A
Health apps tracking steps and heart rate, or national statistics websites reporting COVID-19 cases.
9
Q
What is a “population” in statistics?
A
The entire group you wish to study (often too large to measure every member).
10
Q
What is a “sample”?
A
A representative subset of the population used to infer properties about the whole.
11
Q
Define an “event” in probability.
A
A set of possible outcomes from an experiment (e.g., rolling a die and getting a “4”).
12
Q
How is probability of an event calculated?
A
Number of favorable outcomes ÷ total number of possible outcomes.
13
Q
What range of values can probability take?
A
From 0 (impossible) to 1 (certain).
14
Q
If a fair die is rolled, what’s the probability of getting a “2”?
A
1/6 ≈ 0.167.
15
Q
What are “odds”?
A
Probability of event occurring divided by probability of it not occurring (p/(1–p)).
16
Q
Convert probability p=0.2 to odds.
A
Odds = 0.2 / 0.8 = 0.25 (or 1:4).
17
Q
How do you find probability from odds?
A
p = odds / (1 + odds).
18
Q
What is a “random variable”?
A
A mapping from each outcome in the sample space to a numerical value.
19
Q
Name three common probability distributions.
A
Normal (Gaussian), binomial, Poisson.
20
Q
What percentage of data falls within ±1 standard deviation in a normal distribution?
A
About 68.3%.
21
Q
What is a “diagnostic test” in biostatistics?
A
A procedure to determine presence/absence of a disease in subjects.
22
Q
Define “sensitivity” of a test.
A
True positives / (True positives + False negatives): ability to detect disease when present.
23
Q
Define “specificity” of a test.
A
True negatives / (True negatives + False positives): ability to rule out disease when absent.
24
Q
What is Positive Predictive Value (PPV)?
A
True positives / (True positives + False positives): probability you have disease if test is positive.
25
What is Negative Predictive Value (NPV)?
True negatives / (True negatives + False negatives): probability you don’t have disease if test is negative.
26
Name two main categories of study design.
Experimental (e.g., RCT) and observational (e.g., cohort or case-control).
27
What is a randomized controlled trial (RCT)?
An experimental study with intervention vs. control (placebo) groups to test effects.
28
What’s the difference between prospective and retrospective studies?
Prospective: collect data going forward; Retrospective: analyze existing past data.
29
Define cross-sectional vs. longitudinal data.
Cross-sectional: data at one point in time; Longitudinal: repeated measurements over time.
30
What’s quantitative (numerical) data?
Data that can be measured and expressed numerically (e.g., weight, height).
31
What’s qualitative (categorical) data?
Non-numerical data describing characteristics (e.g., blood type, survey responses).
32
Differentiate nominal vs. ordinal data.
Nominal: categories without order (e.g., pizza vs. pasta); Ordinal: ordered categories (e.g., rating scales).
33
What’s the difference between discrete and continuous quantitative data?
Discrete: countable values (e.g. number of students); Continuous: any value in a range (e.g. temperature).
34
What is the arithmetic mean?
Sum of observed values ÷ number of observations.
35
What symbol denotes the sample mean?
\(\bar x\).
36
When calculating variance, why divide by (n–1) for a sample?
To correct bias since the sample mean is estimated, giving an unbiased estimator of population variance.
37
Define standard deviation (SD).
The square root of the variance; measures spread of data around the mean.
38
What unit is SD expressed in?
Same unit as the original data (e.g. kg, minutes).
39
What is standard error of the mean (SEM)?
SD of sample means over repeated samples; \( \text{SEM} = s / \sqrt{n} \).
40
In skewed distributions, which measure of central tendency is preferred?
Median, because the mean is pulled by extreme values.
41
What does “kurtosis” describe?
How heavy or light the tails of a distribution are compared to normal.
42
What is a Pearson correlation?
Measure of linear relationship between two quantitative variables (−1 to 1).
43
What is a Spearman correlation?
Measure of monotonic relationship (based on ranks); useful for non-normal or ordinal data.
44
Why can a Spearman correlation be higher in presence of outliers than Pearson?
Because Spearman uses ranks, making it less sensitive to extreme values.
45
What does a correlation coefficient of 0 imply?
No linear correlation—but there may still be non-linear association.
46
Name three graphical ways to display data distribution.
Histogram, boxplot, density plot.
47
In a boxplot, what does the box represent?
The interquartile range (25th to 75th percentile), with the median as a central line.
48
How are outliers shown in a boxplot?
As points beyond 1.5×IQR from the quartiles.
49
What’s the purpose of descriptive statistics?
To summarize and describe the main features of a dataset without making inferences.
50
When choosing a statistical test, what key distinction must you consider?
Whether data are descriptive (summarizing) or inferential (testing hypotheses).
51
What is “inferential statistics”?
Methods to analyze relationships between variables based on formal hypotheses and statistical tests.
52
List the six main steps in performing a statistical test.
1) Formulate H₀ and H₁; 2) Choose appropriate test; 3) Perform test; 4) Obtain test statistic & P-value; 5) Evaluate significance; 6) Reject or accept H₀.
53
What defines the null hypothesis (H₀)?
Statement assumed true unless data provide sufficient evidence to reject it.
54
How does a one-tailed test differ from a two-tailed test?
One-tailed tests look for change in one direction; two-tailed tests look for any change (up or down).
55
Give an everyday analogy illustrating hypothesis rejection.
“No person taller than 2.30 m” remains unproven until someone over 2.30 m is found—then H₀ is rejected.
56
What does the P-value represent?
Probability of observing this result or more extreme if H₀ is true.
57
What P-value threshold is commonly used to call a result “statistically significant”?
p < 0.05 for significance; p < 0.01 very significant; p < 0.001 highly significant.
58
What are Type I and Type II errors?
Type I (α): false positive (rejecting true H₀); Type II (β): false negative (failing to reject false H₀).
59
When is a one-tailed test appropriate?
When prior data, physical limits, or common sense indicate any effect can only go in one direction.
60
In Example 1 (FTO & BMI), why was a one-tailed test used?
H₁ predicted BMI would increase; direction specified, so left-sided one-tailed test.
61
In Example 2 (FTO & BMI), why was a two-tailed test used?
H₁ allowed BMI to change either up or down; no direction, so two-tailed test.
62
Why must we be cautious interpreting large P-values?
They don’t prove absence of effect, only insufficient evidence to detect it.
63
What factors determine choice of statistical test?
Data type (categorical vs. continuous, normal vs. non-normal), research question, sample size.
64
What’s the difference between Pearson and Spearman correlation tests?
Pearson tests for linear relationships (normal data); Spearman tests for monotonic relationships using ranks (non-normal or ordinal).
65
What is the Chi-square (χ²) test used for?
To check if observed frequencies deviate from expected frequencies in categorical data.
66
How do you compute degrees of freedom (df) for a χ² contingency table?
df = (rows – 1) × (columns – 1).
67
When should Fisher’s exact test be used instead of χ²?
When any expected cell count is < 5, for small sample contingency tables.
68
What distinguishes unpaired vs. paired Student’s t-tests?
Unpaired compares two independent groups; paired compares repeated measures on same subjects.
69
What assumption underlies Student’s t-test?
Data are normally distributed with equal variances (Welch’s test if variances unequal).
70
Name two ways to test data normality.
Visual inspection (histogram) and D’Agostino-Pearson normality test.
71
What is the Mann-Whitney U-test?
Non-parametric test comparing central tendency of two groups using ranks (alternative to unpaired t-test).
72
What is the ’Agostino-Pearson normality test?
A statistical test used to assess whether a given dataset follows a normal distribution.
73
When do you use one-way ANOVA?
To compare means across more than two groups for a single categorical predictor.
74
What additional factor does two-way ANOVA include?
Tests effects of two categorical predictors and their interaction on a continuous response.
75
Why perform post-hoc tests after ANOVA?
ANOVA tells you groups differ but not which; post-hoc (e.g. Tukey HSD) pinpoints pairwise differences.
76
What is the Kruskal-Wallis test?
Non-parametric extension of Mann-Whitney for comparing medians across > 2 groups using ranks.
77
How can sample size affect statistical significance?
Larger samples can make small effects statistically significant; always consider effect size and design.
78
What is a “borderline significant” P-value?
0.05 < p < 0.1 — suggests a trend but may need larger sample to confirm.
79
Why is 0.05 considered an arbitrary cutoff?
Originated by Fisher; widely accepted but arbitrary threshold for significance.
80
What is α in hypothesis testing?
Significance level equal to the probability of Type I error, often set to 0.05.
81
Why might one prefer median over mean for skewed data?
Median is robust to outliers and better represents central tendency in asymmetric distributions.
82
What is a “statistical model”?
A simplified representation of reality that describes the association structure in data.
83
What does a regression model represent?
How a dependent (outcome) variable (Y) depends linearly on one or more independent (predictor) variables (X).
84
What distinguishes simple from multiple linear regression?
Simple uses one predictor; multiple uses two or more predictors.
85
In linear regression, what is assumed about the expected value of (Y)?
It is a linear function of the covariates: (E[Y]=alpha+eta X).
86
How is linear regression a generalisation of t-tests and ANOVA?
It can compare group means (categorical predictors) or quantify associations between continuous variables under one framework.
87
What criterion does linear regression use to fit the line?
It minimises the sum of squared residuals (distances between observed and predicted values).
88
What is the coefficient of determination ((R^2))?
The proportion of variability in (Y) explained by the fitted model.
89
What does the p-value in regression indicate?
The statistical significance of the fitted model or individual coefficients.
90
What assumption must hold for the relationship in linear regression?
The relationship between dependent and independent variables is linear.
91
What distribution is assumed for the error term (epsilon)?
Normal distribution with mean 0 and variance (sigma^2).
92
What does homoscedasticity mean?
The residuals have constant variance across all levels of (X).
93
Why must errors be independent in regression?
To ensure valid inference; correlated errors violate model assumptions.
94
What is multicollinearity and why is it a concern?
High correlation among predictors makes coefficient estimates unstable.
95
Write the general form of a multiple linear regression equation.
(Y=alpha + eta_1X_1 + eta_2X_2 + dots + epsilon).
96
In regression, what does the intercept ((alpha)) represent?
The expected value of (Y) when all (X)s are zero.
97
What does a regression slope ((eta_i)) represent?
The unit change in (Y) per one-unit increase in (X_i).
98
What does (eta_i=0), (eta_i>0), or (eta_i<0) imply?
No association, positive association, or negative association, respectively.
99
What is the standard error (SE) of a coefficient?
The estimated SD of the coefficient’s sampling distribution, measuring precision.
100
Which test statistics are used in regression analysis?
t-tests for individual coefficients and F-test for overall model significance.
101
Why use adjusted (R^2) instead of (R^2) alone?
Adjusts for the number of predictors to penalise overfitting.
102
In the height-prediction example, what predictors were used?
Weight (kg) and sex (coded as 1=male, 2=female).
103
How do you predict height for a female weighing 65 kg if (hat Y=175.81+0.17W-11.05S)?
Plug in (W=65), (S=2): (hat Y=175.81+0.17·65-11.05·2≈157.76) cm.
104
What is a confidence interval (CI)?
A range around a point estimate likely to contain the true parameter with specified confidence.
105
Give the formula for a 95% CI of a regression coefficient (eta).
(eta pm Z_{0.025} imes SE(eta)) where (Z_{0.025}=1.96).
106
Why is (Z_{0.025}=1.96) used for 95% CIs?
Because (P(|Z|ge1.96)=0.05) under the standard normal distribution.
107
How do you tell if an association is significant from a CI?
A 95% CI that excludes the null value (0 for (eta), 1 for OR) indicates (p<0.05).
108
What percentage of data falls within ±2 SD in a normal distribution?
Approximately 95.4% (three-sigma rule context).
109
What is a standard (calibration) curve in analytical chemistry?
A plot of known concentrations vs. measured response, fitted by linear regression to determine unknowns.
110
How is a standard curve used to find an unknown concentration?
Measure its response, locate on the fitted line, and read off the corresponding concentration.
111
When is logistic regression used?
For binary (yes/no) outcomes, modelling the log-odds of an event as a function of predictors.
112
What link function does logistic regression use?
The logit: (logigl( frac{p}{1-p}igr)), mapping probabilities (0–1) to (mathbb R).
113
How are logistic regression coefficients interpreted?
As log-odds; exponentiating gives odds ratios (ORs).
114
What does OR=1, OR>1, OR<1 indicate?
No association, positive association, negative association, respectively.
115
How do you assess significance of an OR?
A 95% CI for the OR that excludes 1 signifies (p<0.05).
116
Define odds ratio (OR) vs. risk ratio (RR).
OR = (a/b)/(c/d); RR = [a/(a+b)]/[c/(c+d)] from a 2×2 table.
117
Why are OR and RR similar when events are rare?
Because when a, c ≈ 0, odds ≈ probability, so RR ≈ OR.
118
Why can’t RR be calculated in case-control studies?
The total number at risk (a+b and c+d) is unknown, making RR undefined.
119
What is the key caution about association vs. causation?
Correlation or association does not prove one variable causes the other.
120
How is “correlation” more specific than “association”?
Correlation implies a linear or monotonic trend, whereas association is any dependence.
121
What study design is the gold standard for establishing causality?
Randomised controlled trials (RCTs).
122
What are the learning outcomes for the renal function lecture?
Describe the functional role of the kidney in blood composition, blood pressure maintenance, and hormone production; identify clinically relevant readouts of kidney function; explain why they are relevant and how they are measured.
123
What are the key anatomical parts of the kidney?
Hilum (entry/exit for vessels and ureter), cortex (outer pale layer), medulla (inner pink area) containing nephrons.
124
What is the functional unit of the kidney and its main components?
The nephron, comprising the renal corpuscle, renal tubule, and collecting duct; two types exist: cortical and juxtamedullary.
125
How is the filtrate refined along the nephron?
Proximal tubule fluid is isosmotic; descending limb reabsorbs only water (concentrates filtrate); thick ascending limb reabsorbs solutes but is water‐impermeable (dilutes filtrate); distal tubule and collecting duct have hormone‐regulated permeability for final adjustment.
126
What proportion of plasma is filtered and excreted in glomerular filtration?
~20% of plasma filters into Bowman's capsule, ~80% continues in capillaries; >99% of filtered fluid is reabsorbed along the nephron and <1% is excreted.
127
Which pressures determine net glomerular filtration?
Glomerular hydrostatic pressure – Bowman’s capsule hydrostatic pressure – glomerular colloid osmotic pressure.
128
How is GFR controlled at the level of the nephron?
By net filtration pressure, renal blood flow/blood pressure, filtration coefficient, and by changing afferent/efferent arteriole diameters.
129
What mechanisms modulate arteriole tone to regulate GFR?
Hormonal (angiotensin II, prostaglandins), nervous (sympathetic noradrenaline), and autoregulatory (myogenic response, tubuloglomerular feedback via macula densa).
130
What is the clinical use of measuring GFR?
To assess kidney filtering capacity and nephron function; GFR does not alone diagnose specific kidney disorders.
131
What is the fundamental equation for estimating GFR using a substance S?
GFR × [S]ₚₗₐₛₘₐ = [S]ᵤᵣᵢₙₑ × urinary flow rate; GFR varies with body size (10–200 mL/min).
132
Which exogenous substances give the most accurate GFR measurements?
Inulin (nonradioisotope) and radioisotopes such as ⁵¹Cr-EDTA; both are freely filtered, not reabsorbed or secreted.
133
Why is creatinine used as an endogenous marker for GFR?
It is a stable breakdown product of creatine phosphate, produced at a relatively constant rate; freely filtered, minimally secreted, and not reabsorbed.
134
What factors affect creatinine clearance estimates of GFR?
~7–10% tubular secretion (↑ in insufficiency), age.
135
What factors affect creatinine clearance estimates of GFR?
~7–10% tubular secretion (↑ in insufficiency), age, exercise, diet (meat intake), muscle mass, and drugs (e.g., trimethoprim, cimetidine).
136
What are the basic functions of the kidney?
Control blood composition (fluid/electrolyte balance), blood volume, blood pressure; produce hormones (erythropoietin, calcitriol); excrete waste (urea, urate, creatinine).
137
How can blood osmolarity and volume change independently?
Dehydration ↓ volume, ↑ osmolarity; compensation via cardiovascular responses, angiotensin II, vasopressin, and thirst.
138
How does pH regulation by the kidney impact protein function and the nervous system?
H⁺ concentration alters protein three-dimensional structure; acidosis depresses CNS excitability, alkalosis causes hyperexcitability; pH disturbances often link to K⁺ imbalances.
139
What enables the kidney to generate a medullary osmotic gradient?
Countercurrent exchange in vasa recta flowing opposite the Loop of Henle and countercurrent multiplication by selective water/solute transport.
140
Which hormones control water and solute permeability in the nephron?
ADH (vasopressin) controls collecting duct water permeability; aldosterone controls Na⁺ transport (and indirectly water) in the distal nephron and descending limb.
141
How does the proximal tubule achieve acid‐base balance?
NHE exchanger secretes H⁺ into filtrate; filtered HCO₃⁻ combines to form CO₂ (diffuses back), is converted by CA to HCO₃⁻ (reabsorbed) and H⁺ (secreted again); glutamine metabolism yields NH₄⁺ and HCO₃⁻.
142
How does the kidney conserve blood volume?
By adjusting GFR (arteriolar tone) and regulated reabsorption in proximal and distal segments; only external fluid intake can restore lost volume.
143
What is the role of vasopressin in renal fluid balance?
ADH increases water reabsorption by inserting aquaporin channels in the collecting duct epithelium.
144
How does the kidney contribute to blood pressure regulation?
Through RAAS (renin-angiotensin-aldosterone system), autoregulation, and modulation of vascular resistance.
145
Which hormones are produced by the kidney and their functions?
Erythropoietin (stimulates RBC production in response to ↓PO₂) and calcitriol (active vitamin D for GI Ca²⁺ absorption; made via C1-α-hydroxylase; ~14 day half-life).
146
What distinguishes acute from chronic kidney disease and common causes?
Acute: rapid metabolic imbalance, high mortality, often reversible; chronic: progressive, linked to diabetes, hypertension, glomerulonephritis, polycystic kidney disease.
147
What is the primary purpose of urinalysis test strips?
A near-patient clinical diagnostic tool to screen for various conditions by detecting chemical markers in urine.
148
Which conditions are these test strips intended to investigate?
Liver function, kidney function, urinary tract infections, diabetes mellitus, and other metabolic diseases.
149
How many samples are provided for the practical, and what special sample is included?
Six labeled patient samples plus one control/blank sample, making seven total.
150
What are the first steps for using the urinalysis strips?
Mix the urine by inverting the sample; remove a strip, recap the bottle; label the strip with the sample ID; dip so all test pads are submerged.
151
After dipping the strip, what should you do next?
Immediately remove the strip, drag its edge against the container rim to remove excess, then start the timer and compare each pad to the colour blocks on the bottle.
152
How should you record and interpret your results?
Record every analyte’s result (even negatives) in the results table, then match each sample’s profile to the corresponding patient data.
153
Which ten analytes do the test strips measure?
Protein, blood (erythrocytes/hemoglobin), leukocytes, nitrite, glucose, ketone bodies, pH, specific gravity, bilirubin, and urobilinogen.
154
What does a urine pH result indicate?
Normal is slightly acidic (pH 5–6); deviations can signal metabolic disorders.
155
What does a positive glucose result in urine suggest?
Abnormal glucose excretion, often indicating type 1 or type 2 diabetes mellitus.
156
What does the presence of ketone bodies in urine indicate?
Fat breakdown; high levels may indicate ketosis, commonly seen in type 1 diabetes.
157
What is inferred by bilirubin in the urine?
Impaired bilirubin metabolism by the liver, indicative of liver disease.
158
What do elevated leukocytes in urine indicate?
An infection or inflammatory condition in the urinary tract.
159
What does nitrite in urine signify?
Conversion of nitrate to nitrite by bacteria, suggesting a urinary tract infection.
160
What does protein in the urine indicate?
Glomerular filtration failure in the kidney, pointing to kidney disease.
161
What is signaled by erythrocytes or hemoglobin in urine?
Possible urinary tract trauma (bleeding) or infection.
162
Which brand of test strips is used in this practical?
Siemens Multistix 10 SG.
163
What sections must be included in the urinalysis report write-up?
Introduction (aims & objectives), results table, discussion & conclusions, further tests, limitations, and bibliography.
164
What should the discussion section of the report contain?
A reasoned linkage of each patient’s clinical details to their urinalysis results.
165
What is expected in the “Further Tests” section?
Suggested additional diagnostic tests with justification and references.
166
What should the “Limitations” section address?
The test strips’ shortcomings and potential sources of error, explained with references.
167
How is the urinalysis report marked?
0–59% for factual content; 60–100% for critical thinking, writing style, extra reading, and quality of references.
168
What common issues have arisen in past urinalysis reports?
Leaving result boxes empty, omitting patient identifiers, declining reasoning in later samples, and fact overload with little critical analysis.
169
Why is a control (blank) sample included?
To establish a baseline, verify the strips are functioning properly, and detect any faulty reagents or technique.
170
What is chromatography?
A technique for analyzing or separating mixtures (gases, liquids or dissolved substances) by passing a mobile phase over a stationary phase.
171
What are the two phases in chromatography?
The stationary phase (matrix) and the mobile phase (gas or liquid).
172
How does chromatography achieve separation?
Components in the sample partition between mobile and stationary phases according to their relative affinities, causing different elution times.
173
Who first coined the term “chromatography” and when?
The term (“colour writing”) arose in the early 1900s to describe plant pigment separations.
174
Name three applications of chromatography.
Forensics (blood/arson), food regulation (horse-meat scandal), pharmaceutical purification (antibodies/vaccines).
175
What distinguishes column from planar chromatography?
Column chromatography uses a packed column; planar (e.g. TLC) uses a flat plate or paper matrix.
176
List the five main types of chromatography covered.
Gas chromatography (GC), liquid chromatography (LC), high-performance liquid chromatography (HPLC), thin-layer chromatography (TLC), exchange/affinity chromatography.
177
What makes a separation “good” in chromatography?
Distinct, well-resolved peaks with minimal overlap, balanced against run time and cost.
178
What are “small molecules” in this context?
Compounds of low molecular weight (<1,000 Da), e.g. amino acids, lipids, sugars, fatty acids, alkaloids.
179
Why can separating small molecules be more challenging than proteins?
Their small size (10–1,000 Da) means decreased physical separation; advanced chromatographic methods are required.
180
What detection methods follow chromatography?
NMR spectroscopy (robust, costly), mass spectrometry (complex samples), UV/Vis spectroscopy (simplest).
181
How does HPLC differ from simple liquid chromatography?
HPLC applies high pressure to push mobile phase through a tightly packed column for faster, higher-resolution separations.
182
In reverse-phase HPLC, what is the main factor determining elution order?
The polarity of the stationary phase (e.g. C₁₈ hydrophobic chains) with hydrophilic compounds eluting first.
183
How does normal-phase HPLC differ from reverse-phase?
Normal-phase uses a polar stationary phase; non-polar compounds elute first, then polar ones.
184
What is HILIC chromatography?
Hydrophilic Interaction LC: polar stationary phase with an aqueous layer between it and organic mobile phase.
185
Which mobile phase composition separated three cyclic compounds most effectively?
Method 2 (30% ACN / 70% H₂O at pH 4.0): good separation with short run time.
186
What is gas chromatography (GC) best suited for?
Volatile mixtures, using an inert carrier gas and coated column, with high sensitivity and specificity.
187
Name three GC detector types.
Flame Ionization Detector (FID), Mass Spectrometer (MS), Electron Capture Detector (ECD).
188
How do column length and internal diameter affect GC resolution?
Longer columns and smaller diameters increase resolution but also analysis time and pressure requirements.
189
What is temperature programming in GC?
Gradually increasing the oven temperature during the run to separate analytes with a wide range of boiling points.
190
What defines a supercritical fluid in chromatography?
A substance above its critical temperature and pressure, behaving between a gas and a liquid.
191
Give one advantage of SFC for decaffeination.
Supercritical CO₂ penetrates intact coffee beans, dissolves caffeine efficiently, and evaporates at ambient conditions.
192
What is the principle behind UV/Vis detection?
Measurement of absorbance at a predefined wavelength via a monochromator and photodetector.
193
How does DAD (Diode Array Detection) improve on UV/Vis?
Records full absorbance spectra simultaneously across many wavelengths, without selecting one in advance.
194
Outline the basic steps in LC–MS/MS with a triple quadrupole.
Ionisation → Q1 isolation → collision-induced fragmentation in Q2 → Q3 filtering → detection of fragments.
195
What advantage does Time-of-Flight (TOF) MS offer?
High accuracy and fast scan rates for detecting many ions simultaneously, though expensive.
196
How does an Orbitrap mass analyser work?
Ions orbit in an.
197
How does an Orbitrap mass analyser work?
Ions orbit in an electrostatic trap; their oscillation frequency, read via image currents, is proportional to m/z.
198
Why might MS detect more compounds than UV in a cannabis extract?
MS captures all ionisable species (e.g. THC, THCA, CBD, CBDA) while UV only detects those absorbing at the set wavelength.
199
What three factors determine the best chromatographic method?
Speed/efficiency, sample matrix complexity, and number of compounds to detect.
200
What are the five broad application areas of small-molecule chromatography?
Pharmaceuticals, environmental monitoring, food & beverage, clinical diagnostics, and chemical synthesis.
201
How is chromatography used in drug discovery?
To isolate active compounds from complex mixtures (natural or synthetic), quantify them, and guide early-stage screening.
202
What role does chromatography play in pharmacokinetics studies?
Identifying and quantifying drug metabolites in biological matrices to define absorption, distribution, metabolism, and excretion (ADME) profiles.
203
Why is chromatography essential for drug formulation and stability testing?
It assesses compatibility with excipients and detects degradation products under various conditions, ensuring safety and shelf-life.
204
What quality control functions does chromatography serve in pharmaceuticals?
Verifying purity of raw materials, intermediates, and final products; batch release testing; and validating manufacturing consistency.
205
How does chromatography help detect counterfeit drugs?
By comparing chemical profiles to pharmacopeial standards to reveal wrong ingredients, dosages, or harmful contaminants.
206
In environmental monitoring, what samples are commonly analysed?
Water (surface, groundwater, wastewater), air (VOCs/POPs), soil, and industrial effluents for pollutants like pesticides, PFAS, and hydrocarbons.
207
Why are PFAS called 'forever chemicals'?
They do not break down, accumulate in the environment, and pose long-term health risks such as cancer and thyroid disease.
208
Which regulatory bodies enforce environmental standards using chromatography?
Agencies like the US EPA, EA, and EEA, under acts such as the Clean Water Act and Clean Air Act.
209
How is chromatography applied in food safety testing?
Detecting pesticides, herbicides, mycotoxins, heavy metals, veterinary drug residues, and processing contaminants in agricultural and processed foods.
210
What nutritional analyses can chromatography perform on food?
Quantifying vitamins, minerals, fatty acid profiles (e.g., omega-3/6), and authenticating geographical origin and product purity.
211
How does chromatography support flavour and aroma control?
By profiling flavour compounds and off-flavours to ensure product consistency and guide new formulation development.
212
What clinical diagnostics use chromatography for therapeutic drug monitoring?
Measuring blood or plasma levels of antibiotics, antiepileptics, chemotherapy agents, and immunosuppressants to stay within therapeutic ranges.
213
How is chromatography used in clinical toxicology?
To detect and quantify toxins, drugs of abuse, and poisons in biological samples, e.g., blood alcohol or opioid screens via GC or LC-MS.
214
What metabolic disorders can be screened by chromatography?
Inborn errors of metabolism in neonates, by measuring abnormal amino acids, organic acids, or fatty acids in blood/urine.
215
How does chromatography facilitate hormone analysis?
Quantifying steroid, thyroid, and peptide hormones in blood/urine to diagnose endocrine disorders and monitor therapy.
216
What is a key advantage of LC–MS/MS in clinical diagnostics?
High specificity and sensitivity for multiple analytes in complex biological matrices, supporting biomarker discovery and monitoring.
217
For nutritional deficiency testing, which analytes are measured?
Vitamins (e.g., D, B12), trace elements (iron, zinc), and fatty acids to assess patient nutritional status.
218
How does chromatography underpin pharmacokinetic studies in drug trials?
By quantifying drug and metabolite levels in plasma/tissues at multiple time points post-dose to model ADME dynamics.
219
What chromatography techniques monitor reaction progress in synthesis?
TLC for quick checks and HPLC for precise monitoring of reactant consumption and product formation over time.
220
Describe 'flash purification' in peptide synthesis.
Normal-phase column chromatography using silica gel to separate a highly polar target peptide from less polar impurities.
221
Why is method validation important in chemical synthesis QC?
To prove chromatographic methods are accurate, precise, specific, and robust for regulatory compliance and batch consistency.
222
How does stationary/mobile phase choice affect purification?
Based on compound polarity, selecting the right solid phase (e.g., silica) and solvent system ensures efficient separation of target molecules.
223
What information does HPLC-MS provide about a synthesized compound?
Molecular weight and structural fragments, confirming correct synthesis and identifying by-products.
224
In environmental PFAS analysis, what sample prep is used?
Solid-phase extraction to concentrate analytes from water, followed by LC-MS/MS detection.
225
What makes GC-MS suitable for volatile analytes in clinical toxicology?
Its ability to separate and identify small, volatile molecules with high sensitivity after derivatisation if needed.
226
How does DAD differ from single-wavelength UV detection?
DAD records full spectra across wavelengths, enabling peak purity checks and retrospective analysis.
227
What are the three criteria to select an optimal chromatographic method?
Speed/efficiency, sample matrix complexity, and number of analytes to detect.
228
What is electrophoresis?
A technique that uses an electric field to separate charged molecules by their mobility through a matrix.
229
State Ohm’s law as it applies to electrophoresis.
V/I = R; constant voltage and decreasing resistance increase current, generating heat that can melt gels.
230
What factors determine a molecule’s migration speed?
Its charge-to-size ratio (mobility) and the pore size/ionic strength of the medium.
231
Name three common electrophoresis media and their typical uses.
Agarose for DNA/RNA, polyacrylamide (PAGE) for proteins or small nucleic acids, capillaries for high-resolution separations.
232
Why is agarose gel typically run horizontally?
Its large pore sizes (0.5–2%) and horizontal format accommodate DNA’s native structure and prevent sample distortion.
233
What is the purpose of loading dye in agarose gels?
To visualize sample migration (e.g., bromophenol blue) and increase sample density so it sinks into wells.
234
How are DNA bands visualized after agarose electrophoresis?
By staining with ethidium bromide or non-toxic fluorescent dyes and viewing under UV light.
235
What is a Southern blot?
Transfer of DNA fragments from a gel to membrane, then hybridization with labeled probes to detect specific sequences.
236
What key reagents denature proteins in SDS-PAGE?
SDS detergent, β-mercaptoethanol (or DTT), and heat (boiling) to linearize proteins.
237
Why are SDS-PAGE gels 'discontinuous'?
They use a stacking gel (pH 6.8) and resolving gel (pH 8.8) to concentrate proteins before separation for sharper bands.
238
What determines separation in SDS-PAGE?
Protein chain length, since SDS coats proteins with uniform charge per mass, so mobility ≈ size.
239
How is protein molecular weight estimated from an SDS-PAGE gel?
Plot log(MW) of standards versus relative migration distance and interpolate the unknown’s migration.
240
What is Western blotting?
Transfer of proteins from gel to membrane, then detection with primary and enzyme-linked secondary antibodies for amplification.
241
Why use both primary and secondary antibodies in a Western blot?
Secondary antibodies amplify signal by binding multiple epitopes on the primary, enhancing detection sensitivity.
242
What is isoelectric focusing?
Separation of proteins by their isoelectric point on a pH gradient gel, migrating until net charge is zero.
243
How does 2D electrophoresis combine separation principles?
First dimension separates by pI (isoelectric focusing), second by size (SDS-PAGE), yielding high-resolution protein maps.
244
What advantage does capillary electrophoresis offer for DNA sequencing?
Automated, high-resolution separation of fluorescently labeled fragments in narrow capillaries, replacing manual slab gels.
245
Contrast manual versus automated Sanger sequencing.
Manual uses four separate ddNTP reactions on large gels; automated mixes fluorescent ddNTPs in one run through capillaries.
246
Beyond DNA, how can capillary electrophoresis be applied?
Separation and detection of metabolites or small molecules by migration time and detector response.
247
In electrophoresis, why must gels be buffered?
To maintain pH, conductivity, and prevent overheating that would distort or melt the gel matrix.
248
Name two safety cautions when performing electrophoresis.
Always use buffer to avoid gel melting and never touch electrodes or buffer to prevent electric shock.
249
What is the main purpose of protein chromatography?
To purify proteins by exploiting differences in their size, charge, binding affinity, or hydrophobicity using a stationary matrix and mobile phase
250
Name four preliminary steps in preparing a protein sample for chromatography.
Homogenisation, sonication, freeze–thaw cycles, and organic solvent extraction
251
What is “salting out” and how does it aid protein purification?
Addition of salts (e.g., ammonium sulfate) lowers protein solubility, causing selective precipitation and easier recovery by centrifugation
252
Why might detergents like Triton X-100 or CHAPS be used in protein purification?
To dissolve membranes and keep proteins soluble without denaturing them (unlike harsher detergents such as SDS)
253
What is the role of ultracentrifugation in fractionation?
It separates cellular components or organelles by density, enriching specific fractions before chromatography
254
How does dialysis contribute to protein purification?
By placing sample in semipermeable tubing to remove small solutes, retaining proteins in the tubing while impurities diffuse out
255
List the five main chromatographic methods for protein purification.
Size-exclusion, ion-exchange, affinity, hydrophobic interaction, and chromatofocusing
256
In ion-exchange chromatography, what dictates whether cation or anion exchange is used?
The net charge of the target protein at the chosen pH: cation exchange binds positively charged proteins; anion exchange binds negatively charged ones
257
What is gradient elution in ion-exchange chromatography?
Gradually changing buffer conditions (e.g., increasing salt) to sequentially elute bound proteins based on binding strength
258
How do void volume and total volume relate to size-exclusion chromatography?
Void volume is the mobile-phase volume eluting unretained molecules; total volume is all accessible stationary-phase volume
259
Which proteins elute first in size-exclusion chromatography?
The largest proteins, because they cannot enter pores and thus travel straight through the column
260
What is the “exclusion limit” in size-exclusion chromatography?
The molecular weight above which molecules are too large to enter the stationary-phase pores and elute at void volume
261
Describe the principle of affinity chromatography.
A specific ligand is covalently attached to beads; target proteins bind via high affinity and are eluted by competing ligand or changing buffer conditions
262
Why is a spacer arm used in affinity chromatography matrices?
To reduce steric hindrance between immobilised ligand and target protein, improving binding efficiency
263
How does hydrophobic interaction chromatography (HIC) separate proteins?
By binding proteins to a hydrophobic stationary phase under high-salt conditions, then eluting them as salt concentration is lowered
264
What is chromatofocusing?
A variant of ion-exchange where proteins are eluted by creating a pH gradient, separating them at their isoelectric points
265
How can genetic engineering aid protein purification?
By fusing a gene for a target protein to a tag gene (e.g., GST), enabling affinity purification via the tag’s known ligand
266
What advantage does a GST tag offer?
GST binds glutathione on beads; elution with free glutathione yields high-purity fusion protein, which can be proteolytically cleaved to remove the tag
267
Why must the tag often be removed before functional studies?
Tags can alter protein folding or function, so protease cleavage is used to recover the native protein
268
What key variables can be adjusted in column chromatography?
Column dimensions, packing material and particle size, mobile-phase composition, flow rate, temperature, and pressure
269
Why is column equilibration important before sample loading?
To stabilise the stationary phase in the chosen buffer, ensuring reproducible binding and elution properties
270
What role do detectors (e.g., spectrophotometers, mass spectrometers) play in chromatography?
They monitor eluent composition in real time, allowing detection and quantification of separated proteins
271
How does protein concentration measurement follow chromatography?
By collecting fractions, measuring absorbance or using specific assays, then pooling those containing the target protein
272
What is the final step after eluting the target protein from a column?
Cleaning and re-equilibrating the column for reuse, and analysing yield and purity of the recovered protein
273
What are the three main sections required in your LC–MS practical report?
Introduction, Results, Discussion & Conclusion (plus Bibliography).
274
In the Introduction, what should you explain about LC–MS?
That liquid chromatography separates analytes in a column (by gravity or high pressure for HPLC) and mass spectrometry detects them.
275
Which two analytes did this practical focus on?
Nortriptyline and 10-hydroxy-nortriptyline.
276
What two standard curves must you produce in your results?
One for nortriptyline and one for 10-OH-nortriptyline, each with properly labelled axes, units, title, R², p-value and equation.
277
How should your graphs be formatted?
Include a descriptive title, labelled axes (with units), legend if needed, and overlay of fitted line with R², p-value, and regression equation.
278
What should you comment on besides the curve fit?
The meaning of the p-value (significance) and R² (goodness of fit).
279
When analysing group comparisons, what should you include?
Bar or scatter plots of metabolite peak areas for each patient group, annotated with statistical significance markers.
280
What two questions should your Discussion address?
What type of chromatography was used (e.g. reverse-phase) and why group metabolite levels differ (relate to metabolism pathways).
281
What general conclusion should you draw?
The usefulness of LC–MS for small-molecule quantification and interpretation of metabolic differences between groups.
282
Where can you find help with statistics and GraphPad Prism?
Lecture 4 in week 2 of this module (and BMS1054 materials).
283
What style and depth should your report’s writing follow?
Formal scientific language, sufficient detail in discussion (longer for higher-mark sections), and use of precise terminology.
284
What are best practices for selecting references?
Use peer-reviewed academic sources (PubMed, journals), avoid over-reliance on websites, ensure citations are accurate and complete.
285
What warning signs suggest AI-generated or incorrect references?
Unusual journal titles or volumes, incorrect author lists, missing authors, or bibliographic details not matching the source.
286
In obstructive jaundice, why is ALP elevated?
Blocked bile ducts prevent ALP secretion into bile, causing it to be reabsorbed into blood.
287
Why are blood glucose tubes coated with fluoride?
Fluoride inhibits red-cell glycolysis, preventing post-collection glucose degradation.
288
What causes polydipsia and polyuria in uncontrolled diabetes?
Blood glucose exceeds renal threshold, leading to osmotic diuresis and dehydration.
289
What compensatory mechanisms occur in diabetic ketoacidosis?
Hyperventilation (to blow off CO₂) and renal acid–base adjustments (HCO₃⁻ reclamation and H⁺ excretion).
290
Why does diabetic ketoacidosis give breath a fruity smell?
Accumulation of acetone and other ketones, which are volatile acids.
291
Which additional lab tests are recommended in diabetic patients?
Kidney function tests and serum electrolytes to check for dehydration and renal impairment.
292
What are the clinical signs of diabetic ketoacidosis?
Confusion, rapid breathing, low blood pH (<7.35), low bicarbonate, high blood glucose, and ketonuria.
293
What two questions assess protein purity and characteristics after purification?
“How pure is our protein?” and “What is its isoelectric point (pI) and approximate molecular weight?”
294
In PAGE gels, what two factors determine a protein’s migration?
Its charge-to-mass ratio and, to a lesser extent, its shape.
295
How does SDS-PAGE ensure proteins separate by size only?
SDS binds proteins in a 1:1 ratio per amino acid, denaturing them into rod-shapes and equalising charge-to-mass.
296
What stain is commonly used to visualise proteins in SDS-PAGE?
Coomassie Brilliant Blue.
297
How is a protein’s isoelectric point (pI) defined?
The pH at which its net charge is zero.
298
In isoelectric focusing, how are proteins separated by pI?
A pH gradient is applied in a gel; each protein migrates until the local pH equals its pI.
299
What key advantage does chromatofocusing offer?
Final-polishing separation of proteins by very small differences in pI using a continuous pH gradient.
300
Does chromatofocusing use an electric field?
No—proteins elute by changing buffer pH in a Fast Protein Liquid Chromatography (FPLC) column.
301
In chromatofocusing, which proteins elute first?
Those with the highest pI (they become negatively charged later and migrate fastest).
302
What risk must be managed during chromatofocusing at high protein concentration?
Protein aggregation that can block the column.
303
How does two-dimensional (2D) gel electrophoresis combine techniques?
First dimension separates by pI (isoelectric focusing), second by size (SDS-PAGE) for high resolution.
304
Name two methods for desalting protein samples post-chromatography.
Dialysis and commercially available desalting columns.
305
List four ways to concentrate protein samples after purification.
Lyophilisation (freeze-drying), ultrafiltration, chromatographic concentration, precipitation.
306
What assays assess the yield and purity of purified protein?
Enzyme activity assays, total protein assays, SDS-PAGE analysis, and Western blotting.
307
In Western blotting, why use a secondary antibody?
To amplify signal by binding multiple labels per primary antibody, increasing sensitivity.
308
What is the first step in producing a recombinant protein?
Isolate or PCR-amplify the gene encoding the protein of interest.
309
Why convert mRNA to cDNA when making recombinant proteins?
cDNA lacks introns, making it suitable for expression in bacterial or other host systems.
310
What is the purpose of an expression vector?
To provide promoter, ribosome-binding site, and terminator sequences that drive protein production in the host.
311
Give three types of host cells used for recombinant protein expression.
Bacteria (e.g. E. coli), yeast, and mammalian cell cultures.
312
Name three laboratory applications of recombinant proteins.
ELISA standards, Western blot controls, and enzyme assays.
313
Name three therapeutic applications of recombinant proteins.
Synthetic insulin, monoclonal antibody treatments, and clotting factors for haemophilia.
314
What industrial use do recombinant proteins have in agriculture?
Enhancing crop resistance to pests or improving animal-feed nutritional value.
315
List two ethical or safety concerns with recombinant protein use.
Potential misfolding leading to toxicity or immune reactions, and unintended environmental spread of GM material.
316
What is an “inclusion body” in recombinant protein expression?
Aggregated, insoluble protein deposits formed when host folding machinery is inadequate.
317
Why might recombinant proteins lack post-translational modifications?
Prokaryotic hosts often cannot perform eukaryotic-specific modifications like glycosylation.
318
What does “spectroscopy” literally mean and what does it study?
From Latin “ghost/spirit” and Greek “to see”; it studies matter’s interaction with light across the electromagnetic spectrum.
319
What are the main types of spectroscopy covered in this lecture?
UV/Vis absorbance, photoluminescence (fluorescence), bio- and chemo-luminescence, X-ray crystallography, and NMR.
320
In absorbance spectroscopy, what causes the decrease in transmitted light?
Sample molecules absorb photons, reducing light intensity reaching the detector.
321
State the Beer–Lambert law and its components.
A = ε·c·l, where A is absorbance, ε the molar extinction coefficient, c concentration, and l pathlength.
322
Name two real-world factors that violate the ideal Beer–Lambert situation.
Sample turbidity (scattering) and stray reflections or refractions in the cuvette.
323
How does polarisation spectroscopy provide information on molecular structure?
By measuring the rotation or phase shift of polarised light passing through chiral or anisotropic samples.
324
Give one kinetic application of UV/Vis absorbance.
Monitoring NADH absorbance at 340 nm to follow enzyme reactions over time.
325
How is DNA purity assessed using UV absorbance?
By the A₂₆₀/A₂₈₀ ratio—pure DNA yields approximately 1.8.
326
What distinguishes photoluminescence from thermal emission?
Photoluminescence emits light after photon absorption without requiring heat.
327
Compare fluorescence and phosphorescence lifetimes.
Fluorescence: 10⁻¹⁰–10⁻⁸ s (stops immediately when excitation stops); phosphorescence: 10⁻³–10² s (persists after excitation).
328
Define quantum yield in fluorescence.
The fraction of excited molecules that return to ground state by emitting a photon (range 0–1).
329
What is an excitation spectrum versus an emission spectrum?
Excitation spectrum: emitted light intensity monitored at fixed emission wavelength while varying excitation; emission spectrum: emission intensity vs. emission wavelength at fixed excitation.
330
List two advantages of fluorescence over UV/Vis absorbance.
Higher sensitivity at low concentrations and ability to distinguish overlapping species via characteristic emission peaks.
331
Why can’t fluorescence be used with turbid or opaque samples?
Scattering and absorption by particulates prevent accurate measurement of emitted light.
332
How does quenching affect fluorescence measurements?
Quenching reduces fluorescence intensity through collisional or static interactions, often exploited in FRET assays.
333
What is Förster Resonance Energy Transfer (FRET)?
Nonradiative energy transfer from an excited donor fluorophore to an acceptor when they are within ~1–10 nm and spectral overlap exists.
334
Describe a typical FRET microscopy setup.
Excitation light illuminates the donor; emitted light from the acceptor is detected at 90° to minimise scattered excitation.
335
What information does Fluorescence Recovery After Photobleaching (FRAP) provide?
The kinetics of molecular diffusion or mobility in membranes or cytoplasm, by measuring fluorescence recovery in a bleached region.
336
Give one example of bioluminescence in nature and one laboratory use.
Nature: firefly flashes (luciferase reaction); Lab: luciferase reporter assays for gene expression.
337
How does chemiluminescence with luminol detect blood traces?
Luminol is oxidised (e.g., by H₂O₂ with Fe²⁺ catalyst), forms an excited intermediate that emits blue light as it returns to ground state.
338
What is epifluorescence microscopy?
A technique where excitation and emission light paths share the same objective lens, allowing high-contrast fluorescence imaging.
339
How do fluorescent proteins like GFP aid microscopy?
They can be genetically fused to proteins of interest, providing intrinsic fluorescence without external dyes.
340
What advantage does confocal fluorescence microscopy offer?
Optical sectioning for high-resolution, three-dimensional reconstructions by excluding out-of-focus light.
341
What is near-infrared (NIR) absorption spectroscopy useful for?
Monitoring overtone and combination vibrations (800–2500 nm), e.g., fermentation process control or water content analysis.
342
Name two structural biology techniques beyond spectroscopy.
X-ray crystallography for electron density mapping and NMR for atomic‐level interactions in solution.
343
In X-ray crystallography, what is measured to determine atomic positions?
The angles and intensities of X-ray diffraction spots from a crystal lattice.
344
What limitation does X-ray crystallography have?
The requirement for high‐quality crystals, which some proteins or complexes cannot form.
345
How does NMR spectroscopy reveal molecular structure?
It measures resonance frequencies of nuclear spins (e.g., ¹H, ¹³C, ¹⁵N) in a magnetic field, reflecting chemical environment and distances.
346
What is the basic NMR experiment sequence?
Align nuclear spins in a strong magnetic field, apply an RF pulse to excite them, then detect emitted signals as they relax.
347
Give one application of NMR in biochemistry.
Determining protein folding states, conformational changes, and interactions with ligands in solution.
348
What is the difference between analytical chemistry and clinical biochemistry?
Analytical chemistry focuses on screening, diagnosis, prognosis, and monitoring using techniques like chromatography, electrophoresis, and spectroscopy; clinical biochemistry applies these analyses to bodily fluids for diagnostic and therapeutic purposes.
349
Name three sub-specialties of clinical biochemistry.
Special chemistry (elaborate techniques), clinical endocrinology (hormone disorders), and therapeutic drug monitoring (optimizing medication levels).
350
Which four electrolytes are measured in a basic metabolic panel?
Sodium (Na⁺), potassium (K⁺), chloride (Cl⁻), and bicarbonate (HCO₃⁻).
351
What markers are included in liver function tests (LFTs)?
Albumin, total protein, A/G ratio, bilirubin (direct & indirect), AST, ALT, GGT, and ALP.
352
List two cardiac markers and their clinical use.
Troponin (heart muscle injury) and CK-MB (acute myocardial infarction).
353
What are the advantages and disadvantages of point-of-care testing?
Advantages: faster results, smaller samples; Disadvantages: often less accurate than central lab tests.
354
Give two examples of near-patient tests.
Blood glucose meters and rapid strep tests.
355
What is therapeutic drug monitoring (TDM)?
Measuring drug concentrations in body fluids to optimize dosing for efficacy and safety.
356
Name two factors that affect drug availability in the body.
Liver enzyme activity (e.g., CYP450 induction/inhibition) and patient compliance.
357
What distinguishes Phase 1 from Phase 2 drug metabolism?
Phase 1 introduces or exposes functional groups (e.g., via CYP450); Phase 2 conjugates these groups (e.g., glucuronidation).
358
Define the therapeutic window.
The range between the minimum effective dose (MED) and the maximum tolerated dose (MTD).
359
What is the purpose of quality control in clinical biochemistry?
To monitor analytical performance through internal controls and external proficiency testing.
360
Why can genetic variation influence drug response?
Differences in metabolizing enzymes (e.g., CYP2D6 polymorphisms) alter drug clearance and effects.
361
What are the two phases of chromatography?
The mobile phase (gas or liquid carrying sample) and the stationary phase (solid or coated matrix).
362
List three main types of small-molecule chromatography.
HPLC (reverse- or normal-phase), GC (volatile compounds), and SFC (supercritical fluids).
363
Why is UV/Vis–DAD detection advantageous?
It records absorbance across multiple wavelengths simultaneously for spectral analysis.
364
What three column variables affect chromatographic separation?
Column dimensions, packing particle size, and mobile-phase composition.
365
In protein purification, what are “salting out” and dialysis used for?
“Salting out” precipitates proteins selectively; dialysis removes small solutes and exchange buffers.
366
How do ion-exchange and size-exclusion chromatography differ?
Ion-exchange separates by charge under controlled pH/salt gradients; size-exclusion separates by molecular size via pore exclusion.
367
What is the principle of SDS-PAGE?
SDS denatures proteins and gives them uniform charge-to-mass, so they separate by size in an electric field.
368
How does isoelectric focusing separate proteins?
Proteins migrate in a pH gradient until they reach their isoelectric point (net zero charge).
369
What does 2D electrophoresis combine?
First-dimension isoelectric focusing (separation by pI), second-dimension SDS-PAGE (separation by size).
370
What are three common spectroscopic methods in clinical biochemistry?
UV/Vis absorbance (quantitation), fluorescence (sensitivity and specificity), and NMR (structural analysis).
371
State the Beer–Lambert law.
A = ε·c·l (absorbance equals molar extinction coefficient times concentration times pathlength).
372
What is FRET used to measure?
Nanoscale distances between fluorophores, indicating molecular interactions (1–10 nm).
373
Give one application of bioluminescence assays.
Luciferase reporter assays for monitoring gene expression.
374
How does chemiluminescence with luminol detect blood?
Oxidation produces an excited intermediate that emits blue light upon returning to ground state.
